Universal clinical DNT cell as well as preparation method and application thereof

Abstract

The invention provides a technology for preparing universal DNT cells. In the invention, the prepared raw materials are derived from'white filtering residues' after white filtering treatment is carried out to avoid adverse blood transfusion reaction, and the DNT cells extracted from the white filtering residues can be a mixed culture of one or more healthy donors. The universal DNT cell obtained by the method has the characteristics of high yield, high purity, high in-vitro tumor killing activity, suitability for large-scale production and the like. The initial raw material can be fresh cells or re-thawed cells after cryopreservation, the raw material source bottleneck of large-scale preparation of cell drugs is fundamentally solved, the value of leukocytes filtered out in the production process of blood products is re-excavated, and great medical value and commercial application prospects are achieved.

Description

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 and effective tumor treatment model and a new immune anti-cancer treatment method. It uses biotechnology and biological agents to culture and amplify immune cells collected from patients or healthy donors in vitro, and then infuse them back into the patient's body to stimulate and enhance the patient's own immune function, thereby achieving the purpose 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 using immune cell therapy.

[0004] Currently, immune cell therapy mostly involves collecting autologous immune cells, culturing and expanding them in vitro, and then infusing them. If multiple infusions are required, immune cells from human peripheral blood must be collected multiple times for culturing and expansion to have enough cells for infusion. Due to the severity of the disease, most patients who need treatment often cannot provide a sufficient number of immune cells for culture. In addition, due to the patient's physical condition and various medications, it is very difficult or even impossible to culture and expand their immune cells in vitro.

[0005] In recent years, there has been a treatment plan to collect immune cells from healthy donors, culture them in vitro, and then infuse them. Generally, 200-400 ml of whole blood can be collected from a healthy donor at a time, so the cell raw materials for each production batch are limited to 200-400 ml of whole blood and the production scale cannot be expanded.

[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. Summary of the invention

[0007] 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 a preparation method and application thereof.

[0008] The first aspect of the present invention provides a method for preparing universal clinical DN T cells, comprising the steps of:

[0009] (i) providing a peripheral blood starting sample I from a healthy donor;

[0010] (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;

[0011] (iii) removing CD4 + and CD8 + T cells, thereby obtaining sample III;

[0012] (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;

[0013] (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;

[0014] (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;

[0015] (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;

[0016] 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.

[0017] In another preferred embodiment, the amount of the sample I is 10-500 ml, preferably 40-400 ml.

[0018] In another preferred embodiment, the sample III is a cell solution that is thawed after being frozen.

[0019] In another preferred embodiment, in the step (ii), the backwashing fluid is sterile saline.

[0020] 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.

[0021] In another preferred embodiment, 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.

[0022] In another preferred embodiment, in the culture system of steps (iv) to (vi), the culture conditions are 37° C. and 5% CO 2 .

[0023] In another preferred embodiment, the culture medium in steps (iv) to (vi) further includes cytokines selected from the following group: IL-7, IL-12, IL-15, or a combination thereof.

[0024] In another preferred embodiment, the T cell mitogen is selected from the following group: antibodies binding to CD3, lectins, compounds capable of stimulating the expansion of DNT cells, or a combination thereof.

[0025] 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.

[0026] In another preferred embodiment, the compound capable of stimulating DNT cell expansion is selected from the following group: IPP, Pamidronate, Zoledronic Acid, or a combination thereof.

[0027] In another preferred embodiment, the T cell mitogen is an antibody that binds to CD3.

[0028] In another preferred embodiment, the culture system in step (iv) is located in a culture bottle, preferably a T25 culture bottle.

[0029] In another preferred embodiment, the fixed T cell mitogen refers to a T cell mitogen coated on a culture flask or a microplate.

[0030] In another preferred embodiment, in step (v), the initial concentration of the DNT cells to be amplified in the culture system is 0.1×10 6 Up to 1×10 6 cells / mL.

[0031] In another preferred embodiment, the culturing time of step (iv) is 24-72 hours, preferably 36-60 hours, and more preferably 48 hours.

[0032] 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.

