Method for producing universal immune cells and its application

Abstract

The present invention provides a simple method for producing universal immune cells and its applications. The method involves co-culturing allogeneic immune cells with specific cell mitogens, cytokines, and immune adjuvants in a liquid cell culture medium in a culture vessel to obtain a universal immune cell culture with high immune activity. The method is simple and easy to operate, and employs allogeneic immune cells to improve the quality of immune cell preparations, reduce the cost of cell therapy, and enhance safety. Furthermore, numerous long-term clinical applications have proven that the universal immune cells are safe, reliable, durable, and non-rejection-prone. Therefore, they can be used as parent cells for other types of universal immune cell preparations, such as CAR-T, TCR-T, or other immune cell types, in the field of adoptive cellular immunotherapy. This method solves the high cost, poor safety, and low efficiency of current cellular immunotherapies, providing a new avenue for the application and promotion of cellular immunotherapy.

Description

[Technical field]

[0001] The present invention relates to the field of biomedicine, in particular to a method for preparing universal immune cells and its application. [Background technology]

[0002] In recent decades, the field of cancer therapy has made great progress. In particular, cell-based therapies targeting tumor-associated antigens (TAA) have been rapidly developed. T cells have been engineered to have the ability to attack tumor cells by generating CAR constructs consisting of genes encoding scFv, costimulatory domains (CD28 or TNFRSF9), and CD247 signaling domains for T cell proliferation and activation. In principle, CAR-T cells are activated by the recognition of TAA by scFv on the surface of T cells, followed by activation of intracellular signaling domains linked by scFv, inducing downstream signaling involved in T cell proliferation, activation, and cytokines.

[0003] Currently, the effective function of CAR-T cell therapy has been demonstrated in various diseases, including hematological malignancies, solid tumors, autoimmune diseases, and allergic diseases such as asthma. Furthermore, antigen-specific regulatory T cells (Tregs) and gene-edited T cells appear to be beneficial in controlling inflammation in allergic asthma.

[0004] Unlike conventional drugs, CAR-T cell therapy is mainly an autologous T cell therapy incorporating CAR genes. This therapy requires rapid completion of blood collection, isolation, activation, gene transfer, proliferation, preparation, release, freezing, transportation, and drug administration. Therefore, CAR-T cell therapy is a rather expensive and personalized process that requires expensive measures to collect the patient's cells, manipulate these cells, and reinject the cells into the patient at each stage, followed by appropriate quality control. In addition, conventional character antigen receptors (CARs) are fixed in design, and one type of CAR-T cell can only target one antigen epitope. Such a strict design limits clinical applications and leads to very high manufacturing costs.

[0005] In 2017, two CAR-T cell therapies (Yescarta and Kymriah) were approved by the FDA. CAR-T cell therapy has become one of the most promising treatments for certain types of cancer, providing an opportunity to optimize the production of CAR-T cells and making them more accessible to patients. However, the currently approved T cell products with two types of character antigen receptors (CARs) are derived from autologous T cells. These clinically approved CAR-T cells need to be generated on a custom basis. This autologous T cell production platform remains a key limiting factor for large-scale clinical application due to its high cost and lengthy production process. In addition to the inherent risk of production failure, the production process of personalized and tailored autologous CAR-T cells also affects the scope of application in various tumor types.

[0006] To make advanced cell therapy more widely available and reduce costs, it is advantageous to use allogeneic "universal" cell therapy products. The above allogeneic "universal" cell therapy products can be stored in cell banks in a similar manner to biopharmaceuticals and made available upon request. Therefore, universal allogeneic T cells can be used to manufacture universal CAR-T cells, which can be used as "off the shelf" ready-to-use biotherapeutics for large-scale clinical applications. This will significantly reduce the cost and time required for traditional CAR-T cell therapy. At present, the research and development of universal CAR-T mainly involves the type of immune cells and their production, as well as the carrier and structure of CAR-T. The main technical routes are erasure of the expression of endogenous αβ T cell receptor (TCR) to prevent graft-versus-host reaction, genome editing technology, other types of immune cells, and iPSC technology.

[0007] When injecting allogeneic CAR-T cells, it is necessary to avoid host-versus-graft and graft-versus-host rejection reactions to prevent rejection of adoptively transferred cells and damage to host tissues, and to elicit significant antitumor effects. Currently, the editing method employed involves knocking out genes such as endogenous T cell receptors that prevent allogeneic therapy while introducing CAR, thereby eliminating the presence of TCRαβ receptors on the cell surface and preventing alloreactivity. This technology obviously further increases the complexity of the manufacturing process, and also has the potential risk of incompletely knocking out the TCRαβ receptor.

[0008] Because it can edit multiple gene targets simultaneously, the CRISPR / Cas9 system has been adopted to improve CAR-T cells in some clinical trials. The combination of these powerful technologies is expected to provide a new generation of CAR-T cell-based immunotherapy for clinical application; that is, knocking out endogenous TCRs by gene editing technology improves the antitumor activity and safety of T cells. However, this method also further increases the complexity of manufacturing operations and increases production costs.

[0009] Natural killer (NK) cells can be xenotransplanted and potentially off-the-shelf, making CAR-NK cell therapy a versatile product. These products may be safer than CAR-T cell therapy. Scientists reported a novel PCa treatment based on CARNK-92 cell engineering, using a CAR that recognizes human prostate-specific membrane antigen (PSMA), which is overexpressed on prostate tumor cells. After CAR transduction, NK-92 / CAR cells acquire high specificity and lytic activity against PSMA-expressing prostate cancer cells in vitro, degranulate in response to antigen recognition, and produce high levels of IFN-γ. However, this technology requires lethal irradiation of effectors. This is a necessary safety measure for clinical application of targeted NK-92 cells, which can completely erase replication without affecting their phenotype or short-term function.

