A universal and efficient in vitro expansion method for allogeneic DNT cells for multiple clinical transfusions

Abstract

The present invention relates to a universal method for efficient in vitro expansion of allogeneic DNT cells for multiple clinical transfusions. In the method of the present invention, the dosage of T cell activator is gradually reduced during continuous culture without the need for additional addition of interleukin-4 or AB serum.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a universal and efficient in vitro amplification method for allogeneic DNT cells for multiple clinical transfusions. Background Art

[0002] Immune cell therapy is an emerging, highly effective cancer treatment modality and a novel immune-based anti-cancer treatment. It utilizes biotechnology and biologics to culture and expand immune cells collected from patients or healthy donors in vitro, then infuse them back into the patient to stimulate and enhance the body's own immune function, thereby achieving the goal of treating tumors.

[0003] Immune cell therapy is an important treatment for many serious diseases that threaten humans, including cancer, autoimmune diseases, organ transplant rejection, severe allergic diseases, and even severe viral infections. There are successful cases of immune cell therapy.

[0004] Currently, immune cell therapy mostly involves collecting autologous immune cells, culturing and expanding them in vitro, and then re-infusing them. If multiple re-infusions are required, immune cells must be collected from human peripheral blood for multiple culturing and expansion to meet clinical re-infusion needs. Due to the severity of their condition, most patients requiring treatment are often unable to provide a sufficient number of immune cells for culture. Furthermore, due to their physical condition and various medications, culturing and expanding immune cells in vitro is extremely difficult, or even impossible.

[0005] Therefore, there is a need in the art for a universal in vitro expansion method for obtaining immune cells from healthy donors and for multiple transfusions, which is convenient for clinical treatment and avoids the inconvenience caused by multiple collections and multiple transfusions in the clinic, while eliminating the risk of tumor cells in the peripheral blood of cancer patients due to poor quality or even the presence of tumor cells. Summary of the Invention

[0006] The purpose of the present invention is to provide a new and universal method for efficient in vitro expansion of allogeneic DNT cells for multiple clinical transfusions.

[0007] In a first aspect of the present invention, a universal in vitro expansion method for DNT cells is provided, characterized in that it comprises the steps of:

[0008] (i) providing a peripheral blood starting sample I obtained from a donor;

[0009] (ii) Removal of CD4 from the starting sample + and CD8 + T cells, thereby obtaining sample II;

[0010] (iii) culturing sample II in a culture system containing a culture medium suitable for the growth of DNT cells, thereby obtaining sample III; wherein the culture system includes an immobilized T cell mitogen;

[0011] (iv) culturing sample III in a culture system containing a medium suitable for the growth of DNT cells, thereby obtaining sample IV; wherein the culture medium contains a soluble T cell mitogen at a concentration of 30-80 ng / mL (preferably 40-60 ng / mL, more preferably 50 ng / mL);

[0012] (v) culturing sample IV in a culture system containing a culture medium suitable for the growth of DNT cells, wherein the culture medium contains a soluble T cell mitogen at a concentration of 15-40 ng / mL (preferably 20-30 ng / mL, more preferably 25 ng / mL, and more preferably 1 / 2 of the amount of the T cell mitogen added in sub-step (va)), thereby obtaining a desired amount of universal DNT cells, which is sample V; and

[0013] (vi) collecting a sample V in a solution system suitable for preserving DNT cells; wherein the solution contains human serum albumin, thereby obtaining universal DNT cells for clinical use, which is a DNT cell preparation;

[0014] The culture systems in steps (iii) to (v) all contain 200-1000 IU / mL (preferably 300-700 IU / mL, more preferably 500 IU / mL) of recombinant human interleukin-2, and do not contain recombinant human interleukin-4 and AB serum.

[0015] In another preferred embodiment, in step (i), the donor is a healthy donor.

[0016] In another preferred embodiment, the amount of the sample I is 10-300 ml, preferably 20-200 ml.

[0017] In another preferred embodiment, the sample II is a cell solution that has been revived after freezing.

[0018] In another preferred embodiment, the step (ii) comprises: removing CD4 + and CD8 + T cells, thereby obtaining sample IIa (enriched DNT cells); after collecting sample IIa (enriched DNT cells) by centrifugation, resuspend it in freezing solution, cool it under program control and store it in liquid nitrogen, which is sample IIb (enriched frozen DNT cells); after freezing sample IIb, thaw it at 37°C, wash it once with culture medium, and resuspend it in culture medium, which is sample IIc (enriched frozen and revived DNT cells).

[0019] In another preferred embodiment, in the culture system of steps (iii) to (v), the culture conditions are 37° C. and 5% CO 2 .

[0020] In another preferred embodiment, the culture medium in steps (iii) to (v) further comprises a cytokine selected from the group consisting of IL-7, IL-12, IL-15, or a combination thereof.

[0021] In another preferred embodiment, before step (iii), the method further comprises step (iia): washing sample II with 0.9% physiological saline.

[0022] In another preferred embodiment, the T cell mitogen is selected from the following group: an antibody that binds to CD3, a lectin, a compound that can stimulate the expansion of DNT cells, or a combination thereof.

[0023] In another preferred embodiment, the lectin is selected from the group consisting of plant lectin concanavalin A (ConA), plant lectin (PHA), soybean agglutinin (SBA), or a combination thereof.

[0024] In another preferred embodiment, the compound capable of stimulating the expansion of DNT cells is selected from the following group: IPP, pamidronate, zoledronic acid, or a combination thereof.

[0025] In another preferred embodiment, the T cell mitogen is an antibody that binds to CD3.

[0026] In another preferred embodiment, the culture system in step (iii) is located in a culture flask, preferably a T25 culture flask.

[0027] In another preferred embodiment, the immobilized T cell mitogen refers to a T cell mitogen coated on a culture flask or a microplate.

[0028] In another preferred embodiment, in step (iii), the initial concentration of the DNT cells to be expanded in the culture system is 1×10 6 to 4×10 6 cells / mL.