[0033] In another preferred embodiment, in the sub-step (iva), the initial concentration of the DNT cells to be amplified in the culture system is 1×10 6 ~5×10 6 cells / mL.

[0034] 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 Up to 1×10 6 cells / mL.

[0035] 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.

[0036] In another preferred embodiment, the culturing time of step (iv) is 96-192 hours, preferably 120-168 hours, and more preferably 144 hours.

[0037] In another preferred embodiment, in step (v), the DNT cells are amplified 2-5 times (preferably 3-4 times, more preferably 3 times), and 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.

[0038] In another preferred embodiment, step (v) further includes detecting the phenotype and viability of DNT cells.

[0039] In another preferred embodiment, the culturing time of step (v) is 72-168 hours, preferably 96-144 hours, and more preferably 120 hours.

[0040] In another preferred embodiment, 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 6Up to 3×10 6 The culture time for each expansion is 24-72 hours, preferably 36 hours.

[0041] In another preferred embodiment, step (vi) further includes detecting the phenotype and viability of DNT cells.

[0042] In another preferred embodiment, the culturing time of step (vi) is 48-96 hours, preferably 72 hours.

[0043] 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%.

[0044] In another preferred embodiment, in step (vii), the steps are included:

[0045] (viia) collecting DNT cells in sample VI by centrifugation;

[0046] (viib) washing the DNT cells with sodium chloride injection solution containing 2.5% human albumin;

[0047] (viic) DNT cells were adjusted to 0.5×10 8 -1×10 8 The concentration of cells / mL.

[0048] In another preferred embodiment, the universal clinical DN T cells have the following characteristics:

[0049] (1) Expression of CD3 + But it does not express CD4- and CD8 - ;

[0050] (2) will not cause graft-versus-host disease (GvHD);

[0051] (3) Can be used for allogeneic cell therapy.

[0052] The second aspect of the present invention provides 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:

[0053] (a) prevention and / or treatment of tumors;

[0054] (b) prevention and / or treatment of infectious diseases;

[0055] (c) prevention and / or treatment of autoimmune diseases;

[0056] (d) prevention and / or treatment of allergic reactions;

[0057] (e) prevention and / or treatment of graft-versus-host disease;

[0058] (f) Modulation of immune response.

[0059] In another preferred embodiment, the tumor is a tumor allogeneic to the DNT cells.

[0060] In another preferred embodiment, the tumor is selected from the following group: a blood tumor, a solid tumor, or a combination thereof.

[0061] 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 lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome (MDS), or a combination thereof.

[0062] In another preferred embodiment, the solid tumor is selected from the following group: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, melanoma, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon 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.

[0063] 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, exophthalmic goiter, habitual gray hair abortion, 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.

[0064] 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.

[0065] 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 specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 The process flow chart of preparing DNT cell preparation from the filtration residue is shown.

[0067] Figure 2 The flowchart of the operation of filter white backwashing to obtain filter white residue is shown.

[0068] Figure 3 A comparison chart of the changes in DNT cell viability between the process of the present invention and the existing process is shown. The figure mainly compares the DNT cell viability obtained by the process of the present invention and the DNT cell viability obtained by the existing process on day 0 and after 14 days of in vitro culture. The results show that there is no statistically significant difference between the DNT cell viability on day 0 and the DNT cell viability obtained by the two processes on day 14 of culture.

[0069] Figure 4 The figure shows the comparison of the purity changes of DNT cells cultured in vitro by the process of the present invention and the existing process. The figure shows that the DNT cells obtained by the process of the present invention were expanded in vitro, and the purity of the DNT cells was detected on days 0, 7, and 14. There was no statistically significant difference in the purity of the DNT cells obtained by the process of the present invention and the existing process.

[0070] Figure 5 The figure shows a comparison of the expansion multiples of DNT cells cultured in vitro by the process of the present invention and the existing process. The figure shows that there is no significant difference between the fresh culture group and the existing process in the in vitro expansion of DNT cells obtained by the process of the present invention, and the expansion ability of DNT cells in the frozen, thawed and then cultured group is significantly better than that of the existing process.