[0010] Other researchers have explored technological routes to develop off-the-shelf cell therapies based on induced pluripotent stem cell (iPSC)-derived natural killer (NK) cells and proposed strategies to engineer iPSC-derived NK (iPSC-NK) cells to enhance immune cell function, prolong persistence, and promote cell homing. However, to date, there have been no reports of iPSC-derived CTLs (iPSC-CTLs).

[0011] The present invention relates to the antigen-specific immune cell drug (CN101626781B, CN1028471438, CN102847144B) previously applied for in clinical use for nearly 10 years, which has treated approximately 800 tumor patients ranging in age from 18 months to 93 years, with the maximum number of treatments being more than 500, and the maximum number of treatments being more than 10 years. The total number of peripheral blood leukocytes was 0.9-30.0×10 9 / L. Compared to the 50-60% incidence of graft-versus-host disease (GVHD) in patients treated with conventional DLI, multicenter clinical trials have demonstrated that DLI is highly safe and does not cause GVHD or autoimmune diseases.

[0012] Therefore, based on the previously filed patent, the applicant has designed a method for producing universal allogeneic T cells, which provides parent cells for other types of universal immune cell preparations, such as CAR-T, TCR-T, or other immune cell types, for use in fields such as adoptive immune cell therapy, which can improve the quality of immune cells, reduce the cost of cell therapy, and improve safety. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] CN101626781B [Patent Document 2] CN1028471438 [Patent Document 3] CN102847144B Summary of the Invention

[0014] The objective of the present invention is to overcome the shortcomings of the prior art and provide a simple method for preparing universal immune cells and its application, which are safe, reliable, long-lasting, effective and free of graft-versus-host rejection.

[0015] In order to solve the above problems, the present invention employs the following technical solutions.

[0016] A first embodiment of the present invention provides a method for producing universal immune cells, comprising: Step S1 of co-culturing allogeneic immune cells (e.g., mononuclear cells, etc.), cell mitogens, cytokines, and immune adjuvants in a liquid cell culture medium in a culture vessel to obtain a universal immune cell culture; A step S2 of isolating a cell population from the culture obtained in step S1, Here, the cytokine is selected from any one or more of lymphokines, monokines, cytokines that activate inflammation, and cytokines that stimulate hematopoiesis, produced by lymphocytes, monocytes, and other cells.

[0017] Further, the cytokines are selected from any one or more of the following: interleukins, interferons, tumor necrosis factors, colony stimulating factors, chemotactic cytokines, and transforming growth factors.

[0018] Further, the cytokine is one or more of interleukin-2, interleukin-6, interleukin-15 and interferon.

[0019] Furthermore, the concentration of the above cytokines is 200,000 to 5,000,000 units / L.

[0020] Furthermore, the concentration of the above cytokines is 500,000 to 2,000,000 units / L.

[0021] Further, the mitogen is selected from any one or more of concanavalin, phytohemagglutinin, pokeweed, lipopolysaccharide, and dextran.

[0022] Furthermore, the concentration of the above mitogen is 100,000 to 10 million units / L.

[0023] Furthermore, the concentration of the mitogen is 0.1 mg to 10 mg / L.

[0024] Furthermore, the above-mentioned immune adjuvant is selected from any one or more of biological adjuvants, inorganic adjuvants, organic adjuvants, synthetic adjuvants, oils and Freund's adjuvants, and has a concentration of 0.01 mL to 1 mL / L.

[0025] Furthermore, the immune cells are derived from peripheral blood or umbilical cord blood.

[0026] Furthermore, the immune cells can be allogeneic or autologous cells.

[0027] Furthermore, the concentration of the above immune cells was 1 × 10 3 ~1×10 11 pieces / mL.

[0028] Furthermore, the above cell population includes any one or more of cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes (TILs), cytokine-activated killer cells (CIKs), lymphokine-activated killer cells (LAKs), natural killer cells (NKs), tumor-associated macrophages (TAMs), activated killer monocytes (AKMs), and dendritic cells (DCs).

[0029] Furthermore, the above-mentioned cell population having immune activity is lymphocytes such as CTL, NK, CIK and B cells, and most preferably cytotoxic T lymphocytes.

[0030] Furthermore, the co-culture time in step S1 is 3 to 180 days.

[0031] Furthermore, the above-mentioned culture vessel is a three-dimensional large-volume, high-density cell culture vessel.

[0032] The method further includes a cloning step of performing cloning in a liquid cell culture medium using the culture obtained in step S1 or the cell population having immune activity obtained in step S2 as a starting material to obtain a cell line having immune activity, the cloning being performed by either an intermittent cyclic stimulation method or a continuous stimulation method.

[0033] A second embodiment of the present invention provides a universal immune cell produced by the above method, the cell comprising any one or more of cytotoxic T lymphocytes (CTL), tumor infiltrating lymphocytes (TIL), cytokine-activated killer cells (CIK), lymphokine-activated killer cells (LAK), natural killer cells (NK), tumor-associated macrophages (TAM), activated killer monocytes (AKM), and dendritic cells (DC).

