Universal DNT cell for clinical use, and preparation method therefor and use thereof

Abstract

Provided is a technique for preparing a universal DNT cell. The prepared raw materials are derived from "leucocyte filtration residues" after leucocyte filtration treatment in order to avoid adverse blood transfusion reactions, and the DNT cell extracted therefrom can be a mixed culture of one or more healthy donors. The universal DNT cell obtained using the provided method has the characteristics of high yield, high purity, high in-vitro tumor killing activity, suitability for large-scale production, etc. The starting raw material of the cell can be a fresh cell or a cryopreserved re-thawed cell, the bottleneck of the raw material source for large-scale preparation of cell drugs is fundamentally solved, and the value of the filtered leucocyte in the blood product production process is re-mined. The cell has great medical value and commercial application prospects.

Description

A universal clinical DNT cell and its preparation method and application Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a universal clinical DNT cell and a preparation method and application thereof. 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 before reinfusing them into the patient to stimulate and enhance the patient's own immune function, thereby achieving the goal of treating tumors.

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

[0004] Currently, immune cell therapies mostly involve collecting autologous immune cells, culturing and expanding them in vitro, and then infusing them back into the patient. If multiple infusions are required, immune cells must be collected from the patient's peripheral blood for multiple culturing and expansion to ensure sufficient cells for infusion. Due to the severity of their illness, most patients requiring treatment are often unable to provide a sufficient number of immune cells for culture. Furthermore, due to their underlying medical conditions and various medications, culturing and expanding their immune cells in vitro is extremely difficult, or even impossible.

[0005] In recent years, treatment options have emerged that involve collecting immune cells from healthy donors, expanding them through in vitro culture, and then transfusing them back into the body. However, since a typical healthy donor can collect 200-400 ml of whole blood at a time, the cell raw material for each production batch is limited to 200-400 ml of whole blood, hindering scalable production.

[0006] Therefore, there is a need in the art for a method for obtaining immune cells from one or more healthy donors for large-scale in vitro expansion of immune cells, which is not limited to whole blood collection from a single healthy donor.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to provide a new universal allogeneic double negative T (DNT) cell for multiple clinical transfusions, as well as its preparation method and application.

[0009] The first aspect of the present invention provides a method for preparing universal clinical DN T cells, comprising the steps of:

[0010] (i) providing a peripheral blood starting sample I from a healthy donor;

[0011] (ii) filtering sample I through a filter to retain leukocytes, backflushing the filter with a backflushing solution from the "liquid outlet" of the filter, and collecting the backflushed leukocytes from the "liquid inlet" of the filter, thereby obtaining sample II;

[0012] (iii) removing CD4 from sample II of one or more donors + and CD8 + T cells, thereby obtaining sample III;

[0013] (iv) mixing samples III from multiple donors and culturing them in a culture system containing a culture medium suitable for the growth of DNT cells, thereby obtaining sample IV; wherein the culture system includes a fixed T cell mitogen;

[0014] (v) culturing sample IV in a culture system containing a culture medium suitable for the growth of DNT cells, thereby obtaining sample V; wherein the culture medium contains a soluble T cell mitogen at a concentration of 50-150 ng / mL;

[0015] (vi) culturing sample V 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 25-75 ng / mL, thereby obtaining a desired amount of universal DNT cells, which is sample VI;

[0016] (vii) collecting sample VI in a solution system containing a solution suitable for DNT cell preservation, wherein the solution is a cell freezing solution; then storing the sample in a liquid nitrogen tank after programmed cooling, thereby obtaining a universal clinical DNT cell freezing preparation;

[0017] The culture systems in steps (iv) to (vi) all contain 200-600 IU / mL of recombinant human interleukin-2, and do not contain recombinant human interleukin-4, and do not contain serum or plasma.

[0018] In another preferred embodiment, the amount of the sample I is 10-500 ml, preferably 40-400 ml.

[0019] In another preferred embodiment, the sample III is a cell solution that is thawed after being frozen.

[0020] In another preferred embodiment, in step (ii), the backwash fluid is sterile physiological saline.

[0021] In another preferred embodiment, the backflushing step in step (ii) specifically includes: heat-sealing the two ends of the filter and removing them, connecting sterile saline from the "liquid outlet" of the filter through a sterile pipe, pulse-pressing the connected sterile saline bag and tapping or kneading the filter, and collecting the white blood cells obtained by backflushing from the "liquid inlet" of the filter.

[0022] In another preferred embodiment, the step (iii) comprises: removing CD4 + and CD8 + T cells, thereby obtaining sample IIIa, which was resuspended in freezing medium, cooled by programmed cooling, and stored in liquid nitrogen. This was sample IIIb. The frozen sample IIIb from one or more donors was thawed at 37°C, washed once with culture medium, and then resuspended in culture medium and mixed. This was sample III, i.e., DNT derived from donor leukocytes that were frozen and thawed.

[0023] In another preferred embodiment, in the culture system of steps (iv) to (vi), the culture conditions are 37° C. and 5% CO 2 .

[0024] In another preferred embodiment, the culture medium in steps (iv) to (vi) further comprises a cytokine selected from the group consisting of IL-7, IL-12, IL-15, or a combination thereof.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] In another preferred embodiment, the T cell mitogen is an antibody that binds to CD3.

[0029] In another preferred embodiment, the culture system in step (iv) is located in a culture flask, preferably a T25 culture flask.

[0030] In another preferred embodiment, the immobilized T cell mitogen refers to a T cell mitogen coated on a culture flask or a microplate.

[0031] In another preferred embodiment, in step (v), the initial concentration of the DNT cells to be expanded in the culture system is 0.1×10 6 to 1×10 6 cells / mL.