[0071] Figure 6 The figure shows the comparison of the killing ability of DNT cells cultured in vitro by the process of the present invention and the existing process on target cells. 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 on target cells after 14 days of amplification.

[0072] Figure 7 The comparison chart of key indicators of single donor and multiple donors in the process of the present invention is shown. Under the process of the present invention, the viability, cell purity and biological efficacy of multiple donor mixed culture and single donor culture are consistent.

[0073] Figure 8The figure shows the comparison of the expansion multiples of DNT cells in different groups (single donor and multiple donors) of the existing process and the process of the present invention. Due to individual differences between healthy donors, the expansion of each donor is different. Mixed culture of multiple donors can balance the expansion of each DNT cell to a certain extent, thereby better ensuring the yield of the preparation. DETAILED DESCRIPTION

[0074] After extensive and in-depth research and a large number of process optimization experiments, the inventors have developed for the first time an allogeneic universal DNT cell prepared from peripheral blood of one or more donors for multiple clinical transfusions and a preparation method thereof. Specifically, the present invention optimizes the processing process of the starting sample, and can simultaneously extract leukocytes from peripheral blood of one or more donors. The extraction process does not require centrifugation, and a large amount of peripheral blood can be processed in a short time. At the same time, the culture formula and culture process of DNT cells are optimized, so that DNT cells mixed from one or more donors can be expanded and cultured in vitro.

[0075] Compared with the existing method of obtaining mononuclear cells (PBMC) using peripheral blood as the starting material, the method of the present invention uses the leukoplakia residue as the starting material, which is more convenient and easier to obtain. In addition, the DNT cell preparation method of the present invention can be suitable for mixed culture of DNT cells from multiple donors, and the production batch can be enlarged according to demand to obtain a large number of high-purity universal DNT cells with broad-spectrum tumor-killing ability.

[0076] 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 backwashing to enrich and efficiently expand DNT cells in vitro. 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 regulations 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 from 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 produce commercial universal cell drugs. At the same time, the DNT cells obtained by the method of the present invention have a broader spectrum of tumor killing efficacy, which is beneficial to improving clinical efficacy.

[0077] On this basis, the present invention has been completed.

[0078] the term

[0079] 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.

[0080] 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.

[0081] As used herein, the terms "administering", "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.

[0082] DNT cell preparation method of the present invention

[0083] In the present invention, a method for preparing universal clinical DN T cells is provided, wherein the method comprises the steps of:

[0084] (i) providing a peripheral blood starting sample I obtained from a donor;

[0085] (ii) filtering the whole blood with a filter to retain the leukocytes, and backflushing the filter to obtain a leukocyte suspension, thereby obtaining sample II;

[0086] (iii) removing CD4 + and CD8 + T cells, thereby obtaining sample III; specifically, comprising the following steps:

[0087] Remove CD4 from one or more donor samples II + and CD8 + T cells, thereby obtaining sample IIIa, resuspending with freezing solution, cooling by program control and storing in liquid nitrogen, which is sample IIIb, rethawing the frozen sample IIIb from one or more donors at 37°C, washing once with culture medium, resuspending with culture medium and mixing, which is sample III, i.e. DNT derived from donor leukocytes that were frozen and rethawed;

[0088] (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 a fixed T cell mitogen;

[0089] (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;

[0090] (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;

[0091] (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;

[0092] 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.

[0093] In another preferred embodiment, in step (i), the donor is a healthy donor.

[0094] In another preferred embodiment, the amount of the sample I is 10-500 ml, preferably 40-400 ml.

[0095] In another preferred embodiment, the sample II is a cell solution that is thawed after being frozen.

[0096] In another preferred embodiment, in the culture system of steps (iv) to (vi), the culture conditions are 37° C. and 5% CO 2 .

[0097] In another preferred embodiment, the culture medium in steps (iv) to (vi) further comprises cytokines selected from the following group: IL-7, IL-12, IL-15, or a combination thereof.

[0098] In another preferred embodiment, the T cell mitogen is selected from the following group: antibodies binding to CD3, lectins, compounds capable of stimulating the expansion of DNT cells, or a combination thereof.