[0034] The third embodiment of the present invention provides the application of the universal immune cells in the second embodiment in the preparation of an immune cell preparation, in which the parent cells of the immune cell preparation are the universal immune cells.

[0035] Furthermore, the above immune cell preparations include CAR-T cells or TCR-T cells.

[0036] The fourth embodiment of the present invention provides an immune cell preparation, which is produced using the universal immune cell of the second embodiment as a parent cell.

[0037] A fifth embodiment of the present invention provides a biological agent for adoptive cellular immunotherapy, comprising a container and the immune cell preparation of the fourth embodiment disposed in the container.

[0038] Additionally, the above biological formulations may further include saline, albumin and other stabilizers.

[0039] The sixth embodiment of the present invention provides an application of the immune cell preparation according to the fourth embodiment of the present invention in the manufacture of a pharmaceutical for treating and preventing a disease associated with abnormalities in the function and quantity of lymphocytes.

[0040] Furthermore, the route of administration of the pharmaceutical agent may be intravenous, thoracic, intraperitoneal, intraspinal, intradermal, subcutaneous or intratumoral injection.

[0041] The present invention adopts the above technical solutions, and compared with the prior art, has the following technical advantages:

[0042] The method for producing universal immune cells adopted in the present invention is simple and easy to operate, and can adopt allogeneic immune cells to improve the quality of immune cell preparations, reduce the cost of cell therapy, and improve safety. In addition, a large number of clinical applications over a long period of time have proven that the universal immune cells are safe, reliable, durable, and free of rejection. Therefore, they can be used as parent cells for other types of universal immune cell preparations, such as CAR-T, TCR-T, or other immune cell types, in the field of adoptive cell immunotherapy. This solves the problems of high cost, low safety, low efficiency, etc. of current cell immunotherapy, and provides a new way for the application and promotion of cell immunotherapy.

[0043] In addition, the pharmaceutical manufacturing method of the present invention has changed the mechanism of action of traditional vaccines. Traditional vaccines are made of pathogens or their antigenic substances, and after being injected into the human body, the immune system produces protective substances (such as antibodies, etc.), and the immune system produces more protective substances according to the original memory to prevent the damage of pathogens. However, the present invention injects therapeutic vaccines containing cells with immune response reactions into the body, directly improving the body's immune function, and not only enhancing the resistance to tumors, pathogen infections such as HIV and hepatitis viruses, coronaviruses and other types of viruses, but also can be used to treat and prevent cancer, AIDS, and viral hepatitis, and is also effective in delaying aging. [Brief description of the drawings]

[0044] [Figure 1] FIG. 1 is a distribution diagram of significantly differential gene pathways in cell 1 based on pathway analysis in one embodiment of the present invention. [Diagram 2] FIG. 2 is an activation interaction diagram between signaling pathways in cell 1 and cell 2 in one embodiment of the present invention, where the shaded circles are unchanged signaling pathways, the shaded circles are activated signaling pathways in cell 1, and the blank circles are activated signaling pathways in cell 2. [Diagram 3] FIG. 3 is a distribution diagram of significantly differential gene pathways in cell 2 based on pathway analysis in one embodiment of the present invention.

[0045] (Specific embodiment) After extensive and detailed research, through numerous experiments and explorations, the present inventors have found that by co-culturing allogeneic mononuclear cells with cell mitogens, cytokines and immune adjuvants, universal immune cells can produce safe, reliable, long-lasting and non-rejection universal immune cells (especially CTLs), which can provide low-cost, safe and reliable parent cells for cellular immunotherapy of other types of universal immune cell preparations, such as CAR-T, TCR-T or other immune cell types.

[0046] The first embodiment of the present invention provides a simple method for preparing universal immune cells, which is obtained by co-culturing allogeneic mononuclear cells, cell mitogens, cytokines and immune adjuvants to obtain a large number of universal immune cells. The method specifically includes the following steps: Step S1: co-culturing allogeneic immune cells (e.g., mononuclear cells, etc.), cell mitogens, cytokines, and immune adjuvants in a liquid cell culture medium in a culture vessel to obtain a universal immune cell culture with high immune activity; and a step S2 of isolating a cell population having immune activity from the culture obtained in step S1. Here, cytokines may be derived from any one or more of lymphokines, monokines, cytokines that activate inflammation, and cytokines that stimulate hematopoiesis, produced by lymphocytes, monocytes, and other cells.

[0047] In one preferred embodiment of the present invention, the cytokine is derived from any one or more of interleukins, interferons, tumor necrosis factors, colony stimulating factors, chemotactic cytokines, and transforming growth factors, and in one preferred embodiment of the present invention, the cytokine is selected from one or more of interleukin-2, interleukin-6, interleukin-15, and interferons.

[0048] In one preferred embodiment of the invention, said cytokines are interleukin-2 and / or interferon.

[0049] During the co-culture process, allogeneic mononuclear cells can secrete IL-2 and other cytokines under the stimulation of cell mitogens, cytokines, and immune adjuvants, so they are less dependent on IL-2 and do not require IL-2. Of course, exogenous IL-2 can be added simultaneously during the culture to further promote the massive proliferation of activated T lymphocytes.

[0050] In one preferred embodiment of the present invention, the concentration of the above cytokines during culture is 200,000 to 5,000,000 units / L to obtain a culture of a cell population having immune activity. In one preferred embodiment of the present invention, the concentration of the above cytokines is 500,000 to 2,000,000 units / L.