[0032] In another preferred embodiment, the culture time in step (iv) is 24-72 hours, preferably 36-60 hours, and more preferably 48 hours.

[0033] In another preferred embodiment, in step (iv), it is divided into sub-steps (iva), (ivb) and (ivc), and the sub-steps (iva), (ivb) and (ivc) are three amplifications of the DNT cells in sample III.

[0034] In another preferred embodiment, in the sub-step (iva), the initial concentration of the DNT cells to be expanded in the culture system is 1×10 6 ~5×10 6 cells / mL.

[0035] In another preferred embodiment, in sub-steps (ivb) and (ivc), the initial concentration of the DNT cells to be expanded in the culture system is independently 0.1×10 6 to 1×10 6 cells / mL.

[0036] In another preferred embodiment, the culture time of the sub-steps (iva), (ivb) and (ivc) is independently 24-72 hours, preferably 36-60 hours, and more preferably 48 hours.

[0037] In another preferred embodiment, the culturing time in step (iv) is 96-192 hours, preferably 120-168 hours, and more preferably 144 hours.

[0038] In another preferred embodiment, in step (v), the DNT cells are expanded 2-5 times (preferably 3-4 times, more preferably 3 times), and in each expansion, the initial concentration of the DNT cells to be expanded is independently 0.1×10 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.

[0039] In another preferred embodiment, step (v) further comprises detecting the phenotype and viability of DNT cells.

[0040] In another preferred embodiment, the culturing time in step (v) is 72-168 hours, preferably 96-144 hours, and more preferably 120 hours.

[0041] In another preferred embodiment, step (vi) comprises 2-4 (preferably 2) expansions of DNT cells, 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.

[0042] In another preferred embodiment, step (vi) further includes detecting the phenotype and viability of DNT cells.

[0043] In another preferred embodiment, the culture time in step (vi) is 48-96 hours, preferably 72 hours.

[0044] In another preferred embodiment, the culture systems in steps (iv) to (vi) all contain serum replacement, and the volume ratio of serum replacement in the culture system is 8%-15%, preferably 10%.

[0045] In another preferred embodiment, step (vii) comprises the steps of:

[0046] (viia) collecting DNT cells in sample VI by centrifugation;

[0047] (viib) washing the DNT cells with sodium chloride injection containing 2.5% human albumin;

[0048] (viic) DNT cells were adjusted to 0.5×10 8 -1×10 8 The concentration of cells / mL.

[0049] In another preferred embodiment, the universal clinical DN T cells have the following characteristics:

[0050] (1) Expression of CD3 + But does not express CD4 - With CD8 - ;

[0051] (2) will not cause graft-versus-host disease (GvHD);

[0052] (3) Can be used for allogeneic cell therapy.

[0053] A second aspect of the present invention provides a use of an effective amount of DN T cells obtained using 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:

[0054] (a) prevention and / or treatment of tumors;

[0055] (b) prevention and / or treatment of infectious diseases;

[0056] (c) prevention and / or treatment of autoimmune diseases;

[0057] (d) prevention and / or treatment of allergic reactions;

[0058] (e) prevention and / or treatment of graft-versus-host disease;

[0059] (f) Modulation of immune response.

[0060] In another preferred embodiment, the tumor is a tumor allogeneic to the DNT cells.

[0061] In another preferred embodiment, the tumor is selected from the following group: hematological tumors, solid tumors, or a combination thereof.

[0062] 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.

[0063] 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.

[0064] 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 graying of the hair, Raynaud's syndrome, glomerulonephritis, dermatomyositis, chronic active hepatitis, celiac disease, tissue-specific autoimmunity, degenerative autoimmune delayed hypersensitivity, autoimmune complications of AIDS, atrophic gastritis, ankylosing spondylitis, Addison's disease, or a combination thereof.

[0065] 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.

[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 described in detail here. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] FIG1 shows a process flow chart for preparing DNT cell preparations from the white filtration residue.

[0068] FIG2 shows a flow chart of the operation of obtaining filter white residue by backwashing the filter white.

[0069] Figure 3 shows a comparison of DNT cell viability changes using the process of the present invention and existing processes. This figure primarily compares the DNT cell viability obtained using the process of the present invention and using existing processes on day 0 and after 14 days of in vitro culture. The results show that there was no statistically significant difference in DNT cell viability on day 0 or after 14 days of culture between the two processes.

[0070] Figure 4 shows a comparison of the purity of DNT cells cultured in vitro using the process of the present invention and existing processes. This figure demonstrates that the purity of DNT cells obtained using the process of the present invention was measured on days 0, 7, and 14 after in vitro expansion. There was no statistically significant difference in the purity of DNT cells obtained using the process of the present invention and existing processes.

[0071] Figure 5 shows a comparison of the expansion multiples of DNT cells cultured in vitro using the process of the present invention and the existing process. This figure shows that there is no significant difference in the expansion of DNT cells obtained by the process of the present invention in the fresh culture group and the existing process, while the expansion capacity of DNT cells in the frozen, thawed and then cultured group is significantly better than that of the existing process.

[0072] Figure 6 shows a comparison of the killing ability of DNT cells cultured in vitro by the process of the present invention and the existing process. There is no statistically significant difference in the killing ability of DNT cells obtained by the process of the present invention and the existing process after 14 days of expansion.

[0073] Figure 7 shows a comparison of key indicators between single-donor and multi-donor cultures using the inventive process. Under the inventive process, multi-donor mixed culture maintained consistency with single-donor culture in terms of viability, cell purity, and biological efficacy.