[0099] 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.

[0100] In another preferred embodiment, the compound capable of stimulating DNT cell expansion is selected from the following group: IPP, Pamidronate, Zoledronic Acid, or a combination thereof.

[0101] In another preferred embodiment, the T cell mitogen is an antibody that binds to CD3.

[0102] In another preferred embodiment, the culture system in step (iv) is located in a culture bottle, preferably a T25 culture bottle.

[0103] In another preferred embodiment, the fixed T cell mitogen refers to a T cell mitogen coated on a culture flask or a microplate.

[0104] In another preferred embodiment, in step (v), the initial concentration of the DNT cells to be amplified in the culture system is 1×10 6 Up to 4×10 6 cells / mL.

[0105] In another preferred embodiment, the culturing time of step (iv) is 24-72 hours, preferably 36-60 hours, and more preferably 48 hours.

[0106] 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.

[0107] In another preferred embodiment, in the sub-step (iva), the initial concentration of the DNT cells to be amplified in the culture system is 1×10 6 ~5×10 6 cells / mL.

[0108] 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 0.5×10 6 Up to 1×10 6 cells / mL.

[0109] 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.

[0110] In another preferred embodiment, the culturing time of step (iv) is 96-192 hours, preferably 120-168 hours, and more preferably 144 hours.

[0111] In another preferred embodiment, in step (v), the DNT cells are amplified 2-5 times (preferably 3-4 times, more preferably 3 times), and in each amplification, the initial concentration of the DNT cells to be amplified is independently 1×10 6Up to 3×10 6 The culture time for each expansion is 24-72 hours, preferably 36-60 hours, and more preferably 48 hours.

[0112] In another preferred embodiment, step (v) further includes detecting the phenotype and viability of DNT cells.

[0113] In another preferred embodiment, the culturing time of step (v) is 72-168 hours, preferably 96-144 hours, and more preferably 120 hours.

[0114] In another preferred embodiment, 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.

[0115] In another preferred embodiment, step (vi) further includes detecting the phenotype and viability of DNT cells.

[0116] In another preferred embodiment, the culturing time of step (vi) is 48-96 hours, preferably 72 hours.

[0117] In another preferred embodiment, in step (vii), the steps are included:

[0118] (viia) collecting DNT cells in sample VI by centrifugation;

[0119] (viib) washing the DNT cells with sodium chloride injection solution containing 2.5% human albumin;

[0120] (viic) Adjust the number of DNT cells to 0.5-1×10 using CS5 cryopreservation medium. 8 The concentration of cells / mL.

[0121] 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.

[0122] 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 refrigerated blood, bone marrow, lymphoid tissue, thymus, liver, spleen, lymph node tissue, tumor tissue, fetal tissue, and fractions or enriched parts thereof, and can also be derived from induced pluripotent stem cells (iPSC).

[0123] In a preferred embodiment, the starting sample is blood, preferably human blood, more preferably human peripheral blood.

[0124] Before culturing the starting sample or its fractions, sample III was substantially depleted of CD4 + and CD8 + T cells. "Substantially" means that the majority of these cells are removed but does not exclude that a small portion of these cells still remain.

[0125] These cell types in the sample can be removed 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.

[0126] In another preferred embodiment, magnetic beads that specifically bind to CD4 and CD8 are added to sample II.

[0127] 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.

[0128] 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 cryopreserved, and DNT cells from one or more donors are rethawed on a production scale and used in the preparation method of the present invention alone or in combination.

[0129] 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 T cells are removed, they can be cultured directly, or they can be frozen in liquid nitrogen and then thawed and cultured as needed.

[0130] 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 amplification method of the present invention, or can be frozen in liquid nitrogen and then thawed and then cultured; after sample IIIa (enriched DNT cells) is collected by centrifugation, it is resuspended in freezing solution, and after program-controlled cooling, it is placed in liquid nitrogen for storage, which is sample IIIb (enriched frozen DNT cells); according to the production scale, one or more frozen samples IIIb from donors are selected, thawed at 37°C, mixed, washed once with culture medium, and then resuspended with culture medium, which is sample III (enriched, frozen, and then thawed multi-donor DNT cells).