[0051] In one preferred embodiment of the present invention, the mitogen is selected from any one or more of the following: concanavalin, phytohemagglutinin, pokeweed, lipopolysaccharide, and dextran.

[0052] In one preferred embodiment of the present invention, the concentration of the mitogen during culture is 100,000 to 10 million units / L to obtain a culture of a cell population having immune activity. In one preferred embodiment of the present invention, the concentration of the mitogen is 500,000 to 5 million units / L.

[0053] In one preferred embodiment of the present invention, the concentration of the mitogen during culture is 0.1 mg to 10 mg / L to obtain a culture of a cell population having immune activity. In one preferred embodiment of the present invention, the concentration of the mitogen during culture is 0.5 mg to 5 mg / L.

[0054] In one preferred embodiment of the present invention, the immune adjuvant is selected from any one or more of biological adjuvants, inorganic adjuvants, organic adjuvants, synthetic adjuvants, oils and Freund's adjuvants, and the immune adjuvant concentration during the culture is 0.01 mL-1 mL / L to obtain a culture of a cell population having immune activity. In one preferred embodiment of the present invention, the immune adjuvant concentration is 0.1 mL-0.5 mL / L.

[0055] In one preferred embodiment of the invention, the immune cells are derived from peripheral blood or umbilical cord blood. In one preferred embodiment of the invention, the immune cells are derived from peripheral blood.

[0056] In one preferred embodiment of the present invention, the immune cells may be allogeneic or autologous cells. In one preferred embodiment of the present invention, the immune cells are allogeneic cells.

[0057] In one preferred embodiment of the present invention, in order to obtain a culture of a cell population having immune activity, the concentration of the immune cells during the culture is 1×10 3 ~1×10 11 In one preferred embodiment of the present invention, the concentration of the immune cells is 1×10 5 ~1×10 9 pieces / mL.

[0058] However, different types of immune cells (i.e., allogeneic mononuclear cells) will require different ratios of cell mitogens, cytokines, and immune adjuvants, and one of skill in the art can determine the appropriate ratios based on testing and the characteristics of the immune cells.

[0059] In one preferred embodiment of the present invention, the immune activity cell population comprises cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes (TILs), cytokine-activated killer cells (CIKs), lymphokine-activated killer cells (LAKs), natural killer cells (NKs), tumor-associated macrophages (TAMs), activated killer monocytes (AKMs) and dendritic cells (DCs).

[0060] In one preferred embodiment of the present invention, the above-mentioned immune activity cell population is lymphocytes such as CTL, NK, CIK and B cells, and in a more preferred embodiment, the above-mentioned immune activity cell population is cytotoxic T lymphocytes.

[0061] In one preferred embodiment of the present invention, the co-culture time in step S1 is 3 to 180 days, in one preferred embodiment of the present invention, the co-culture time in step S1 is 3 to 60 days, in one preferred embodiment of the present invention, the co-culture time in step S1 is 7 to 28 days, and in a more preferred embodiment, the co-culture time in step S1 is 14 to 21 days.

[0062] In one preferred embodiment of the invention, the culture vessel is a three-dimensional large volume, high density cell culture vessel, although other known culture vessels, including bioreactors, are also possible.

[0063] In one preferred embodiment of the present invention, the method also includes a cloning step of performing cloning in a liquid cell culture medium using the culture obtained in step S1 or the cell population having immune activity obtained in step S2 as a starting material to obtain a cell line having immune activity, the cloning being performed by either an intermittent cyclic stimulation method or a continuous stimulation method.

[0064] A second embodiment of the present invention provides a universal immune cell produced by the above method, the cell comprising one or more of cytotoxic T lymphocytes (CTL), tumor infiltrating lymphocytes (TIL), cytokine-activated killer cells (CIK), lymphokine-activated killer cells (LAK), natural killer cells (NK), tumor-associated macrophages (TAM), activated killer monocytes (AKM), and dendritic cells (DC).

[0065] In one preferred embodiment of the present invention, the universal immune cells are lymphocytes, such as CTL, NK, CIK and B cells. In a more preferred embodiment, the universal immune cells are cytotoxic T lymphocytes.

[0066] The third embodiment of the present invention provides the application of the universal immune cells in the second embodiment in the preparation of an immune cell preparation, in which the universal immune cells serve as parent cells for the immune cell preparation.

[0067] In one preferred embodiment of the present invention, the above-mentioned immune cell preparation comprises CAR-T cells or TCR-T cells, and the immune cell preparation can be used for adoptive cellular immunotherapy and can be clinically used as a biopharmaceutical for the treatment and prevention of various blood diseases and various solid tumors.

[0068] In one preferred embodiment of the present invention, the blood disorders include red blood cell disorders such as aplastic anemia and paroxysmal nocturnal hemoglobinuria (PNH), and white blood cell disorders such as various leukemias, malignant lymphomas, malignant lymphoreticular disease, malignant lymphoreticular cytosis, plasma cell dyscrasias, histiocytoses, and myelodysplastic syndromes (polycythemia vera, essential thrombocythemia, myelofibrosis).