[0074] Figure 8 compares the expansion fold of DN T cells from a single donor using the existing process and different groups (single and multiple donors) using the present invention. Due to individual differences between healthy donors, the expansion of each donor varies. Multi-donor mixed culture can balance the expansion of individual DN T cells to a certain extent, thereby ensuring the yield of the preparation. DETAILED DESCRIPTION

[0075] After extensive and in-depth research and numerous process optimization experiments, the inventors have developed, for the first time, a method for preparing allogeneic universal DNT cells, prepared from the peripheral blood of one or more donors for multiple clinical transfusions. Specifically, the present invention optimizes the initial sample processing process, enabling simultaneous leukocyte extraction from the peripheral blood of one or more donors. This extraction process eliminates the need for centrifugation, allowing for the processing of large volumes of peripheral blood in a short period of time. Furthermore, the present invention optimizes the DNT cell culture formulation and process, enabling the in vitro expansion and culture of DNT cells derived from a mixture of one or more donors.

[0076] Compared to existing methods that use peripheral blood as a starting material for obtaining mononuclear cells (PBMCs), the present method uses leukocyte filtration residue as a starting material, which is more convenient and more readily available. Furthermore, the present method for preparing DNT cells is suitable for co-culturing DNT cells from multiple donors, allowing for scale-up of production batches as needed to obtain large quantities of highly pure, universal DNT cells with broad-spectrum tumorigenicity.

[0077] The experimental results show that the method of the present invention can be used to quickly prepare a filter residue from one or more donor peripheral blood by backflushing for the enrichment and efficient in vitro expansion of DNT cells. The total number of DNT cells expanded using the process of the present invention is high, and the number of CD3 + CD4 - CD8 -The purity of T cells is high (>85%). Due to the existing regulatory restrictions on whole blood collection, the collected whole blood or single-collected cells cannot be directly used for the industrial production of commercial universal cell drugs, and the number of whole blood or single-collected cells obtained in a single collection is limited, which cannot meet the needs of large-scale cell preparation. Therefore, the use of the DNT cell preparation method of the present invention can expand the source of raw materials for cell preparation, and at the same time, it can process samples more quickly and efficiently, and mix and culture DNT cells from different donors. The DNT cells prepared by the method of the present invention are no longer limited by the number of white blood cells in the raw materials, and can truly be used for the production of commercial universal cell drugs. At the same time, the DNT cells obtained by the method of the present invention have a broader spectrum of tumoricidal efficacy, which is conducive to improving clinical efficacy.

[0078] On this basis, the present invention was completed.

[0079] the term

[0080] 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.

[0081] 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.

[0082] 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.

[0083] DNT cell preparation method of the present invention

[0084] In the present invention, a method for preparing universal clinical DN T cells is provided, wherein the method comprises the steps of:

[0085] (i) providing a peripheral blood starting sample I obtained from a donor;

[0086] (ii) filtering the whole blood through a filter to retain leukocytes, and backflushing the filter to obtain a leukocyte suspension, thereby obtaining sample II;

[0087] (iii) removing CD4 from sample II of one or more donors + and CD8 + T cells, thereby obtaining sample III; specifically, comprising the following steps:

[0088] Remove CD4 from one or more donor samples II + and CD8 + T cells, thereby obtaining sample IIIa, which is resuspended in freezing solution, cooled by programmable cooling, and then stored in liquid nitrogen. This is sample IIIb. Frozen sample IIIb from one or more donors is thawed at 37°C, washed once with culture medium, and then resuspended in culture medium and mixed. This is sample III, i.e., frozen and thawed donor leukocyte-derived DNT;

[0089] (iv) mixing samples III from one or more donors and culturing them in a culture system containing a culture medium suitable for the growth of DNT cells, thereby obtaining sample IV; wherein the culture system includes an immobilized T cell mitogen;

[0090] (v) culturing sample IV in a culture system containing a culture medium suitable for the growth of DNT cells, thereby obtaining sample V; wherein the culture medium contains a soluble T cell mitogen at a concentration of 30-80 ng / mL;

[0091] (vi) culturing sample V 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, thereby obtaining a desired amount of universal DNT cells, which is sample VI;

[0092] (vii) collecting sample VI in a solution system containing a solution suitable for DNT cell preservation, wherein the solution is a cell freezing solution; then storing the sample in a liquid nitrogen tank after programmed cooling, thereby obtaining a universal clinical DNT cell freezing preparation;

[0093] The culture systems in steps (iv) to (vi) all contain 200-600 IU / mL of recombinant human interleukin-2, and do not contain recombinant human interleukin-4, and do not contain serum or plasma.

[0094] In another preferred embodiment, in step (i), the donor is a healthy donor.

[0095] In another preferred embodiment, the amount of the sample I is 10-500 ml, preferably 40-400 ml.

[0096] In another preferred embodiment, the sample II is a cell solution that is thawed after being frozen.

[0097] In another preferred embodiment, in the culture system of steps (iv) to (vi), the culture conditions are 37° C. and 5% CO 2 .

[0098] In another preferred embodiment, the culture medium in steps (iv) to (vi) further comprises a cytokine selected from the group consisting of IL-7, IL-12, IL-15, or a combination thereof.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] In another preferred embodiment, the T cell mitogen is an antibody that binds to CD3.

[0103] In another preferred embodiment, the culture system in step (iv) is located in a culture flask, preferably a T25 culture flask.

[0104] In another preferred embodiment, the immobilized T cell mitogen refers to a T cell mitogen coated on a culture flask or a microplate.

[0105] In another preferred embodiment, in step (v), the initial concentration of the DNT cells to be expanded in the culture system is 1×10 6 to 4×10 6 cells / mL.

[0106] In another preferred embodiment, the culture time in step (iv) is 24-72 hours, preferably 36-60 hours, and more preferably 48 hours.