[0029] In another preferred embodiment, the culture time in step (iii) is 24-72 hours, preferably 36-60 hours, and more preferably 48 hours.

[0030] In another preferred embodiment, in step (iii), 15%-25% (preferably 18%-22%, more preferably 20%) of the donor's plasma is added to the culture medium.

[0031] In another preferred embodiment, step (iii) is divided into sub-steps (iiia), (iiib) and (iiic), and the sub-steps (iiia), (iiib) and (iiic) are three expansions of the DNT cells in sample III.

[0032] In another preferred embodiment, in sub-step (iiia), the initial concentration of the DNT cells to be expanded in the culture system is 1×10 6 ~4×10 6 cells / mL.

[0033] In another preferred embodiment, in sub-steps (iiia) and (iiib), the initial concentration of the DNT cells to be expanded in the culture system is independently 0.5×10 6 to 1×10 6 cells / mL.

[0034] In another preferred embodiment, the culture time of the sub-steps (iiia), (iiib) and (iiic) is independently 24-72 hours, preferably 36-60 hours, and more preferably 48 hours.

[0035] In another preferred embodiment, the culturing time in step (iii) is 96-192 hours, preferably 120-168 hours, and more preferably 144 hours.

[0036] In another preferred embodiment, step (iv) comprises 2-5 times (preferably 3-4 times, more preferably 3 times) of amplification of DNT cells. In each amplification, the initial concentration of the DNT cells to be amplified is independently 1×10 6 to 3×10 6 cells / mL, and the culture time for each expansion is 24-72 hours, preferably 36-60 hours, and more preferably 48 hours.

[0037] In another preferred embodiment, step (iv) further comprises detecting the phenotype and viability of DNT cells.

[0038] In another preferred embodiment, the culturing time in step (iv) is 72-168 hours, preferably 96-144 hours, and more preferably 120 hours.

[0039] In another preferred embodiment, in step (v), the DNT cells are expanded 2-4 times (preferably 2 times), and in each expansion, the initial concentration of the DNT cells to be expanded is independently 1×10 6 to 3×10 6 cells / mL, and the culture time for each expansion is 24-72 hours, preferably 36 hours.

[0040] In another preferred embodiment, step (v) further comprises detecting the phenotype and viability of DNT cells.

[0041] In another preferred embodiment, the culturing time in step (v) is 48-96 hours, preferably 72 hours.

[0042] In another preferred embodiment, step (vi) comprises the steps of:

[0043] (via) collecting DNT cells in sample V by centrifugation;

[0044] (vib) washing the DNT cells with physiological saline containing 2.5% human albumin (or "vehicle"); and

[0045] (vic) DNT cells were adjusted to 1×10 8 The concentration of cells / mL.

[0046] In a second aspect of the present invention, there is provided a use of an effective amount of DN T cells obtained by the method according to the first aspect of the present invention for preparing a pharmaceutical composition or preparation, wherein the pharmaceutical composition or preparation is used for:

[0047] (a) prevention and / or treatment of tumors;

[0048] (b) prevention and / or treatment of infectious diseases;

[0049] (c) prevention and / or treatment of autoimmune diseases;

[0050] (d) prevention and / or treatment of allergic reactions;

[0051] (e) preventing and / or treating graft-versus-host disease; and / or

[0052] (f) Modulation of immune response.

[0053] In another preferred embodiment, the tumor is a tumor allogeneic to the DNT cells.

[0054] In another preferred embodiment, the tumor is selected from the following group: a blood tumor, a solid tumor, or a combination thereof.

[0055] In another preferred embodiment, the blood tumor is selected from the following group: lymphoma (Hodgkins and non-Hodgkins), acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome (MDS), or a combination thereof.

[0056] In another preferred embodiment, the solid tumor is selected from the group consisting of gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, melanoma, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, head and neck cancer, pancreatic cancer, or a combination thereof.

[0057] In another preferred embodiment, the autoimmune diseases include: diabetes, arthritis, multiple sclerosis, lupus erythematosus, inflammatory bowel disease, dermatitis, meningitis, thrombotic thrombocytopenic purpura, syndrome, encephalitis, uveitis, leukocyte adhesion defect, rheumatic fever, Reiter's syndrome, progressive systemic sclerosis, primary biliary cirrhosis, necrotizing vasculitis, myasthenia gravis, polymyositis, sarcoidosis, granulomatous disease, vasculitis, pernicious anemia, CNS inflammatory disorders, antigen-antibody complex-mediated diseases, autoimmune hemolytic anemia, lymphomatous goiter, exophthalmos goiter, habitual white hair abortion, Raynaud's syndrome, glomerulonephritis, dermatomyositis, chronic active hepatitis, lacteal diarrhea, tissue-specific autoimmunity, degenerative autoimmune delayed hypersensitivity, autoimmune complications of AIDS, atrophic gastritis, ankylosing spondylitis, Addison's disease, or a combination thereof.

[0058] In another preferred embodiment, the allergic reaction includes hay fever, asthma, atopic eczema, allergic reaction to poison ivy and poison ivy, house dust mites, bee pollen, nuts, crustaceans, penicillin, or a combination thereof.

[0059] In a third aspect of the present invention, there is provided a use of DNT cells obtained by the method according to the first aspect of the present invention, characterized in that they are used for:

[0060] (a) prevention and / or treatment of tumors;

[0061] (b) prevention and / or treatment of infectious diseases;

[0062] (c) prevention and / or treatment of autoimmune diseases;

[0063] (d) prevention and / or treatment of allergic reactions;

[0064] (e) preventing and / or treating graft-versus-host disease; and / or

[0065] (f) Modulation of immune response.