[0069] In a preferred embodiment of the present invention, the solid tumor is selected from nasopharyngeal cancer, esophageal cancer, gastric cancer, liver cancer, breast cancer, colorectal cancer, prostate cancer, lung cancer, cervical cancer, leukemia, oral cancer, salivary gland tumors, malignant tumors of the nasal cavity and paranasal sinuses, laryngeal cancer, ear tumors, eye tumors, thyroid tumors, mediastinal tumors, chest wall and pleural tumors, small intestinal tumors, biliary tract tumors, pancreatic and periampullary tumors, mesenteric and retroperitoneal tumors, kidney tumors, adrenal tumors, bladder tumors, prostate cancer, testicular tumors, penile cancer, endometrial cancer, ovarian malignant tumors, malignant trophoblastic tumors, vulvar and vaginal cancer, malignant lymphoma, multiple myeloma, soft tissue tumors, bone tumors, skin and adnexal tumors, malignant melanoma, nervous system tumors, various childhood tumors, etc.

[0070] The fourth embodiment of the present invention provides an immune cell preparation, which is produced using the universal immune cell of the second embodiment as a parent cell.

[0071] A fifth embodiment of the present invention provides a biological agent for adoptive cellular immunotherapy, comprising a container and the immune cell preparation of the fourth embodiment disposed in the container.

[0072] In one preferred embodiment of the present invention, the biological preparation further comprises saline, albumin and other stabilizers.

[0073] The sixth embodiment of the present invention provides an application of the immune cell preparation of the fourth embodiment of the present invention in a pharmaceutical manufacturing process for treating and preventing diseases associated with abnormalities in lymphocyte function and quantity, including, but not limited to, tumor immunity, transplant immunity, hypersensitivity immunity, autoimmune diseases, immunoproliferative diseases, immunodeficiency diseases, infectious diseases and immunity, aging and immunity, reproduction and immunity, reproductive organs and immunity, immunohematological diseases, respiratory diseases and immunity, renal diseases and immunity, digestive diseases and immunity, endocrine diseases and immunity, cardiovascular diseases and immunity, connective tissue diseases, immunodermatology, trauma and immunity, parasitic diseases and immunity, for example, treating and preventing tumors, AIDS, viral hepatitis, coronavirus and other types of viruses, or delaying aging, etc.

[0074] Here, the above medicine may be a vaccine that improves the immune function of the body, or a medicine that treats a disease by improving the immune function of the body. The vaccine and medicine may be prepared into any available dosage form.

[0075] In one preferred embodiment of the present invention, the administration route of the above medicine is intravenous, thoracic, intraperitoneal, intraspinal, intradermal, subcutaneous or intratumoral injection, etc.

[0076] The present invention will now be described with reference to specific examples and drawings to facilitate a better understanding of the present invention, but is not intended to limit the scope of the present invention.

[0077] The methods used in the examples are conventional methods unless otherwise specified, and the reagents used are either commercially available or prepared by conventional methods unless otherwise specified.

[0078] Example 1 This example provides a simple method for producing universal immune cells, which includes the following steps:

[0079] Allogeneic mononuclear cells derived from normal human peripheral blood were co-cultured with a cell mitogen, a cytokine and an immune adjuvant in liquid medium, the amount of the cell mitogen concanavalin in the medium being 100,000 units / L, the amount of the cytokine interleukin-2 in the medium being 500,000 units / L and the amount of the immune adjuvant (5% Tween-80) being 0.5 mL / L.

[0080] Allogeneic mononuclear cells were co-cultured with cell mitogens, cytokines, and immune adjuvants for 30 days to obtain immune cell cultures with immune activity, primarily cytotoxic T lymphocytes.

[0081] Example 2 This example provides a simple method for producing universal immune cells, which includes the following steps:

[0082] Allogeneic peripheral blood-derived mononuclear cells were co-cultured with cell mitogens, cytokines and immune adjuvants in liquid medium, the culture medium containing 0.5 mg / L of the cell mitogen phytohemagglutinin, 5 million units / L of the cytokine interferon and 0.1 mL / L of the immune adjuvant (5% Tween 80).

[0083] Allogeneic mononuclear cells were co-cultured with cell mitogens, cytokines, and immune adjuvants for 30 days to obtain immune cell cultures with immune activity, primarily cytotoxic T lymphocytes.

[0084] Example 3 This example provides a simple method for producing universal immune cells, which includes the following steps:

[0085] Normal human umbilical cord blood-derived mononuclear cells were co-cultured with a cell mitogen, a cytokine and an immune adjuvant in a liquid medium, the amount of the cell mitogen concanavalin in the medium being 1 million units / L, the amount of the cytokine interleukin-2 in the medium being 1 million units / L, and the amount of the immune adjuvant (5% Tween-80) being 0.1 mL / L.

[0086] Allogeneic mononuclear cells were co-cultured with cellular mitogens, cytokines and immune adjuvants for 30 days to obtain immune cell cultures with immune activity, primarily cytotoxic T lymphocytes.

[0087] Example 4 This example provides a simple method for producing universal immune cells, which includes the following steps:

[0088] Allogeneic peripheral blood-derived mononuclear cells were co-cultured with cell mitogens, cytokines and immune adjuvants in liquid medium, the culture medium containing 1 mg / L of cell mitogen phytohemagglutinin, 3 million units / L of cytokine interferon and 0.5 mL / L of immune adjuvant (5% Tween 80).

[0089] Allogeneic mononuclear cells were co-cultured with cell mitogens, cytokines, and immune adjuvants for 30 days to obtain immune cell cultures with immune activity, primarily cytotoxic T lymphocytes.

[0090] Example 1 Taking T lymphocytes as an example, a large number of activated and proliferated immune cells can be obtained by using the above method. Using combinatorial barcoding, single-cell whole gene expression sequencing and whole gene gene expression sequencing are used. Two different groups of immune effector cells are characterized by gene screening analysis, cell 2 is co-cultured with immune cells, cell mitogens, cytokines, and immune adjuvants for 8 days, and cell 1 is co-cultured with immune cells, cell mitogens, cytokines, and immune adjuvants for 26 days. Below are the results of the joint expression of the whole gene characteristics of immune cells by the two detection analysis methods.