[0131] Cultured in culture medium that has essentially eliminated CD4 + and CD8 + A sample of cells, the culture medium comprising fixed T cell mitogen and an agent capable of stimulating the growth of DNT cells.

[0132] 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 Zoledronate. Preferably, the T cell mitogen is an antibody to CD3, such as OKT3.

[0133] The T cell mitogen can be fixed using techniques known in the art. Preferably, the T cell mitogen is coated on 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 fixed on a microplate.

[0134] 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.

[0135] It is particularly noteworthy that in the present invention, the culture medium used for expansion culture does not contain IL-4 and serum.

[0136] 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.

[0137] The culture medium may 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.

[0138] In one embodiment, the cells can be co-cultured with DC cells or stimulated with antigens and cytokines when cultured with agents capable of stimulating the growth of DNT cells and fixed T cell mitogens. In the preparation of anti-tumor DNT cells, inactivated tumor cells or peptides or neoantigens from tumor-specific or tumor-associated antigens can be used.

[0139] 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.

[0140] The purity of the DNT cells prepared by the present method can be confirmed using techniques known in the art, such as flow cytometry.

[0141] Use of the DNT cells prepared by the present invention

[0142] The present invention also includes the use of universal double negative T cells (DNT) obtained by the method of the present invention in any and all applications. The multi-donor allogeneic T cells obtained by the method of the present invention can be used for the treatment of various tumors, can be prepared on a large scale, have stable and controllable quality, and can be used in a timely manner for any applicable patient.

[0143] In an embodiment of the present invention, the multi-donor double negative T cells amplified 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 drug for treating tumors.

[0144] 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, such as treating cancer.

[0145] The term "animal" as used herein includes all members of the animal kingdom, including humans. In a preferred embodiment, the animal is a human.

[0146] 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 disease, allergy, infection, etc.), reduction in the severity 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 prolonged survival as compared to expected survival if not receiving treatment.

[0147] 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 into tumor tissues.

[0148] Treatable cancers include tumors that are not vascularized or substantially not yet 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 leukemias or lymphoid malignancies, benign and malignant tumors, and malignancies, such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and childhood tumors / cancers are also included.

[0149] 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, myelo-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.

[0150] Solid tumors are abnormal masses of tissue that usually do not 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, ovarian cancer.

[0151] The present invention also includes other therapeutic uses of the multiple donor DN T cells of the present invention, such as the treatment of infectious diseases and, for example, for regulating immune responses in the treatment of autoimmune diseases, allergies, transplant rejection and graft-versus-host disease. In this case, the method for preparing multiple donor DN T cells may include adding a suitable infectious agent, allergen cell or tissue as an antigen.

[0152] 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 to treat infectious diseases. 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 in the preparation of a medicament for treating infectious diseases.

[0153] In a further embodiment, the present invention provides a method for regulating 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 regulating 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 in the preparation of a medicament for regulating an immune response.

[0154] 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, exophthalmic goiter, habitual gray hair abortion, 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.

[0155] In another embodiment, multiple donor 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.

[0156] In further embodiments, multi-donor DN T cells can be used to treat allergies. In allergies, the immune system mounts an attack against normally harmless antigens or allergens. 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.

[0157] The multi-donor DNT cells prepared by the method of the present invention can be formulated into a pharmaceutical composition that is administered 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 in an appropriate manner, preferably by injection, such as intravenous, subcutaneous, intramuscular, etc.

[0158] 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 as 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). On this basis, compositions include, but are not limited to, solutions of substances combined with one or more pharmaceutically acceptable carriers or diluents, containing physiological solutions in a buffer solution having a suitable pH and isotonicity.

[0159] 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.

[0160] 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.

[0161] 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 such factors as the patient's condition, the type and severity of the patient's disease, although appropriate dosage can be determined by clinical trials.