[0066] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The cell growth curves of application examples 1-4 and comparative examples 1 and 2 are shown; specifically, the cells of the application examples and comparative examples were sampled on the 1st, 7th, 14th and 17th day of culture to detect the total cell number and compare the changes in the total cell number. DETAILED DESCRIPTION

[0068] After extensive and in-depth research and numerous process optimization experiments, the inventors have developed, for the first time, a practical, efficient in vitro expansion method for allogeneic DNT cells, harvested from donor peripheral blood once for multiple clinical transfusions. Specifically, the present invention optimizes the culture formulation and process for DNT cells. Compared to existing in vitro expansion methods for DNT cells, the present method does not require the addition of IL-4 and utilizes a phased reduction in the dosage of the T cell activator (here, an anti-human CD3 antibody) during the continuous culture process. This method produces highly pure, universal DNT cells, significantly reducing the impurity content in the final product.

[0069] The results showed that the method of the present invention can be used to efficiently expand DNT cells freshly collected and enriched from human peripheral blood in vitro. It can also be used to freeze the above-mentioned DNT cells and then resuscitate them in vitro. The number of DNT cells expanded using the process of the present invention is large, and the characteristic surface marker of DNT cells is CD3. + CD4 - CD8 - The purity of T cells is high (>85%). Since the tumoricidal activity of DNT cells does not depend on T cell receptors, DNT cells prepared from the same donor can be provided to different patients for clinical treatment.

[0070] On this basis, the present invention was completed.

[0071] the term

[0072] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0073] As used herein, the term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0074] As used herein, the terms "administering" and "administering" are used interchangeably and refer to the physical introduction of the product of the present invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

[0075] In vitro amplification method of the present invention

[0076] In the present invention, a method for in vitro large-scale expansion of universal DNT cells is provided, wherein the method comprises the steps of:

[0077] (i) providing a peripheral blood starting sample I obtained from a donor;

[0078] (ii) Removal of CD4 from the starting sample + and CD8 + T cells, thereby obtaining sample II;

[0079] (iii) culturing sample II in a culture system containing a culture medium suitable for the growth of DNT cells, thereby obtaining sample III; wherein the culture system includes an immobilized T cell mitogen;

[0080] (iv) culturing sample III in a culture system containing a medium suitable for the growth of DNT cells, thereby obtaining sample IV; wherein the culture medium contains a soluble T cell mitogen at a concentration of 30-80 ng / mL (preferably 40-60 ng / mL, more preferably 50 ng / mL);

[0081] (v) culturing sample IV in a culture system containing a culture medium suitable for the growth of DNT cells, wherein the culture medium contains a soluble T cell mitogen at a concentration of 15-40 ng / mL (preferably 20-30 ng / mL, more preferably 25 ng / mL, and more preferably 1 / 2 of the amount of the T cell mitogen added in sub-step (va)), thereby obtaining a desired amount of universal DNT cells, which is sample V; and

[0082] (vi) collecting a sample V in a solution system suitable for preserving DNT cells; wherein the solution contains human serum albumin, thereby obtaining universal DNT cells for clinical use, which is a DNT cell preparation;

[0083] The culture systems in steps (iii) to (v) all contain 200-1000 IU / mL (preferably 300-700 IU / mL, more preferably 500 IU / mL) of recombinant human interleukin-2, and do not contain recombinant human interleukin-4 and AB serum.

[0084] As used herein, the terms "double negative T cells" and "DNT cells" are used interchangeably and refer to a subpopulation of T lymphocytes that express CD3 molecules on their cell surface but lack CD4, CD8 molecules, and CD16 / CD56 molecules. Among them, CD4 is a characteristic surface molecule of helper T cells, CD8 is a characteristic surface molecule of cytotoxic T cells, and CD16 / CD56 is a characteristic surface molecule of NK cells. DNT cells express T cell receptors (TCRs), which can be αβ or γδ TCRs. The DNT cell subpopulations amplified by the method of the present invention can include αβ + and γδ + In a preferred embodiment, the DNT cells are cells derived from human peripheral blood.

[0085] The starting sample can be derived from any biological sample containing double-negative T cells or their precursors. Such samples include, but are not limited to, fresh or cryopreserved blood, bone marrow, lymphoid tissue, thymus, liver, spleen, lymph node tissue, tumor tissue, fetal tissue cells, and fractions or enriched portions thereof, and can also be derived from induced pluripotent stem cells (iPSCs).

[0086] In a preferred embodiment, the starting sample is blood, preferably human blood, more preferably human peripheral blood.

[0087] Before culturing the starting sample or its fractions, the starting sample is substantially depleted of CD4 + and CD8 + "Substantially" means that the majority of these cells are removed, but does not exclude that a small portion of these cells still remain.

[0088] These cell types can be removed from the sample using techniques known in the art. Specifically, antibodies that bind to the CD8 + and CD4 +In a preferred embodiment, labeled antibodies that specifically bind to CD4 and CD8 are added to the sample.

[0089] In another preferred embodiment, magnetic beads that specifically bind to CD4 and CD8 are added to the sample.

[0090] Once the starting sample has been depleted of CD4 + and CD8 + T cells, the method of the present invention can be continued immediately or the sample can be frozen and stored, which can be a low-temperature refrigerator or device (below -80 ° C) or liquid nitrogen, for later use. Therefore, when DNT cells are needed, the in vitro expansion of DNT cells can be started from the frozen sample at this time.

[0091] In one embodiment of the present invention, a method is provided for obtaining peripheral blood (100-400 ml) from a healthy donor at one time, enriching DNT cells in vitro and immediately freezing them, and resuscitating the enriched DNT cells according to a clinical transfusion protocol for use in the in vitro expansion method of the present invention.

[0092] Preferably, step (ii) of the method of the present invention may include: removing CD4 + and CD8 + T cells are obtained, thereby obtaining sample IIa (enriched DNT cells); sample IIa can be directly cultured using the in vitro expansion method of the present invention, or it can be frozen in liquid nitrogen and then revived and cultured; sample IIa (enriched DNT cells) is collected by centrifugation, resuspended in freezing solution, and stored in liquid nitrogen after program-controlled cooling, which is sample IIb (enriched frozen DNT cells); after sample IIb is frozen, it is revived and cultured according to clinical needs; after thawing at 37°C, it is washed once with culture medium and then resuspended in culture medium, which is sample IIc (enriched frozen and revived DNT cells).