[0091] [Table 1]

[0092] When studying the differences between two groups of samples, we will take the differential genes as research objects for further study. We use DESeq2 to compare the two groups and obtain the differential fold-change of marker genes between samples. In this analysis, we compared the two groups to screen for differential genes. The screening condition was log-transformed fold change to log2FoldChange, and genes with log2FoldChange>1.9 or <-1.9 were selected as marker genes. It can be seen from Table 1 that the differential expression characteristics of these genes are obvious.

[0093] Example 2 Taking T lymphocytes as an example, by using the above method, the present invention can obtain a large number of activated and proliferated immune cells. Cell 1 was co-cultured with immune cells, cell mitogens, cytokines and immune adjuvants for 26 days, and then pathway analysis was performed.

[0094] Systematic analysis of gene (and its encoded product) relationships, gene functions, and genome information databases helps researchers study genes and expression information as a whole network. The integrated metabolic pathways provided by KEGG include metabolism of carbohydrates, nucleosides, amino acids, etc. and biodegradation of organic matter, and not only provide all possible metabolic pathways but also comprehensively annotate the enzymes that catalyze each step of the reaction. KEGG is a powerful tool for in vivo metabolic analysis and metabolic network research. Currently, KEGG Pathways are classified into eight categories: whole network, metabolic process, genetic information transfer, environmental information transfer, intracellular biological process, living system, human disease, and drug discovery.

[0095] Pathway analysis was based on the KEGG database, and for differential genes, Fisher's exact test and chi-square test were used to perform significance analysis of pathways involved in target genes, and screened according to p-value < 0.05 to obtain significant pathways. According to the significance level (p-value) of each differential gene pathway in the differential gene pathways showing significant up-regulation and down-regulation in the analysis results and the number of differential genes included, a distribution diagram of significant differential gene pathways can be constructed. In the diagram, the vertical axis is the name of the differential gene pathway, and the horizontal axis is the negative logarithm of the p-value (-LgP), the larger it is, the smaller the p-value is, that is, the higher the significance level is, and the size of the bubble represents the number of differential genes included in the pathway. To make it easier for researchers to see, a bubble diagram was created for each of the significantly up-regulated and down-regulated differential gene functions (parts with p-values ​​less than 0.05, and if there are many parts with p-values ​​less than 0.05, only the top 30 items with small p-values ​​are displayed). Taking a path diagram showing a significant increase as an example, the results showing the significant parts are shown in Figure 1.

[0096] From Figure 1, we can see that there are two signaling pathways that are enriched in marker genes. These two pathways are the hematopoietic cell lineage signaling pathway and the cytokine-cytokine receptor interaction signaling pathway, and these two signaling pathways are the main characteristic pathways of cell 1 activation.

[0097] Example 3 Taking T lymphocytes as an example, by using the above method, the present invention can obtain a large number of activated and proliferated immune cells. Cell 1 is co-cultured with immune cells, cell mitogen, cytokine and immune adjuvant for 26 days, and Cell 2 is co-cultured with immune cells, cell mitogen, cytokine and immune adjuvant for 8 days, and then analyze the activation interaction relationship between the signal transduction pathways of Cell 1 and Cell 2. The results are shown in Figure 2.

[0098] In Figure 2, the above effects cause the signal pathways to form an activation delivery network and initiate a cascade that induces the immune cell phenotype. After analyzing all immune signaling pathways, we found that the hematopoietic cell lineage signaling pathway, which is the characteristic pathway of cell 1, is a very important effector signaling pathway that receives stimulation from upstream signaling pathways, and the cytokine-cytokine receptor interaction signaling pathway is the central target of stimulation of more upstream signaling pathways.

[0099] Effect example 4 Taking T lymphocytes as an example, by using the above method, the present invention can obtain a large number of activated and proliferated immune cells. Cell 2 is co-cultured with immune cells, cell mitogens, cytokines and immune adjuvants for 8 days, and pathway analysis as shown in FIG. 3 is performed. Similarly, the main signaling pathway in which cell 2 is activated is the ECM receptor interaction signaling pathway, and its marker gene enrichment is most obvious (-LgP is the largest). According to the significance level (p value) of each differential gene pathway in the differential gene pathway showing significant up-regulation and down-regulation in the analysis results and the number of differential genes contained therein, a distribution diagram of significant differential gene pathways can be constructed. In the diagram, the vertical axis is the name of the differential gene pathway, and the horizontal axis is the negative logarithm of the p value (-LgP), and the larger it is, the smaller the p value is, that is, the higher the significance level is. Here, the color depth represents the degree of up- and down-regulation and the additive effect of genes in a signaling pathway, and the size of the bubble represents the number of differential genes in the pathway.

[0100] Through the signaling pathway activation network, it was found that the ECM receptor interaction signaling pathway was activated in the focal adhesion signaling pathway of cell 2, again reinforcing the focal adhesion signaling pathway.