[0107] In another preferred embodiment, in step (iv), it is divided into sub-steps (iva), (ivb) and (ivc), and the sub-steps (iva), (ivb) and (ivc) are three amplifications of the DNT cells in sample III.

[0108] In another preferred embodiment, in the sub-step (iva), the initial concentration of the DNT cells to be expanded in the culture system is 1×10 6 ~5×10 6 cells / mL.

[0109] In another preferred embodiment, in sub-steps (iva) and (ivb), the initial concentration of the DNT cells to be expanded in the culture system is independently 0.5×106 to 1×10 6 cells / mL.

[0110] In another preferred embodiment, the culture time of the sub-steps (iva), (ivb) and (ivc) is independently 24-72 hours, preferably 36-60 hours, and more preferably 48 hours.

[0111] In another preferred embodiment, the culturing time in step (iv) is 96-192 hours, preferably 120-168 hours, and more preferably 144 hours.

[0112] In another preferred embodiment, step (v) 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.

[0113] In another preferred embodiment, step (v) further comprises detecting the phenotype and viability of DNT cells.

[0114] In another preferred embodiment, the culturing time in step (v) is 72-168 hours, preferably 96-144 hours, and more preferably 120 hours.

[0115] In another preferred embodiment, step (vi) comprises 2-4 (preferably 2) expansions of DNT cells, 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.

[0116] In another preferred embodiment, step (vi) further includes detecting the phenotype and viability of DNT cells.

[0117] In another preferred embodiment, the culture time in step (vi) is 48-96 hours, preferably 72 hours.

[0118] In another preferred embodiment, step (vii) comprises the steps of:

[0119] (viia) collecting DNT cells in sample VI by centrifugation;

[0120] (viib) washing the DNT cells with sodium chloride injection containing 2.5% human albumin;

[0121] (viic) Adjust the number of DNT cells to 0.5-1×10 using CS5 freezing medium. 8 The concentration of cells / mL.

[0122] 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 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 derived from human peripheral blood.

[0123] 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, and fractions or enriched portions thereof, and can also be derived from induced pluripotent stem cells (iPSCs).

[0124] In a preferred embodiment, the starting sample is blood, preferably human blood, more preferably human peripheral blood.

[0125] Sample III is substantially depleted of CD4 before culturing the starting sample or its fractions. + 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.

[0126] These cell types can be removed from the sample using techniques known in the art. Specifically, antibodies can be added to Sample II that bind to the CD8 and CD4 cells to be removed but do not bind to double negative T cells. In a preferred embodiment, labeled antibodies that specifically bind to CD4 and CD8 are added to Sample II.

[0127] In another preferred embodiment, magnetic beads that specifically bind to CD4 and CD8 are added to sample II.

[0128] Once CD4 has been removed from Sample II + and CD8 +T cells, the method of the present invention can be continued immediately or the sample can be frozen and stored for later use. Therefore, when DNT cells are needed, the expansion of DNT cells can be started from the frozen sample at this time.

[0129] In one embodiment of the present invention, a method is provided for obtaining peripheral blood from a healthy donor, filtering the peripheral blood in vitro to obtain leukocytes and removing CD4 + and CD8 + T cells are immediately frozen and thawed according to the production scale, and the DN T cells of one or more donors are used in the preparation method of the present invention alone or in combination.

[0130] Preferably, step (ii) of the method of the present invention may include: filtering the starting sample with a filter to retain leukocytes, backflushing the filter to collect the filtered leukocytes, thereby obtaining sample II; performing step (iii) on the sample II of one or more donors to obtain CD4 + and CD8 + After removal, T cells can be cultured directly, or they can be frozen in liquid nitrogen and then thawed and cultured as needed.

[0131] Preferably, step (iii) of the method of the present invention may include: removing CD4 + and CD8 + T cells, thereby obtaining sample IIIa (enriched DNT cells); after mixing, samples IIIa from multiple donors can be directly cultured using the in vitro expansion method of the present invention, or can be frozen in liquid nitrogen and then thawed and then cultured; sample IIIa (enriched DNT cells) is collected by centrifugation, resuspended in freezing solution, and stored in liquid nitrogen after program-controlled cooling, which is sample IIIb (enriched frozen DNT cells); according to the production scale, frozen samples IIIb from one or more donors are selected, thawed at 37°C, mixed, washed once with culture medium, and then resuspended in culture medium, which is sample III (enriched frozen and then thawed multi-donor DNT cells).

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] It is particularly noteworthy that, in the present invention, the culture medium used for expansion culture does not contain IL-4 and serum.

[0137] The concentration of the agent should be suitable for promoting the expansion of double negative cells. Preferably, the cytokine is administered in a range of about 200 IU / mL to 600 IU / mL. In a particularly preferred embodiment, the concentration of IL-2 in the culture medium is 250 IU / mL.

[0138] The culture medium can be any medium suitable for T cell culture, including but not limited to AIM-V medium, RPMI medium, X-VIVO10 and X-VIVO15, and any other commercial animal serum-free medium.

[0139] 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 peptides or neoantigens derived from tumor-specific or tumor-associated antigens can be used.

[0140] The cells are preferably cultured in any one of steps (iv)-(vi) 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.

[0141] The purity of the DNT cells prepared by this method can be confirmed using techniques known in the art, such as flow cytometry.

[0142] Uses of the DNT cells prepared by the present invention

[0143] The present invention also encompasses the use of universal double-negative T cells (DNTs) obtained by the methods of the present invention in any and all applications. The multi-donor 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.

[0144] In an embodiment of the present invention, the multi-donor double-negative T cells expanded by the method of the present invention have a strong and broad-spectrum anti-tumor effect. Therefore, in one embodiment, the present invention provides a method for treating tumors, comprising administering an effective amount of multi-donor 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 multi-donor 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 multi-donor DNT cells obtained by the method of the present invention in the preparation of a medicament for treating tumors.