[0093] Cultured in a culture medium that has essentially eliminated CD4 + and CD8 + The culture medium comprises a fixed T cell mitogen and an agent capable of stimulating the growth of DNT cells.

[0094] The fixed T cell mitogen can be any reagent capable of stimulating double negative T cells, including but not limited to antibodies and lectins that bind CD3 or T cell receptors, including plant lectins concanavalin A (ConA) and plant lectin (PHA), or any compound capable of stimulating DNT cell expansion, such as but not limited to IPP, pamidronate, and zoledronic acid. Preferably, the T cell mitogen is an antibody to CD3, such as OKT3.

[0095] The T cell mitogen can be immobilized using techniques known in the art. Preferably, the T cell mitogen is coated onto a solid support, including but not limited to a microplate, a culture dish, a culture bag, or a culture flask. Preferably, the T cell mitogen is an immobilized anti-CD3 antibody, more preferably immobilized onto a microplate.

[0096] The agent capable of stimulating the growth of DNT cells can be any suitable agent, preferably a cytokine, such as an interleukin. Preferably, the cytokine comprises interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15) or a mixture of two or more of these.

[0097] It is particularly noteworthy that, in the present invention, the culture medium used for expansion culture does not contain IL-4 and AB serum.

[0098] The concentration of the agent should be suitable for promoting the expansion of double negative cells. Preferably, the cytokine dosage ranges from about 200 IU / mL to 1000 IU / mL. In a particularly preferred embodiment, the concentration of IL-2 in the culture medium is 500 IU / mL.

[0099] The culture medium can be any culture medium suitable for T cell culture, including but not limited to AIM-V medium, RPMI medium, X-VIVOI0 and X-VIVOI5, and any other commercial animal serum-free medium. The culture medium preferably contains other suitable agents including antibiotics.

[0100] In one embodiment, when cultured with reagents capable of stimulating the growth of DNT cells and fixed T cell mitogens, the cells can be co-cultured with DC cells or stimulated with antigens and cytokines. In the preparation of anti-tumor DNT cells, inactivated tumor cells or tumor-specific or tumor-associated antigens, peptides or neoantigens can be used.

[0101] The cells are preferably cultured in any one of steps (ii)-(v) for a period of time ranging from about 2-8 days. Preferably, each step is performed for about 3-7 days, more preferably 4-6 days.

[0102] The purity of the DNT cells prepared by this method can be confirmed using techniques known in the art, such as flow cytometry or other live cell phenotyping techniques.

[0103] Uses of the DNT cells prepared by the present invention

[0104] The present invention also encompasses the use of double-negative T cells obtained by the methods of the present invention in any and all applications. The allogeneic T cells produced by the methods of the present invention can be used to treat a variety of tumors, can be produced on a large scale, have stable and controllable quality, and can be promptly administered to any appropriate patient.

[0105] In an embodiment of the present invention, double-negative T cells expanded by the method of the present invention have a strong anti-tumor effect. Therefore, in one embodiment, the present invention provides a method for treating tumors, comprising administering an effective amount of universal DNT cells obtained by the method of the present invention to an animal in need thereof. The present invention also includes the use of an effective amount of universal DNT cells obtained by the method of the present invention for treating tumors. The present invention also includes the use of an effective amount of universal DNT cells obtained by the method of the present invention in the preparation of a medicament for treating tumors.

[0106] As used herein, the term "effective amount" refers to a dose effective, at dosages and for periods of time necessary, to achieve the desired result, eg, treating cancer.

[0107] The term "animal" as used herein includes all members of the animal kingdom, including humans. In a preferred embodiment, the animal is a human.

[0108] The term "treat" includes, but is not limited to, alleviation or amelioration of one or more symptoms of a disease or condition (e.g., cancer, transplant rejection and graft-versus-host disease, autoimmune diseases, allergies, infections, etc.), reduction in the extent of the disease, stabilization of the disease, prevention of disease spread, delay or slowing of disease progression, and improvement or remission of the disease state, detectable or undetectable relief of symptoms, and / or prolongation of survival as compared to expected survival if not receiving treatment.

[0109] In the treatment of tumors or cancer, any tumor that can be treated is one that can be treated with double-negative T cells alone or in combination with other treatments such as surgery, radiotherapy or chemotherapy, including various targeted therapy drugs, immune checkpoint inhibitors, immune cell enhancers and nanomaterials that enhance the infiltration of DNT cells in tumor tissues.

[0110] Treatable cancers include tumors that are not vascularized or substantially not vascularized, as well as vascularized tumors. Cancers may include non-solid tumors (such as hematological tumors, e.g., leukemias and lymphomas) or may include solid tumors. The types of cancers treated with the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemic or lymphoid malignancies, benign and malignant tumors, and malignancies, e.g., sarcomas, carcinomas, and melanomas. Adult tumors / cancers and childhood tumors / cancers are also included.

[0111] Hematological cancers are cancers of the blood, bone marrow, or lymphoid tissue. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, granulocytic-monocytic, monocytic, and erythroleukemias), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin's lymphomas (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0112] Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcomas, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, and ovarian cancer.

[0113] The present invention also includes other therapeutic uses of DN T cells, such as the treatment of infectious diseases and the regulation of immune responses in the treatment of autoimmune diseases, allergies, transplant rejection, and graft-versus-host disease. In this case, the method for preparing DN T cells can include adding a suitable infectious agent, allergen cell, or tissue as an antigen.

[0114] Therefore, in one embodiment, the present invention provides a method for treating an infectious disease, comprising administering an effective amount of double-negative T cells obtained by the method of the present invention. The present invention also includes the use of an effective amount of double-negative T cells obtained by the method of the present invention for treating an infectious disease. The present invention also includes the use of an effective amount of double-negative T cells obtained by the method of the present invention for preparing a medicament for treating an infectious disease.