[0162] When an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount" is indicated, the exact 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 Up to 10 9 The dosage is preferably 10 cells / kg body weight. 6 Up to 10 8 The multi-donor DNT cell composition can also be administered multiple times at these doses. The cells can be administered using well-known infusion techniques in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a specific patient can be obtained by monitoring the patient's disease condition, so treatment adjustments are easily determined by those skilled in the medical field.

[0163] 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 node, 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.

[0164] In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic doses are administered to patients in combination with any number of related treatment forms (e.g., before, at the same time, or after), including but not limited to treatment with the following agents: 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 can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to patients in combination with bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (e.g., before, at the same time, or after). For example, in one embodiment, the subject can undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. 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 prior to or after surgery.

[0165] 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. The dosage ratio for human administration can be implemented according to practices accepted in the art. Typically, 1×10 9 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.

[0166] The main advantages of the present invention include:

[0167] 1) Improve the formulation and process, expand the source of raw materials for cell products, and develop a method for preparing cell products on a large scale for clinical use using DNT cells from multiple donors.

[0168] 2) Improvements in formulation and process allow for mixed culture of DNT cells from multiple donors.

[0169] 3) The formulation and process have been improved, and no human serum or fetal bovine serum (FBS) is required.

[0170] 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.

[0171] 5) Improvements in formulation and process can achieve one-time collection / freezing, multiple thawing and culture, and provide multiple reinfusions or use by multiple patients.

[0172] 6) Formula and process improvement, cell expansion can reach 1x10 9 cells / mL of whole blood.

[0173] 7) Formula and process improvement to ensure the purity of the final product DNT cells (CD3 + CD4 - CD8-)≥85%.

[0174] 8) Improve the formulation and process to ensure that the in vitro efficient tumor killing activity (biological efficacy) of cells is ≥ 50%.

[0175] 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 where specific conditions are not specified are usually 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 under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0176] Example 1: Direct expansion and culture of DNT cells enriched with filtration residues

[0177] 1.1 Preparation of filter residue

[0178] Collect 200-400ml peripheral blood from healthy donors into a blood bag containing anticoagulant, hang the blood bag high, and use gravity to make the whole blood pass through the filter (Spacell RZ2000) so that the white blood cells are trapped in the filter. Heat-seal the two ends of the filter and remove it, connect sterile saline from the "liquid outlet" of the filter through a sterile pipe, pulse-press the connected saline bag and pat or knead the filter, and obtain the leukocyte residue from the "liquid inlet" of the filter.

[0179] 1.2 Enrichment of DNT cells from filtration residues

[0180] 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, centrifugation to remove CD4 + \CD8 + cells to obtain enriched DNT cells.

[0181] 1.3 In vitro culture of donor DNT cells

[0182] 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 culture medium was adjusted to 1×10 cells / mL with serum-free medium (containing 10% serum replacement and 250 IU / mL recombinant human interleukin-2). 6 ~5×10 6 The cells were placed in the coated T25 culture flask at a concentration of 10 cells / mL and cultured in a 37°C 5% CO2 incubator.

[0183] On the third day, DNT cells were adjusted to 0.1×10 6 ~1×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0184] On day 5, 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 7th day, the phenotype and viability of DNT cells were detected, and 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 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.

[0187] On the 10th day, the phenotype and viability of DNT cells were detected, and DNT cells were adjusted to 1×10 with fresh serum-free medium (containing 100 ng / mL anti-human CD3 monoclonal antibody and 250 IU / mL recombinant human interleukin-2). 6 ~3×10 6 The cells were transferred to a culture bag at a concentration of 10 cells / mL and continued to be cultured.

[0188] 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.

[0189] On the 14th day, 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.

[0190] 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 concentration of DNT cells to 1×10 6 ~3×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0191] On days 17 to 20, DNT cells were harvested as needed.

[0192] 1.4 Harvesting donor DNT cells to prepare DNT cell preparations

[0193] On the 17th to 20th day, 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 concentration of cells / mL is 100 cells / mL. After encapsulation, the cells are cooled by a program and stored in a liquid nitrogen tank. This is the finished product of DNT cell preparation, which can be used clinically after passing the quality inspection.