[0145] 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.

[0146] The term "animal" as used herein includes all members of the animal kingdom, including humans. In a preferred embodiment, the animal is a human.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] Hematological cancers are cancers of the blood, bone marrow, or lymphoid tissue. Examples of hematological (or blood-borne) 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.

[0151] 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.

[0152] The present invention also includes other therapeutic uses of the multi-donor DN T cells of the present invention, such as the treatment of infectious diseases and the modulation of immune responses in the treatment of autoimmune diseases, allergies, transplant rejection, and graft-versus-host disease. In this case, the method for preparing the multi-donor DN T cells may include adding a suitable infectious agent, allergen cell, or tissue as an antigen.

[0153] Therefore, in one embodiment, the present invention provides a method for treating an infectious disease, comprising administering to an animal in need thereof an effective amount of multiple donor double-negative T cells obtained by the method of the present invention. The present invention also includes the use of an effective amount of multiple donor 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 multiple donor double-negative T cells obtained by the method of the present invention for preparing a medicament for treating an infectious disease.

[0154] In a further embodiment, the present invention provides a method for modulating an immune response, comprising administering to an animal in need thereof an effective amount of multiple-donor double-negative T cells obtained by the method of the present invention. The present invention also includes the use of an effective amount of multiple-donor 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 multiple-donor double-negative T cells obtained by the method of the present invention for preparing a medicament for modulating an immune response.

[0155] In one embodiment, multiple donor DN T 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, celiac disease, tissue-specific autoimmunity, degenerative autoimmune delayed hypersensitivity, autoimmune complications of AIDS, atrophic gastritis, ankylosing spondylitis, and Addison's disease.

[0156] In another embodiment, multiple donor DNA cells can be used to treat graft-versus-host disease, in which immune cells in the transplant attack the recipient's normal tissues. This situation 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.

[0157] In a further embodiment, multi-donor DNA cells can be used to treat allergies. In an allergy, the immune system mounts an attack against 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.

[0158] The multi-donor DN T 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.

[0159] The compositions described herein can be prepared by methods known per se for preparing pharmaceutically acceptable compositions that can be administered to a subject by combining an effective amount of cells in a mixture 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). In this regard, compositions include, but are not limited to, solutions of a substance in combination with one or more pharmaceutically acceptable carriers or diluents, including physiological solutions in a buffered solution having an appropriate pH and isotonicity.

[0160] 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.

[0161] 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.

[0162] The pharmaceutical composition of the present invention can be used in a manner suitable for the disease to be treated (or prevented). The quantity and frequency of use will be determined by factors such as the patient's condition, the type and severity of the patient's disease, although appropriate dosage can be determined by clinical trials.

[0163] 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 multiple donor DN 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 6to 10 8 The multi-donor DNT cell composition can be administered at a dose of 10 cells / kg body weight (including all integer values ​​within that range). Multiple donor DNT cell compositions can also be administered multiple times at these doses. 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 determined by monitoring the patient's disease status, so that treatment adjustments are readily determined by those skilled in the medical field.

[0164] 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 composition of the T cell can be injected directly into the tumor, lymph node or infection site.

[0165] 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.

[0166] 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, 1×109 to 1×10 11 The multi-donor double-negative T cells of the present invention are administered to the patient, for example, by intravenous infusion.

[0167] The main advantages of the present invention include:

[0168] 1) Improve the formulation and process, explore the source of raw materials for cell products, and develop a method for large-scale cultivation of DNT cells from multiple donors for clinical use.

[0169] 2) Improvements in formulation and process allow for mixed culture of DNT cells from multiple donors.

[0170] 3) The formula and process have been improved, and no human serum or fetal bovine serum (FBS) is required.

[0171] 4) Formula and process improvements can be used to culture freshly enriched multi-donor DNT cells, as well as multi-donor DNT cells that have been frozen and thawed.

[0172] 5) Improvements in formulation and process enable single-time collection / freezing, multiple thawing and culture, and multiple transfusions or use by multiple patients.

[0173] 6) Formula and process improvements have enabled cell proliferation to reach 1x10 9 cells / mL of whole blood.

[0174] 7) Improve the formula and process to ensure the purity of the final product DNT cells (CD3 + CD4 - CD8 - )≥85%.

[0175] 8) Improve the formulation and process to ensure that the in vitro high-efficiency tumoricidal activity (biological efficacy) of cells is ≥50%.

[0176] 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 present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under 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 by weight.

[0177] Example 1: Direct expansion and culture of DNT cells enriched with filtration residues

[0178] 1.1 Preparation of filter residue

[0179] Collect 200-400ml of peripheral blood from a healthy donor into a blood bag containing an anticoagulant. The bag is then hung high, and gravity forces the whole blood through a filter (Spacell RZ2000), trapping the white blood cells in the filter. Heat-seal the filter at both ends and remove it. Connect sterile saline to the "outlet" end of the filter via a sterile pipette. Pulse pressure is applied to the saline bag, and the filter is tapped or kneaded to obtain the leukocyte residue from the "inlet" end of the filter.

[0180] 1.2 Enrichment of DNT cells from filtration residue

[0181] Place the filtered white residue in a high-speed centrifuge, centrifuge and discard the supernatant, leaving a volume of about 100 ml after centrifugation. Add RosetteSep TM Human CD4 Depletion Cocktail and RosetteSep TM Human CD8 Depletion Cocktail, CD4 removed by centrifugation + \CD8 + cells to obtain enriched DNT cells.