[0115] In a further embodiment, the present invention provides a method for modulating an immune response, comprising administering an effective amount of double-negative T cells obtained by the method of the present invention. The present invention also includes the use of an effective amount of double-negative T cells obtained by the method of the present invention for modulating an immune response. The present invention also includes the use of an effective amount of double-negative T cells obtained by the method of the present invention for preparing a medicament for modulating an immune response.

[0116] In one embodiment, the DNT cells are used to treat autoimmune diseases. Autoimmune diseases that can be treated according to the present invention include, but are not limited to, diabetes, arthritis, multiple sclerosis, lupus erythematosus, inflammatory bowel disease, dermatitis, meningitis, thrombotic thrombocytopenic purpura, syndrome, encephalitis, uveitis, leukocyte adhesion defect, rheumatic fever, Reiter's syndrome, progressive systemic sclerosis, primary biliary cirrhosis, necrotizing vasculitis, myasthenia gravis, polymyositis, sarcoidosis, granulomatous disease, vasculitis, pernicious anemia, CNS inflammatory disorders, antigen-antibody complex-mediated diseases, autoimmune hemolytic anemia, lymphomatous goiter, exophthalmos goiter, habitual graying of the hair, Raynaud's syndrome, glomerulonephritis, dermatomyositis, chronic active hepatitis, lacteal diarrhea, tissue-specific autoimmunity, degenerative autoimmune delayed hypersensitivity, autoimmune complications of AIDS, atrophic gastritis, ankylosing spondylitis, and Addison's disease.

[0117] In another embodiment, DNT cells can be used to treat graft-versus-host disease, in which immune cells in the transplant attack the recipient's normal tissues. This may occur when the transplanted tissue contains immune cells, such as when bone marrow or lymphoid tissue from a healthy donor is transplanted to treat leukemia, aplastic anemia, and enzyme or immune deficiencies.

[0118] In a further embodiment, DNT cells can be used to treat allergies. In an allergy, the immune system attacks a normally harmless antigen or allergen. Allergies that can be prevented or treated using the methods of the present invention include, but are not limited to, hay fever, asthma, atopic eczema, and allergies to poison ivy and poison ivy, house dust mites, bee pollen, nuts, crustaceans, penicillin, and many other substances.

[0119] The DNT cells prepared by the methods of the present invention can be formulated into pharmaceutical compositions for administration to a subject in a biocompatible form suitable for in vivo administration. "Biocompatible form suitable for in vivo administration" refers to a form of the substance being administered in which the therapeutic effect outweighs any toxic effects. The substance can be administered to living organisms, including humans and animals. The composition can be administered by any suitable means, preferably by injection, such as intravenous, subcutaneous, intramuscular, or the like.

[0120] The compositions described herein can be prepared by methods known per se for preparing pharmaceutically acceptable compositions that can be administered to a subject such that an effective amount of cells is combined in admixture with a pharmaceutically acceptable carrier. Suitable carriers are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Company, Easton, Pa., USA 2000). Furthermore, compositions include, but are not limited to, solutions of a substance in combination with one or more pharmaceutically acceptable carriers or diluents, comprising a physiological solution in a buffered solution having an appropriate pH and isotonicity.

[0121] The effective amount of the composition may vary depending on factors such as the disease state, age, sex, and weight of the individual and the ability of the cells to elicit the desired response in the individual. The dosage regimen may be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered weekly, or the dose may be proportionally reduced as indicated by the needs of the therapeutic situation.

[0122] Drugs that can be used in combination with the present invention include other active substances that can be used to treat the disease or condition to be treated. For example, in tumor treatment, other anticancer agents can be administered in the same composition or in separate compositions.

[0123] The pharmaceutical compositions of the present invention can be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, the type and severity of the patient's disease, although appropriate dosages can be determined by clinical trials.

[0124] When an "immunologically effective amount," "anti-tumor effective amount," "tumor-inhibitory effective amount," or "therapeutic amount" is indicated, the precise amount of the composition of the present invention to be administered can be determined by clinical trials, which take into account individual differences in age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). It can be generally stated that the pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 to 10 9 The dose of cells / kg body weight is preferably 10 6 to 10 8The T cell composition can be administered at a dose of 10 cells / kg body weight (including all integer values ​​within those ranges). These doses can also be administered multiple times. The cells can be administered using infusion techniques well known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be obtained by monitoring the patient's disease status, so treatment adjustments are easily determined by those skilled in the medical field.

[0125] Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intralymph nodeally, intraspinal, intramuscularly, by intravenous (iv) injection or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by iv injection. The T cell composition can be injected directly into the tumor, lymph node or infection site.

[0126] In certain embodiments of the present invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic doses are administered to a patient in combination with (e.g., before, simultaneously, or after) any number of relevant treatment modalities, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab treatment for MS patients, or efavirenz treatment for psoriasis patients, or other treatments for PML patients. In further embodiments, the T cells of the present invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to a patient in combination with (e.g., before, simultaneously, or after) bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), or cyclophosphamide. For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In some embodiments, the subject receives an infusion of the expanded immune cells of the invention following transplantation. In an additional embodiment, the expanded cells are administered before or after surgery.

[0127] The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration may be implemented according to practices accepted in the art. Typically, 1×10 9 to 1×10 11The double-negative T cells of the present invention are administered to the patient, for example, by intravenous infusion.

[0128] The main advantages of the present invention include:

[0129] 1) Formula and process improvements to create an efficient in vitro expansion method for human DNT cells suitable for clinical use.

[0130] 2) The formula and process have been improved, eliminating the need for human AB serum and fetal bovine serum (FBS).

[0131] 3) Formula and process improvements can be used to culture freshly enriched DNT cells, as well as DNT cells that have been revived after freezing.