[0101] Effect example 5 Taking T lymphocytes as an example, by using the above method, the present invention can obtain a large number of activated and proliferated immune cells. Using Countstar® Rigel fluorescence analyzer, the cell concentration and viability of PBMC samples are detected by AOPI dye method, and the results are as follows:

[0102] [Table 2]

[0103] Flow cytometer analyzes immune cell subsets, showing that CD8+ T lymphocytes are 95.34%, 96.9%, and 96.2% in samples 1, 2, and 3, respectively. These results are similar to those obtained by previous similar methods, showing that the increase in CD8+ T lymphocytes reached more than 95%, among which were mainly CD45RO positive cells at about 93%, strongly suggesting that the immune effector cells obtained are cytotoxic T lymphocytes (CTLs).

[0104] Effect example 6 The universal immune cells produced by the method according to the present invention were injected into subjects to verify their safety and efficacy.

[0105] Subject information: Mr. Liu, male, 65 years old, received a course of intravenous infusion of allogeneic universal immune cells prepared by the above method every 3 months for the past 2 years. Each course of treatment was divided into 3 intravenous infusions, one infusion every other day, with the number of cells infused each time being 5 × 10 8 ~1×10 10 It is.

[0106] In terms of efficacy, the entire process is well tolerated, and there are no clinical symptoms or signs such as rash, jaundice, or diarrhea associated with rejection in the skin, liver, intestines, or other organs or systems. In addition, after the administration of the allogeneic universal immune cells produced by the above method, the following 10 major effects are observed: (1) blemishes are lightened, (2) skin texture is improved, (3) chronic eczema disappears, (4) gray hair is reduced, (5) memory is improved, (6) blood lipids are slightly reduced, (7) glycosylated hemoglobin returns to normal after a slight increase, (8) there is no oral mucosal ulcer, (9) joint damage is reduced, and (10) energy is increased.

[0107] Effect example 7 The universal immune cells produced by the method according to the present invention were injected into subjects to verify their safety and efficacy.

[0108] Subject information: Mr. Xue, male, 56 years old, a subject with liver cancer. The allogeneic universal immune cells prepared by the above method were infused for nearly 4 years. At first, one course of intravenous injection was given every month, and each course was divided into 5 intravenous injections. After 2 years, it was changed to one course of intravenous injection every other month, and each course was divided into 5 intravenous injections, and the number of cells infused each time was 5 × 10 8 ~1×10 10 It is.

[0109] As for efficacy, the entire process was well tolerated, and no adverse events were observed, except for occasional transient fever. Repeated blood tests, liver function tests, and kidney function tests showed no abnormalities. Various tests, including imaging tests, showed complete tumor regression.

[0110] Effect example 8 The universal immune cells produced by the method according to the present invention were injected into subjects to verify their safety and efficacy.

[0111] Subject information: Ms. Wang, female, 75 years old, with colon cancer. The allogeneic universal immune cells produced by the above method were injected monthly for almost 10 years, with the number of cells injected each time being 5 × 10 8 ~1×10 10 It is.

[0112] In terms of efficacy, the entire process was well tolerated, and no adverse reactions were observed. There were no clinical symptoms or signs, such as rash, jaundice, or diarrhea, associated with rejection in the skin, liver, intestines, or other organs or systems. Repeated blood tests, liver function tests, and kidney function tests showed no abnormalities. Clinically, there were no particular symptoms or signs, and all blood tests and tumor markers showed normal results. Repeat colonoscopy showed no abnormalities. The subject complained of a significant alleviation of common colds.

[0113] Effect example 9 The universal immune cells produced by the method according to the present invention were injected into subjects to verify their safety and efficacy.

[0114] Subject information: Mr. Li, male, 70 years old, subject with malignant melanoma. Allogeneic universal immune cells produced by the above method were infused for 4 years via various routes including intravenous injection, intraperitoneal injection, and subcutaneous (intratumoral) injection. Initially, the treatment course was every 2 weeks, but then moved to monthly, 3-monthly, or 6-monthly treatment courses. Each course of treatment was divided into 4 injections, one injection every other day, with the number of cells injected each time being 5 × 10 8 ~1×10 10 It is.

[0115] As for efficacy, except for pain during subcutaneous injection, the whole process was well tolerated and no adverse reactions were observed. Blood tests, liver function, and kidney function were repeatedly tested and found to be normal. There were no clinical symptoms or signs such as rash, jaundice, or diarrhea associated with rejection in organs and systems such as the skin, liver, or intestines. The subject had advanced malignant melanoma, which recurred and metastasized after surgery, and chemotherapy was ineffective. The allogeneic universal immune cells produced by the above method are monotherapy, and the tumor is well controlled.

[0116] Although the specific embodiments of the present invention have been described in detail above, these are merely examples, and the present invention is not limited to the above specific embodiments. For those skilled in the art, all equivalent modifications and replacements to these embodiments are within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should belong to the scope of the present invention. The invention as originally claimed in the present application is set forth below. [1] Step S1, co-culturing allogeneic immune cells, cell mitogens, cytokines and immune adjuvants in a liquid cell culture medium in a culture vessel to obtain a universal immune cell culture; A step S2 of isolating a cell population from the culture obtained in step S1, The method for producing universal immune cells, wherein the cytokines are derived from one or more of lymphokines, monokines, cytokines that activate inflammation, and cytokines that stimulate hematopoiesis produced by lymphocytes, monocytes, and other cells. [2] A method for producing universal immune cells, characterized in that the cytokines in the method described in [1] are derived from any one or more of interleukins, interferons, tumor necrosis factors, colony-stimulating factors, chemotactic cytokines, and transforming growth factors. [3] A method for producing universal immune cells, characterized in that the cytokines in the method described in [1] are derived from one or more of interleukin-2, interleukin-6, interleukin-15 and interferon. [4] [1] A method for producing universal immune cells, characterized in that the concentration of the cytokine is 200,000 to 5,000,000 units / L. [5] [1] A method for producing universal immune cells, characterized in that the concentration of the cytokine is 500,000 to 2,000,000 units / L. [6] A method for producing universal immune cells, characterized in that the mitogen is selected from any one or more of concanavalin, phytohemagglutinin, pokeweed, lipopolysaccharide, and dextran, in the method described in [1]. [7] [6] A method for producing universal immune cells, characterized in that the concentration of the mitogen is 100,000 to 10 million units / L. [8] [6] A method for producing universal immune cells, characterized in that the concentration of the mitogen is 0.1 mg to 10 mg / L. [9] [1] A method for producing universal immune cells, characterized in that the immune adjuvant is selected from one or more of biological adjuvants, inorganic adjuvants, organic adjuvants, synthetic adjuvants, oils and Freund's adjuvants, and has a concentration of 0.01 mL to 1 mL / L.