[0194] Example 2: Cryopreservation and thawing of DNT cells enriched with filtration residues and then in vitro expansion and culture

[0195] 2.1 Preparation of filter residue

[0196] Same as 1.1 above.

[0197] 2.2 Enrichment of DNT cells from the filtrate residue and cryopreservation

[0198] 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 TMHumanCD8 Depletion Cocktail) was used to remove CD4+\CD8+ cells by centrifugation to obtain enriched DNT cells. The cells were resuspended in CS5 cryopreservation buffer and the cell concentration was adjusted to 3-5×10 6 cells / mL and stored in liquid nitrogen.

[0199] 2.3 Thawing of frozen DNT cells

[0200] Select one or more frozen DNT cells from one or more donors according to the production scale, take out the frozen DNT cells from multiple donors in step 2.2 from liquid nitrogen, thaw them in a constant temperature water bath at 37°C, mix the DNT cells from multiple donors, wash them once with serum-free medium (containing 250 IU / mL recombinant human interleukin 2), and then resuspend them with serum-free medium (containing 10% serum replacement and 250 IU / mL recombinant human interleukin 2).

[0201] 2.4 In vitro culture of donor DNT cells

[0202] Same as 2.3 above.

[0203] 2.5 Harvesting of donor DNT cells to prepare multi-donor DNT cell preparations

[0204] Same as 1.4 above.

[0205] Example 3: Filter residue enriched DNT cells and mixed with multiple donors for expansion and culture

[0206] 3.1 Preparation of filter residue

[0207] Same as 1.1 above.

[0208] 3.2 Filter residue enrichment of DNT cells and multi-donor mixing

[0209] 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 HumanCD8 Depletion Cocktail) was used to remove CD4+\CD8+ cells by centrifugation to obtain enriched DNT cells, which were resuspended in cryopreservation buffer and adjusted to a cell concentration of 3-5×10 6 cells / mL and stored in liquid nitrogen.

[0210] The frozen DNT cells obtained by treating the leukocyte residues from different donors were thawed and mixed for culture.

[0211] 3.3 In vitro culture of donor DNT cells

[0212] 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 bottle according to the production scale requirements, and adjust the culture medium (containing 10% serum replacement and 250 IU / mL recombinant human interleukin 2) to 1×10 6 ~5×10 6 The cells were placed in the coated culture flask at a concentration of 10 cells / mL and cultured in a 37°C 5% CO2 incubator.

[0213] On the third day, DNT cells were adjusted to 0.1×10 6 ~1×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0214] On day 5, 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 7th day, the phenotype and viability of DNT cells were detected, and 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 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.

[0217] On the 10th day, the phenotype and viability of DNT cells were detected, and DNT cells were adjusted to 1×10 with fresh serum-free medium (containing 100 ng / mL anti-human CD3 monoclonal antibody and 250 IU / mL recombinant human interleukin-2). 6 ~3×10 6 The cells were transferred to a culture bag or a reaction tank with a larger volume to continue the culture.

[0218] On day 12, DNT cells were adjusted to 1×106 ~3×10 6 The cells can be transferred to a culture bag or a reaction tank with a larger volume to continue the culture.

[0219] On the 14th day, 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.

[0220] 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 concentration of DNT cells to 1×10 6 ~3×10 6 The cells were cultured at a concentration of 10 cells / mL.

[0221] On days 17 to 20, DNT cells were harvested as needed.

[0222] 3.4 Harvesting of donor DNT cells to prepare multi-donor DNT cell preparations

[0223] On the 17th to 20th day, DNT cells can be harvested as needed. Collect the cells using a 250 ml conical bottom centrifuge bottle or a large volume culture medium concentration device, 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 8 The concentration of cells / mL is 100 cells / mL. After encapsulation, the cells are cooled by a program and stored in a liquid nitrogen tank. This is the finished product of DNT cell preparation, which can be used clinically after passing the quality inspection.

[0224] Application Example 1 (Fresh Culture from a Single Donor)

[0225] 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 anti-human CD3 monoclonal antibody dosage (100 ng / mL in the first stage, 50 ng / mL in the second stage), no interleukin 4 and no AB serum), and the cells were collected on the 14th day to detect cell activity, phenotype and total number of cell expansion.