[0132] 4) Improvements in formulation and process can enable single-time collection / freezing, multiple resuscitation and culture, and multiple transfusions or use by multiple patients.

[0133] 5) Formula and process improvements have enabled cell proliferation to reach 1x10 9 cells / mL of whole blood and above.

[0134] 6) Improve the formulation and process to ensure the purity of the final product DNT cells (CD3 + CD4 - CD8 - )≥85%.

[0135] 7) Improve the formulation and process to ensure that the in vitro high-efficiency tumor killing activity (biological efficacy) of cells is ≥50%.

[0136] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0137] Example 1: Magnetic beads enriched DNT cells in vitro expansion culture

[0138] 1.1 Enrichment of DN T cells from healthy donors using magnetic beads

[0139] Collect 20-200 ml of peripheral blood from healthy donors into a tube containing sodium heparin. Separate the buffy coat cells with lymphocyte separation solution and add CD4 magnetic beads to remove CD4 + cells, and then add CD8 magnetic beads to remove CD8 +The cells thus obtained are CD4-depleted + With CD8 + of DNT cells.

[0140] 1.2 In vitro expansion and culture of DNT cells

[0141] First, coat the T25 culture flask with anti-human CD3 monoclonal antibody (10 μg / mL). Then, adjust the DNT cells obtained in step 1.1 to 1×10 cells / mL with AIM-V medium (containing 10% donor plasma and 500 IU / mL recombinant human interleukin-2). 6 ~4×10 6 The cells were placed into the coated T25 culture flask at a concentration of 10 cells / mL and cultured in a 37°C 5% CO2 incubator.

[0142] On the third day, DNT cells were adjusted to 1×10 6 ~1×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0143] On the 5th day, the DNT cells were adjusted to 1×10 6 ~2×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0144] On the 7th day, the phenotype and viability of DNT cells were detected, and fresh AIM-V medium (containing 50 ng / mL anti-human CD3 monoclonal antibody and 500 IU / mL recombinant human interleukin-2) was used to adjust the DNT cells to 1×10 6 ~3×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0145] On day 9, DNT cells were adjusted to 1×10 6 ~3×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0146] On the 10th day, the phenotype and viability of DNT cells were detected, and fresh AIM-V medium (containing 50 ng / mL anti-human CD3 monoclonal antibody and 500 IU / mL recombinant human interleukin-2) was used to adjust the DNT cells to 1×10 6 ~3×10 6 The cells were transferred to a culture bag at a concentration of 10 cells / mL and continued to be cultured.

[0147] On day 12, DNT cells were adjusted to 1×10 6 ~3×10 6 The cells were transferred to a culture bag at a concentration of 10 cells / mL and continued to be cultured.

[0148] On day 14, the phenotype and viability of DNT cells were detected. To reduce the interference of impurities in the final product and improve the purity of DNT cells, the concentration of anti-human CD3 monoclonal antibody in AIM-V medium was reduced to 25 ng / mL. DNT cells were adjusted to 1×10 6 ~3×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0149] On the 15th to 16th day, if necessary, fresh AIM-V medium (containing 25 ng / mL anti-human CD3 monoclonal antibody and 500 IU / mL recombinant human interleukin-2) was used to adjust the DNT cells to 1×10 6 ~3×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0150] On day 17-18, DNT cells were harvested as needed.

[0151] 1.3 Harvesting DNT cells to prepare DNT cell preparations

[0152] On day 17-18, DNT cells were harvested as needed. The cells were collected in a 250 ml conical bottom centrifuge bottle, centrifuged at 900 × g for 10 minutes, and then washed with solvent. The collected DNT cells were adjusted to 0.5-1 × 10 8 The concentration of cells / mL is 100 cells / mL. This is the finished product of DNT cell preparation and can be used clinically after passing quality inspection.

[0153] Example 2: Magnetic bead-enriched DNT cells cryopreserved and revived for in vitro expansion and culture

[0154] 2.1 Enrichment of DNT cells from healthy donors using magnetic beads

[0155] Same as 1.1 above.

[0156] 2.2 Cryopreservation of the obtained DNT cells

[0157] The DNT cells obtained in step 2.1 were washed once with 0.9% saline, resuspended in freezing buffer and adjusted to a cell concentration of 3-5 × 10 6 cells / mL, cryopreserved in liquid nitrogen, and revived for in vitro expansion culture as needed.

[0158] 2.3 Recovery of frozen DNT cells

[0159] Remove the frozen DNT cells from liquid nitrogen in step 2.2 and thaw in a 37°C incubator. Wash the thawed DNT cells once with AIM-V medium (containing 500 IU / mL recombinant human interleukin-2) and resuspend them in AIM-V medium (containing 10% donor plasma and 500 IU / mL recombinant human interleukin-2).

[0160] 2.4 In vitro expansion and culture of DNT cells

[0161] Same as 1.2 above.

[0162] On day 17-18, DNT cells were harvested as needed.

[0163] 2.5 Harvesting DNT cells to prepare DNT cell preparations

[0164] Same as 1.3 above.

[0165] Example 3: In vitro expansion and culture of sedimentation-enriched DNT cells

[0166] 3.1 Healthy Donor DNT Cells Enrichment

[0167] 20-200 ml of peripheral blood was collected from healthy donors into heparinized tubes. CD4 / CD8 lymphocyte depletion reagent (RossettSep, StemCell) was used to remove CD4 + With CD8 + T cells, and then PBMCs were separated with an equal volume of Ficoll-Hypaque solution. The cells obtained in this way are CD4 + With CD8 + The isolated cells were washed once with 0.9% saline and cultured for in vitro expansion.

[0168] 3.2 In vitro expansion and culture of DNT cells

[0169] Same as 1.2 above.

[0170] On day 17-18, DNT cells were harvested as needed.

[0171] 3.3 Harvesting DNT cells to prepare DNT cell preparations

[0172] Same as 1.3 above.