[10] A method for producing universal immune cells, characterized in that the immune cells are allogeneic cells or autologous cells derived from peripheral blood or umbilical cord blood, in the method described in [1].

[11] [1] A method for producing universal immune cells, characterized in that the concentration of the immune cells is 1 x 103 to 1 x 1011 cells / mL.

[12] [1] A method for producing universal immune cells, characterized in that the cell population comprises one or more of cytotoxic T lymphocytes, tumor-infiltrating lymphocytes, cytokine-activated killer cells, lymphokine-activated killer cells, natural killer cells, tumor-associated macrophages, activated killer monocytes, and dendritic cells.

[13] [1] A method for producing universal immune cells, characterized in that the co-culture time in step S1 is 3 to 180 days.

[14] A method for producing universal immune cells, characterized in that the culture vessel described in [1] is a three-dimensional large-capacity high-density cell culture vessel.

[15] The method for producing universal immune cells according to the above-mentioned method described in [1], further comprising a cloning step of performing cloning in a liquid cell culture medium using the culture obtained in step S1 or the cell population obtained in step S2 as a raw material to obtain a cell line, wherein the cloning is performed by employing an intermittent cyclic stimulation method or a continuous stimulation method.

[16] A universal immune cell produced by the method described above in any one of [1] to

[15] , characterized in that the universal immune cell comprises one or more of cytotoxic T lymphocytes, tumor-infiltrating lymphocytes, cytokine-activated killer cells, lymphokine-activated killer cells, natural killer cells, tumor-associated macrophages, activated killer monocytes, and dendritic cells.

[17] The parent cell of the immune cell preparation is a universal immune cell, as described in

[16] . The universal immune cell is used to produce the immune cell preparation.

[18] In the above application described in

[17] , the immune cell preparation comprises CAR-T cells or TCR-T cells.

[19] An immune cell preparation produced by using the universal immune cell described in

[16] as a parent cell.

[20] A biological agent for adoptive cellular immunotherapy, comprising a container and the immune cell preparation described in

[11] disposed in the container.

[21] The biological preparation described in

[20] , characterized in that it contains physiological saline, albumin and other stabilizers.

[22]

[19] Use of the immune cell preparation described above in the manufacture of a medicine for treating and preventing a disease associated with abnormalities in lymphocyte function and quantity.

[23] The application according to

[22] , characterized in that the administration route of the above-mentioned medicines is intravenous, thoracic, intraperitoneal, intraspinal, intradermal, subcutaneous or intratumoral injection.

Claims

1. Step S1, co-culturing allogeneic mononuclear cells, cell mitogens, cytokines and immune adjuvants in a liquid cell culture medium in a culture vessel to obtain a universal immune cell culture; A step S2 of isolating a cell population from the culture obtained in step S1, wherein the cell mitogen is selected from any one or more of concanavalin and phytohemagglutinin; the cytokine is selected from any one or more of interleukin-2 and interferon; The immune adjuvant is Tween-80. A method for producing universal immune cells, wherein the cell population is cytotoxic T lymphocytes.

2. The method for producing universal immune cells according to claim 1, wherein the concentration of the cytokine is 200,000 to 5,000,000 units / L.

3. The method for producing universal immune cells according to claim 1, wherein the concentration of the cytokine is 500,000 to 2,000,000 units / L.

4. 2. The method for producing universal immune cells according to claim 1, wherein the concentration of the mitogen is 100,000 to 10 million units / L.

5. The method for producing universal immune cells according to claim 1, wherein the concentration of the mitogen is 0.1 mg to 10 mg / L.

6. The method for producing universal immune cells according to claim 1, wherein the mononuclear cells are derived from peripheral blood or umbilical cord blood.

7. 10. The method of claim 1, wherein the concentration of the immune cells is 1×10 ３ ~1×10 １１ A method for producing universal immune cells, characterized in that the number of universal immune cells per mL.

8. The method for producing universal immune cells according to claim 1, wherein the co-culture time in step S1 is 3 to 180 days.

9. The method for producing universal immune cells according to claim 1, wherein the culture vessel is a three-dimensional large-volume high-density cell culture vessel.

10. 2. A method for producing universal immune cells according to claim 1, comprising a cloning step of performing cloning in a liquid cell culture medium using the culture obtained in step S1 or the cell population obtained in step S2 as a raw material to obtain a cell line, wherein the cloning employs an intermittent cyclic stimulation method or a continuous stimulation method.

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