[0182] 1.3 In vitro culture of donor DNT cells

[0183] A sample of DNT cells derived from the leukocyte residue was selected and coated with anti-human CD3 monoclonal antibody (10 μg / mL) in a T25 culture flask. The cells were then adjusted to 1×10 cells / mL in serum-free medium (containing 10% serum replacement and 250 IU / mL recombinant human interleukin-2). 6 ~5×10 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.

[0184] On the third day, the DNT cells were adjusted to 0.1×10 6 ~1×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0185] On the 5th day, the DNT cells were adjusted to 0.1×10 6 ~1×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0186] On the 7th day, the phenotype and viability of DNT cells were detected, and fresh serum-free medium (containing 100 ng / mL anti-human CD3 monoclonal antibody and 250 IU / mL recombinant human interleukin-2) was used to adjust the DNT cells to 0.1×10 6 ~1×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0187] On day 9, DNT cells were adjusted to 0.1×10 6 ~1×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0188] On the 10th day, the phenotype and viability of DNT cells were detected, and fresh serum-free medium (containing 100 ng / mL anti-human CD3 monoclonal antibody and 250 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.

[0189] 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.

[0190] On day 14, the phenotype and viability of DNT cells were tested. 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 the serum-free medium was reduced to 50 ng / mL. DNT cells were adjusted to 1×10 6 ~3×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0191] On the 15th to 16th day, if necessary, fresh serum-free medium (containing 50 ng / mL anti-human CD3 monoclonal antibody and 250 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.

[0192] On days 17 to 20, DNT cells were harvested as needed.

[0193] 1.4 Harvesting Donor DN T Cells to Prepare DN T Cell Preparations

[0194] On days 17 to 20, 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 to 1 × 10 8 The cells are placed in a liquid nitrogen tank at a concentration of 10 cells / mL. After encapsulation, the cells are cooled and stored in a liquid nitrogen tank. This is the finished product of the DNT cell preparation and can be used clinically after passing quality inspection.

[0195] Example 2: Enrichment of DNT cells from filtration residues and cryopreservation followed by in vitro expansion and culture

[0196] 2.1 Preparation of filter residue

[0197] Same as 1.1 above.

[0198] 2.2 Enrichment of DNT cells from the filtration residue and cryopreservation

[0199] Place the filtered white residue in a high-speed centrifuge, centrifuge and discard the supernatant, leaving a volume of about 100 ml after centrifugation. Add CD4\CD8 remover (RosetteSep TM Human CD4 Depletion Cocktail and RosetteSep TM Human CD8 Depletion Cocktail) was used to remove CD4+\CD8+ cells by centrifugation to obtain enriched DNT cells. The cells were resuspended in CS5 freezing medium and the cell concentration was adjusted to 3-5×10 6 cells / mL and stored frozen in liquid nitrogen.

[0200] 2.3 Thawing of frozen DNT cells

[0201] Depending on the production scale, select one or more donors' frozen DNT cells. Remove the DNT cells from liquid nitrogen from multiple donors frozen in step 2.2 and thaw them in a 37°C water bath. Combine the DNT cells from multiple donors, wash them once with serum-free medium (containing 250 IU / mL of recombinant human interleukin-2), and resuspend them in serum-free medium (containing 10% serum replacement and 250 IU / mL of recombinant human interleukin-2).

[0202] 2.4 In vitro culture of donor DNT cells

[0203] Same as 2.3 above.

[0204] 2.5 Harvesting Donor DNT Cells to Prepare Multi-Donor DNT Cell Preparations

[0205] Same as 1.4 above.

[0206] Example 3: Enrichment of DNT cells from leukocyte residues and multi-donor mixing followed by expansion and culture

[0207] 3.1 Preparation of filter residue

[0208] Same as 1.1 above.

[0209] 3.2 Enrichment of DNT cells from leukocyte residues and multi-donor pooling

[0210] Place the filtered white residue in a high-speed centrifuge, centrifuge and discard the supernatant, leaving a volume of about 100 ml after centrifugation. Add CD4\CD8 remover (RosetteSep TM Human CD4 Depletion Cocktail and RosetteSep TM Human CD8 Depletion Cocktail) was used to remove CD4+\CD8+ cells by centrifugation to obtain enriched DNT cells and resuspend them in freezing buffer to adjust the cell concentration to 3-5×10 6 cells / mL and stored frozen in liquid nitrogen.

[0211] The frozen DNT cells obtained by treating the leukocyte residues from different donors were thawed and mixed cultured.

[0212] 3.3 In vitro culture of donor DNT cells

[0213] According to the production scale requirements, take the mixed cells described in 2.2, coat T25 with anti-human CD3 monoclonal antibody (10 μg / mL) or choose a larger culture flask according to the production scale requirements, and adjust the volume to 1×10 6 ~5×10 6 The cells were placed into the coated culture flask at a concentration of 10 cells / mL and cultured in a 37°C 5% CO2 incubator.

[0214] On the third day, the DNT cells were adjusted to 0.1×10 6 ~1×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0215] On the 5th day, the DNT cells were adjusted to 0.1×10 6 ~1×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0216] On the 7th day, the phenotype and viability of DNT cells were detected, and fresh serum-free medium (containing 100 ng / mL anti-human CD3 monoclonal antibody and 250 IU / mL recombinant human interleukin-2) was used to adjust the DNT cells to 0.1×10 6 ~1×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0217] On day 9, DNT cells were adjusted to 0.1×10 6 ~1×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0218] On the 10th day, the phenotype and viability of DNT cells were detected, and fresh serum-free medium (containing 100 ng / mL anti-human CD3 monoclonal antibody and 250 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 or a reaction tank with a larger volume to continue the culture.