[0173] Example 4: Sedimentation-enriched DNT cells were cryopreserved and then revived for in vitro expansion and culture

[0174] 4.1 Healthy Donor DNT Cells Enrichment

[0175] Same as 3.1 above.

[0176] 4.2 Cryopreservation of the obtained DNT cells

[0177] Same as 2.2 above.

[0178] 4.3 Recovery of frozen DNT cells

[0179] Same as 2.3 above.

[0180] 4.4 In vitro expansion and culture of DNT cells

[0181] Same as 1.2 above.

[0182] On day 17-18, DNT cells were harvested as needed.

[0183] 4.5 Harvesting DNT cells to prepare DNT cell preparations

[0184] Same as 1.3 above.

[0185] Example 5: Application examples and effect data

[0186] In this embodiment, CD3 + CD4 - CD8 - Taking T (Double Negative T, DNT) cells as an example, the process and effect of in vitro expansion using the above-mentioned universal clinical human DNT cell efficient in vitro expansion method were verified.

[0187] Application Example 1

[0188] According to the method of Example 1, CD4 + With CD8 + The obtained DNT cells were directly expanded and cultured in vitro using the method of the present invention (recombinant human interleukin-2 concentration of 500 IU / mL, the amount of anti-human CD3 monoclonal antibody was reduced in two stages (50 ng / mL in the first stage and 25 ng / mL in the second stage), and no interleukin-4 or AB serum was used). The cells were collected on days 17 to 20, and the cell activity, phenotype, and total number of expanded cells were detected.

[0189] Application Example 2

[0190] According to the method of Example 2, CD4 + With CD8 +The obtained DNT cells were frozen and then revived and then expanded and cultured in vitro using the method of the present invention (recombinant human interleukin-2 concentration of 500 IU / mL, two-stage reduction of the anti-human CD3 monoclonal antibody dosage (50 ng / mL in the first stage and 25 ng / mL in the second stage), without the use of interleukin-4 and AB serum). The cells were collected on the 17th to 20th day, and the cell activity, phenotype, and total number of cell proliferation were detected.

[0191] Application Example 3

[0192] According to the method of Example 3, CD4 / CD8 lymphocyte removal agent was used to remove CD4 + With CD8 + The obtained DNT cells were directly expanded and cultured in vitro using the method of the present invention (recombinant human interleukin-2 concentration of 500 IU / mL, the dosage of anti-human CD3 monoclonal antibody was reduced in two stages (50 ng / mL in the first stage and 25 ng / mL in the second stage), without the use of interleukin-4 and AB serum). The cells were collected on days 17 to 20, and the cell activity, phenotype, and total number of expanded cells were detected.

[0193] Application Example 4

[0194] According to the method of Example 4, CD4 / CD8 lymphocyte removal agent was used to remove CD4 + With CD8 + The obtained DNT cells were frozen and then revived and then expanded and cultured in vitro using the method of the present invention (recombinant human interleukin-2 concentration of 500 IU / mL, two-stage reduction of the anti-human CD3 monoclonal antibody dosage (50 ng / mL in the first stage and 25 ng / mL in the second stage), without the use of interleukin-4 and AB serum). The cells were collected on the 17th to 20th day, and the cell activity, phenotype, and total number of cell proliferation were detected.

[0195] Comparative Example 1

[0196] According to existing techniques, recombinant human interleukin-2 (500 IU / mL), recombinant human interleukin-4 (2 ng / mL), recombinant human interleukin-7 (8 ng / mL), recombinant human interleukin-12 (5 ng / mL), 5% autologous plasma, and 6% AB serum were added to the culture medium. The specific cell culture process was the same as in the above application example.

[0197] Comparative Example 2

[0198] Referring to the existing literature (Lee JB et al., Clin Cancer Res. 25(7):2241-2253, 2019), 250 IU / mL interleukin-2 and 100 ng / mL anti-human CD3 monoclonal antibody were used. The specific cell culture process was the same as the above application example.

[0199] The cells of the above application examples and comparative examples were sampled on the 1st, 7th, 14th, and 17th day of culture to detect the total cell count. The changes in the total cell count were compared to obtain the cell growth curves of the application examples and comparative examples as shown in the attached figure. Figure 1 . Figure 1 As can be seen, the cell count of the cells in the application example increased significantly starting on day 7, while the cell count in comparative example 1 did not show a significant increase until day 14. In comparative example 2, the cell count increased slightly from day 7 to day 14, before beginning to increase significantly on day 14. This indicates that at the same culture time point, the method of the present invention can expand more DNT cells compared to the comparative example.

[0200] On day 17, DNT cells were collected and the cell viability, phenotypic purity and DNT expansion fold of the application example and the control example were compared. The results are listed in Table 1.

[0201] Table 1 Cell viability, phenotypic purity and amplification times of application examples and comparative examples

[0202]

[0203] In Table 1, Application Examples 1 and 3, as well as Comparative Examples 1 and 2, all used freshly isolated DNT cells for direct culture. Comparing the expansion folds of DNT cells, the expansion folds obtained by magnetic bead enrichment and sedimentation enrichment followed by in vitro expansion using the present method were 15,873.68-fold and 20,948.08-fold, respectively, significantly exceeding the 8,827.93-fold (Comparative Example 1) and 9,402.65-fold (Comparative Example 2), respectively. Cell phenotype and cell viability showed little difference. Therefore, using the present expansion technology, DNT cells enriched using the two different methods can yield a larger DNT cell product.

[0204] The isolated DNT cells were cryopreserved and then revived and expanded in vitro using the method of the present invention, resulting in Application Examples 2 and 4 (Table 1). Compared with Comparative Examples 1 and 2, which were directly cultured without cryopreservation, the DNT cell expansion folds obtained by enriching DNT cells using two different methods, then cryopreserving and reviving the DNT cells and then expanding them in vitro were 10778.95-fold and 10819.41-fold, respectively, comparable to Comparative Example 1 (8827.93-fold) and Comparative Example 2 (9402.65-fold), which were directly cultured without cryopreservation. There was no significant difference in DNT cell purity or cell viability.