[0226] Application Example 2 (Cultivation after reconstitution of a single donor)

[0227] According to the method of Example 2, the single donor-derived leukocyte 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 anti-human CD3 monoclonal antibody dosage (100 ng / mL in the first stage, 50 ng / mL in the second stage), no interleukin 4 and no AB serum), and the cells were collected on the 14th day to detect cell activity, phenotype, and total number of cell proliferation.

[0228] Application Example 3 (Multiple Donors Reconstituted and Cultivated)

[0229] 400 ml of whole blood collected from multiple donors was filtered and the residue was removed to remove CD4 + With CD8 + The obtained DNT cells were frozen for standby use. According to the method of Example 2, the frozen DNT cells were rethawed 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 anti-human CD3 monoclonal antibody dosage (100 ng / mL in the first stage, 50 ng / mL in the second stage), no interleukin 4 and no AB serum were used), and the cells were collected on the 14th day to detect cell activity, phenotype, and total number of cell proliferation.

[0230] Comparative Example 1

[0231] The DNT cells prepared by collecting whole blood from 47 healthy donors according to the existing process (patent number ZL201410259944.0) in the company were summarized and analyzed, and cultured using the method of the present invention. The specific culture process is the same as the above-mentioned application example 1.

[0232] The DNT cells of the aforementioned application examples and comparative examples were sampled on enrichment day 0 and culture day 14 to detect the total number of cells, cell viability, cell phenotype, and biological efficacy, and the DNT cell expansion multiples were compared to obtain the comparative results of the cell-related items of the application examples and comparative examples. Among them, the various results of the comparative example are the average results after aggregation.

[0233] Depend on Figure 3 It can be seen that there is no significant difference between the DNT cell viability on day 0 and the DNT cell viability obtained from the 14th day of culture in Application Examples 1 and 2 and the Comparative Example; Figure 4 It can be seen that there is no significant difference in the purity of DNT cells obtained by culture on days 0, 7, and 14 between Application Examples 1 and 2 and the Comparative Example; Figure 5It can be seen that Application Examples 1 and 2 are superior to the comparative example in terms of DNT cell expansion multiples, especially Application Example 2 (culture after reconstitution); Figure 6 It can be seen that there is no significant difference in the killing ability of the DNT cells obtained in Application Examples 1 and 2 and the comparative example on target cells after 14 days of expansion.

[0234] On the 14th day, DNT cells were collected, and the cell viability, phenotypic purity and DNT expansion multiples of the application example and the control example were compared. The results are listed in Table 1.

[0235] Table 1 Cell viability, phenotypic purity and amplification times of application examples and comparative examples

[0236]

[0237] In Table 1, Application Example 3 and Application Example 2 are DNT cells isolated by the method of the present invention and then thawed and cultured, Application Example 1 is direct culture of DNT cells obtained by the process of the present invention, and Comparative Example 1 is direct culture of freshly isolated DNT cells. Comparing the expansion multiples of DNT cells, the expansion multiples of DNT cells obtained by the method of the present invention using the single donor bleaching residue in Application Example 1 and the DNT cells obtained by mixing multiple donors in Application Example 3 are both 5834.55 times higher than that of Comparative Example 1 produced by the existing process, while the cell phenotype and cell viability are not much different (see Figure 7 and Figure 8 ). Therefore, more DNT cell products can be obtained by using two different methods to enrich DNT cells and the amplification technology of the present invention.

[0238] The above examples prove that the multi-donor DNT cell efficient in vitro expansion process provided by the present invention provides flexibility in cell preparation suitable for clinical treatment programs. On the basis of the existing invention expansion technology, the production process has been further improved, so that the production scale of DNT cells is no longer limited by the blood collection volume of a single donor, and the influence of individual differences of donors on the tumor killing efficacy of the final product can be avoided. The DNT cell preparation can be more stable, efficient, and broad-spectrum, providing more effective clinical treatment products.

[0239] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

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 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.

7. 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.

8. 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.

9. 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%.

10. 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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