[0219] On day 12, DNT cells were adjusted to 1×10 6 ~3×10 6 The cells were transferred to a culture bag or a reaction vessel with a larger volume to continue the culture.

[0220] On day 14, the phenotype and viability of DNT cells were tested. 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 the serum-free medium was reduced to 50 ng / mL. DNT cells were adjusted to 1×10 6 ~3×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0221] On the 15th to 16th day, if necessary, fresh serum-free medium (containing 50 ng / mL anti-human CD3 monoclonal antibody and 250 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.

[0222] On days 17 to 20, DNT cells were harvested as needed.

[0223] 3.4 Harvesting Donor DNT Cells to Prepare Multi-Donor DNT Cell Preparations

[0224] On days 17 to 20, harvest DNT cells as needed. Collect the cells using a 250 ml conical bottom centrifuge bottle or a large volume culture medium concentrator, centrifuge at 900 × g for 10 minutes, and then wash with solvent. Adjust the concentration of the collected DNT cells to 0.5 to 1 × 10 cells using CS5 freezing medium. 8 The cells are placed in a liquid nitrogen tank at a concentration of 10 cells / mL. After encapsulation, the cells are cooled and stored in a liquid nitrogen tank. This is the finished product of the DNT cell preparation and can be used clinically after passing quality inspection.

[0225] Application Example 1 (Single Donor Fresh Culture)

[0226] According to the method of Example 1, 400 ml of whole blood collected from a single donor was filtered and the leukocyte residue was removed 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 250 IU / mL, serum replacement culture volume ratio of 10%, two-stage reduction of the anti-human CD3 monoclonal antibody dosage (100 ng / mL in the first stage and 50 ng / mL in the second stage), without the use of interleukin-4 and AB serum). The cells were collected on the 14th day to detect cell activity, phenotype, and total number of expanded cells.

[0227] Application Example 2 (Cultivation after rethawing of a single donor)

[0228] According to the method of Example 2, the single donor-derived leukocyte filtration residue was removed to remove CD4 + With CD8 + The obtained frozen DNT cells were thawed and cultured in vitro using the method of the present invention (recombinant human interleukin-2 concentration of 250 IU / mL, serum replacement culture volume ratio of 10%, two-stage reduction of the anti-human CD3 monoclonal antibody dosage (100 ng / mL in the first stage and 50 ng / mL in the second stage), without the use of interleukin-4 and AB serum). The cells were collected on the 14th day, and the cell activity, phenotype, and total number of cell proliferation were detected.

[0229] Application Example 3 (Multiple Donor Reconstitution and Culture)

[0230] 400ml of whole blood collected from multiple donors was filtered and the leukocyte residue was removed to remove CD4 + With CD8 +The obtained DNT cells were frozen separately for future use. The frozen DNT cells were rethawed according to the method of Example 2 and cultured in vitro using the method of the present invention (recombinant human interleukin-2 concentration of 250 IU / mL, serum replacement culture volume ratio of 10%, two-stage reduction of the anti-human CD3 monoclonal antibody dosage (100 ng / mL in the first stage and 50 ng / mL in the second stage), without the use of interleukin-4 and AB serum). The cells were collected on day 14 and the cell viability, phenotype, and total number of expanded cells were tested.

[0231] Comparative Example 1

[0232] The DNT cells were prepared by collecting whole blood from 47 healthy donors according to the existing process (Patent No. ZL201410259944.0) and cultured using the method of the present invention. The specific culture process was the same as that of Application Example 1 above.

[0233] The DNT cells from the aforementioned application examples and comparative examples were sampled on enrichment day 0 and culture day 14 to measure total cell count, cell viability, cell phenotype, and biological efficacy. The DNT cell expansion factor was compared to obtain comparative results for cell-related parameters between the application examples and the comparative examples. The results for the comparative example are the averaged results.

[0234] As can be seen from Figure 3, there is no significant difference in the viability of DNT cells on day 0 and the viability of DNT cells obtained on day 14 of culture between Application Examples 1 and 2 and the Comparative Example; as can be seen from Figure 4, there is no significant difference in the purity of DNT cells obtained on days 0, 7, and 14 of culture between Application Examples 1 and 2 and the Comparative Example; as can be seen from Figure 5, Application Examples 1 and 2 are superior to the Comparative Example in terms of DNT cell expansion multiples, especially Application Example 2 (cultured after reconstitution); as can be seen from Figure 6, there is no significant difference in the killing ability of DNT cells obtained from Application Examples 1 and 2 and the Comparative Example against target cells after 14 days of expansion.

[0235] On the 14th day, 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.

[0236] Table 1 Cell viability, phenotypic purity and amplification times of application examples and comparative examples

[0237] In Table 1, Application Examples 3 and 2 represent DNT cells isolated and obtained using the present invention's method, followed by cryopreservation and subsequent thawing and culture. Application Example 1 represents direct culture of DNT cells obtained using the present invention's process, while Comparative Example 1 represents direct culture of freshly isolated DNT cells. Comparing the DNT cell expansion folds, the DNT cells obtained using the present invention's single-donor bleaching residue in Application Example 1 and the DNT cells obtained using a multi-donor mixture in Application Example 3 both exhibited expansion folds 5834.55 times greater than those in Comparative Example 1, produced using existing processes. However, the differences in cell phenotype and cell viability were negligible (see Figures 7 and 8). Therefore, enriching DNT cells using two different methods and then amplifying them using the present invention's technology can yield a larger DNT cell product.

[0238] The above examples demonstrate that the efficient in vitro expansion process for multiple donor DNT cells provided by the present invention provides flexibility in cell preparation to suit clinical treatment protocols. Building on existing expansion technologies, the production process has been further improved, freeing the scale of DNT cell production from the limitations of blood collection from a single donor and minimizing the impact of individual donor differences on the tumoricidal efficacy of the final product. This results in more stable, efficient, and broad-spectrum DNT cell preparations, providing a more effective clinical treatment product.