[0205] Application Example 5

[0206] CD4-depleted peripheral blood + With CD8 +The enriched DNT cells were cryopreserved in liquid nitrogen for 7, 14, and 30 days, then revived and expanded in vitro using the expansion method of the present invention (recombinant human interleukin-2 at a concentration of 500 IU / mL, a two-stage reduction in the amount of anti-human CD3 monoclonal antibody (50 ng / mL in the first stage and 25 ng / mL in the second stage), without the use of interleukin-4 or AB serum). Cells were collected on day 17 and assayed for cell viability, purity, cell biological potency, and DNT cell expansion multiple.

[0207] The cell viability, purity, cell biological efficacy and DNT cell expansion folds of fresh whole blood enriched DNT cells were compared with those of frozen and thawed DNT cells. The results are listed in Table 2.

[0208] Table 2 Comparison of cell purity, biological efficacy and DNT cell expansion times

[0209]

[0210] As shown in Table 2, compared with the DNT cell batches that were freshly enriched and cultured for 17 days, the DNT cell batches that were frozen for different time periods (7 days, 14 days, 30 days) and then revived and cultured in vitro showed significant differences in cell viability (>85%), cell purity (CD3 + CD4 - CD8 - , >90%), tumor killing activity (>50), and in vitro expansion folds showed no significant differences. The differences in expansion folds between batches may be related to the characteristics of DNT cells from different donors, but all meet clinical infusion requirements. Overall, the present invention can be applied to the in vitro expansion of freshly enriched DNT cells as well as DNT cells revived after cryopreservation. Not only does the final product have a DNT cell viability of over 85%, a purity exceeding 90%, and a biological efficacy exceeding 50%, but the DNT cell expansion fold can also reach over 8,000-fold.

[0211] The above examples demonstrate that the present invention's process for cryopreserving DNT cells on day 0 and subsequently expanding them in vitro offers flexibility in cell preparation for clinical treatment protocols. Building on existing patented expansion technology, the production process has been further improved, increasing the in vitro expansion of DNT cells by an order of magnitude of 8,000-fold within 17 days. This allows DNT cells obtained from a single expansion to provide clinical therapeutic doses for multiple patients.

[0212] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A universal in vitro expansion method for DNT cells, characterized in that: Including steps: (i) obtaining a peripheral blood starting sample I; (ii) Removal of CD4 from the starting sample I + and CD8 + T cells, thereby obtaining sample II; (iii) culturing sample II in a culture system containing a culture medium suitable for DNT cell growth, thereby obtaining sample III; wherein the culture system includes an immobilized T cell mitogen; (iv) culturing sample III in a culture system containing a culture medium suitable for DNT cell growth, thereby obtaining sample IV; wherein the culture medium contains a soluble T cell mitogen at a concentration of 40-60 ng / mL; (v) culturing sample IV in a culture system containing a culture medium suitable for the growth of DNT cells, wherein the culture medium contains a soluble T cell mitogen at a concentration of 20-30 ng / mL, thereby obtaining a desired amount of universal DNT cells, which is sample V; and (vi) collecting a sample V in a solution system suitable for preserving DNT cells; wherein the solution contains human serum albumin, thereby obtaining universal DNT cells for clinical use, which is a DNT cell preparation; The culture systems in steps (iii) to (v) all contain 200-1000 IU / mL of recombinant human interleukin-2, and do not contain recombinant human interleukin-4 and AB serum; and the T cell mitogen is an antibody that binds to CD3.

2. The in vitro amplification method according to claim 1, wherein The sample II is a cell solution recovered after freezing.

3. The in vitro amplification method according to claim 1, wherein The culture medium in steps (iii) to (v) further comprises the following cytokines: IL-7, IL-12, and / or IL-15.

4. The in vitro amplification method according to claim 1, wherein In step (iv), the culture medium contains a soluble T cell mitogen at a concentration of 50 ng / mL.

5. The in vitro amplification method according to claim 1, wherein In step (v), the culture medium contains a soluble T cell mitogen at a concentration of 25 ng / mL.

6. The in vitro amplification method according to claim 1, wherein The culturing time of step (iii) is 24-72 hours.

7. The in vitro amplification method according to claim 1, wherein In step (iii), the initial concentration of the DNT cells to be expanded in the culture system is 1×10 6 to 4×10 6 cells / mL.

8. The in vitro amplification method according to claim 1, wherein In step (iv), the DNT cells are expanded 2-5 times. In each expansion, the initial concentration of the DNT cells to be expanded is independently 1×10 6 to 3×10 6 cells / mL, and the culture time for each expansion was 24-72 hours.

9. The in vitro amplification method according to claim 1, wherein In step (v), the DNT cells are expanded 2-4 times. In each expansion, the initial concentration of the DNT cells to be expanded is independently 1×10 6 to 3×10 6 cells / mL, and the culture time for each expansion was 24-72 hours.

10. The in vitro amplification method according to claim 1, wherein In step (iv), the method further comprises detecting the phenotype and viability of the DNT cells.

11. The in vitro amplification method according to claim 1, wherein In step (v), the method further comprises detecting the phenotype and viability of the DNT cells.

12. The in vitro amplification method according to claim 1, wherein The culture systems in steps (iii) to (v) all contain 300-700 IU / mL of recombinant human interleukin-2.

13. The in vitro amplification method according to claim 1, wherein The culture systems in steps (iii) to (v) all contain 500 IU / mL of recombinant human interleukin-2.

14. The in vitro amplification method according to claim 1, wherein In step (vi), the steps include: (via) collecting DNT cells in sample V by centrifugation; (vib) washing DNT cells with physiological saline containing 2.5% human albumin; and (vic) DNT cells were adjusted to 1×10 8 The concentration of cells / mL.

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