[0239] 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 method for preparing universal clinical DN T cells, comprising the steps of: (i) providing a peripheral blood starting sample I from a healthy donor; (ii) filtering sample I through a filter to retain leukocytes, backflushing the filter with a backflushing solution from the "liquid outlet" of the filter, and collecting the backflushing leukocytes from the "liquid inlet" of the filter, thereby obtaining sample II; (iii) removing CD4 + and CD8 + T cells, thereby obtaining sample III; (iv) mixing samples III from multiple donors and culturing them in a culture system containing a culture medium suitable for the growth of DNT cells, thereby obtaining sample IV; wherein, The culture system includes a fixed T cell mitogen; (v) culturing sample IV in a culture system containing a culture medium suitable for the growth of DNT cells, thereby obtaining sample V; wherein the culture medium contains a soluble T cell mitogen at a concentration of 50-150 ng / mL; (vi) culturing sample V 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 25-75 ng / mL, thereby obtaining a desired amount of universal DNT cells, which is sample VI; (vii) collecting sample VI in a solution system containing a solution suitable for DNT cell preservation, wherein the solution is a cell freezing solution; and then storing the sample in a liquid nitrogen tank after programmed cooling, thereby obtaining a universal clinical DNT cell freezing preparation; The culture systems in steps (iv) to (vi) all contain 200-600 IU / mL of recombinant human interleukin 2, and do not contain recombinant human interleukin 4, and do not contain serum or plasma.

2. The method according to claim 1, characterized in that In the step (ii), the backwashing fluid is sterile saline; Furthermore, the backflushing step in step (ii) specifically includes: heat-sealing the two ends of the filter and then removing them, connecting sterile saline from the "liquid outlet" of the filter through a sterile pipe, pulse-pressing the connected sterile saline bag and tapping or kneading the filter, and collecting the white blood cells obtained by backflushing from the "liquid inlet" of the filter.

3. The method according to claim 1, characterized in that The step (iii) comprises: removing CD4 + and CD8 + T cells, thereby obtaining sample IIIa, which was resuspended in freezing solution, stored in liquid nitrogen after programmed cooling, and this was sample IIIb; the frozen sample IIIb derived from one or more donors was thawed at 37°C, washed once with culture medium, and then resuspended in culture medium and mixed, and this was sample III, i.e., DNT derived from donor leukocytes that were frozen and thawed.

4. The method according to claim 1, characterized in that The culture medium in steps (iv) to (vi) further comprises a cytokine selected from the group consisting of IL-7, IL-12, IL-15, or a combination thereof.

5. The method according to claim 1, characterized in that The T cell mitogen is selected from the group consisting of an antibody that binds to CD3, a lectin, a compound that can stimulate the expansion of DNT cells, or a combination thereof.

6. The method according to claim 5, characterized in that The lectin is selected from the group consisting of lectin concanavalin A (ConA), plant lectin (PHA), soybean agglutinin (SBA), or a combination thereof.

7. The method according to claim 5, characterized in that The compound capable of stimulating DNT cell expansion is selected from the following group: IPP, Pamidronate, Zoledronic Acid, or a combination thereof.

8. The method according to claim 1, characterized in that In step (iv), it is divided into sub-steps (iva), (ivb) and (ivc), and the sub-steps (iva), (ivb) and (ivc) are three expansions of the DNT cells in sample III; The culture time of the sub-steps (iva), (ivb) and (ivc) is independently 24-72 hours, preferably 36-60 hours, and more preferably 48 hours.

9. The method according to claim 8, characterized in that In the sub-step (iva), the initial concentration of the DNT cells to be expanded in the culture system is 1×10 6 ~5×10 6 cells / mL.

10. The method according to claim 8, characterized in that In sub-steps (ivb) and (ivc), the initial concentration of the DNT cells to be expanded in the culture system is independently 0.1×10 6 Up to 1×10 6 cells / mL.

11. The method according to claim 1, characterized in that In step (v), the DNT cells are amplified 2-5 times (preferably 3-4 times, more preferably 3 times). In each amplification, the initial concentration of the DNT cells to be amplified is independently 0.1×10 6 Up to 3×10 6 The culture time for each expansion is 24-72 hours, preferably 36-60 hours, and more preferably 48 hours.

12. The method according to claim 1, characterized in that In step (vi), the DNT cells are amplified 2-4 times (preferably 2 times), and in each amplification, the initial concentration of the DNT cells to be amplified is independently 1×10 6 Up to 3×10 6 The culture time for each expansion is 24-72 hours, preferably 36 hours.

13. The method according to claim 1, characterized in that The culture systems in steps (iv) to (vi) all contain serum substitutes, and the volume ratio of serum substitutes in the culture system is 8%-15%, preferably 10%.

14. The method according to claim 1, wherein: The universal clinical DN T cells have the following characteristics: (1) Expression of CD3 + But does not express CD4 - With CD8 - ; (2) will not cause graft-versus-host disease (GvHD); (3) Can be used for allogeneic cell therapy.

15. Use of an effective amount of DNT cells obtained by the method according to claim 1, characterized in that: For preparing a pharmaceutical composition or preparation, the pharmaceutical composition or preparation is used for: (a) prevention and / or treatment of tumors; (b) prevention and / or treatment of infectious diseases; (c) prevention and / or treatment of autoimmune diseases; (d) prevention and / or treatment of allergic reactions; (e) prevention and / or treatment of graft-versus-host disease; (f) Modulation of immune response.

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