In-vitro induction culture method for gamma delta T cells

Abstract

The invention discloses a gamma delta T cell in-vitro induction culture method. According to the method provided by the invention, the gamma delta T (V delta 2 type) cells with the purity of 80% (conservative value) or above can be induced from the peripheral blood of healthy adults within 19 days, 400 (average value) times (nearly 3 times of that of other culture methods) of total cells can be realized in vitro under a serum-free condition, the amplification cost of the gamma delta T cells is greatly saved, the amplification efficiency is improved, and the method is suitable for industrial production. And a foundation is laid for the application of the gamma delta T cell in tumor treatment.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to an in vitro induction and culture method for γδT cells. Background Art

[0002] γδT cells are a conservative group of innate lymphocytes with different structural and functional heterogeneity. They participate in various immune responses during tumor progression. γδT cells perform effective immune surveillance by exerting direct cytotoxicity, strong cytokine production, and indirect anti-tumor immune responses. This role of γδT cells makes them a promising cell therapy method in tumor treatment. The existing in vitro induction culture time of γδT cells is usually 20 days, and the culture process mostly contains FBS (fetal bovine serum), which brings safety risks to the later cell application, and the total cell expansion multiple is usually less than 200 times, which increases the cost of cell therapy and the time patients have to wait for transfusion. Therefore, a culture method for rapid induction and expansion of γδT cells needs to be developed. Summary of the invention

[0003] In order to make up for the deficiencies of the prior art, the present invention provides a method for inducing and culturing γδT cells in vitro.

[0004] To achieve the above purpose, the present invention adopts the following technical solution.

[0005] A first aspect of the present invention provides a composition for inducing and culturing γδT cells, the composition comprising an activation medium, the components of the activation medium comprising a BTN3A1 protein.

[0006] Furthermore, the components of the activation medium also include basal medium, plasma, and interleukin.

[0007] Furthermore, the composition also includes an expansion medium, which includes a basal medium, plasma, interleukin, and antibodies.

[0008] Furthermore, the basal culture medium is selected from GT-T551.

[0009] Furthermore, the plasma is autologous plasma.

[0010] Furthermore, the interleukin is IL-2.

[0011] Furthermore, the antibody is selected from one or more of CD3, CD28 or γδTCR.

[0012] Furthermore, the concentration of the BTN3A1 protein is 1-5 μg / ml.

[0013] Furthermore, the concentration of the BTN3A1 protein is 3 μg / ml.

[0014] Furthermore, the concentration of the autologous plasma is 10%.

[0015] Furthermore, the concentration of IL-2 is 100-500 U / ml.

[0016] Furthermore, the concentration of IL-2 is 500 U / ml.

[0017] Furthermore, the concentration of CD3 is 30-100 ng / ml.

[0018] Furthermore, the concentration of CD3 is 50 ng / ml.

[0019] Furthermore, the concentration of CD28 is 5-10 ng / ml.

[0020] Furthermore, the concentration of CD28 is 10 ng / ml.

[0021] Furthermore, the concentration of the γδTCR is 1-5 μg / ml.

[0022] Furthermore, the concentration of the γδTCR is 3 μg / ml.

[0023] The second aspect of the present invention provides a method for inducing and culturing γδT cells, the method comprising culturing a cell population comprising γδT cells using the composition according to the first aspect of the present invention.

[0024] Further, the method is specifically as follows: 1) activating a cell population including γδT cells using the activation medium described in the first aspect of the present invention; 2) Expanding the cells obtained in 1) using the expansion medium described in the first aspect of the present invention.

[0025] Furthermore, the cell population including γδT cells is PBMC.

[0026] Furthermore, the inoculation density of the PBMC is 1-5×10^6 / ml.

[0027] Furthermore, the inoculation density of the PBMC is 2×10^6 / ml.

[0028] Furthermore, the activation time of step 1) is 1-3 days.

[0029] Furthermore, step 2) also includes a fluid replenishment step.

[0030] Furthermore, rehydration was performed every 2-3 days.

[0031] Furthermore, the density of the γδT cells is 1-3×10^6 / ml.

[0032] Furthermore, the density of the γδT cells is 1×10^6 / ml.

[0033] The third aspect of the present invention provides use of the composition of the first aspect of the present invention or the method of the second aspect of the present invention in inducing and culturing γδT cells.

[0034] The fourth aspect of the present invention provides a γδT cell, which is cultured by the composition of the first aspect of the present invention or the method of the second aspect of the present invention.

[0035] The fifth aspect of the present invention provides a pharmaceutical composition, which comprises the γδT cells described in the fourth aspect of the present invention.

[0036] The sixth aspect of the present invention provides the use of the composition described in the first aspect of the present invention, the method described in the second aspect of the present invention, or the γδT cells described in the fourth aspect of the present invention in the preparation of a pharmaceutical composition for treating a disease, wherein the disease includes cancer, an infectious disease, or an autoimmune disease.

[0037] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0038] Advantages and beneficial effects of the present invention: The method provided in this application can induce γδT (type Vδ2) cells with a purity of more than 80% (conservative value) from the peripheral blood of healthy adults within 19 days, and achieve 400 (average) times the total cells in vitro under serum-free conditions (nearly 3 times that of other culture methods), and greatly save the cost of γδT cell expansion and improve the expansion efficiency, laying the foundation for the application of γδT cells in the treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a schematic diagram of isolating PBMC from peripheral blood; Figure 2 This is the flow cytometric graph of the γδT percentage of donors in batch 1 (the left figure is the γδT percentage in PBMC before induction on Day 0; the right figure is the γδT percentage after induction on Day 19); Figure 3 It is a line graph of the expansion of total cells from batch 1 donors after 19 days of culture; Figure 4 This is a line graph of the expansion of γδT cells from batch 1 donors after 19 days of culture; Figure 5 This is the flow cytometric graph of the γδT percentage of donors in batch 2 (the left picture shows the γδT percentage in PBMC before induction on Day 0; the right picture shows the γδT percentage after induction on Day 19); Figure 6This is the flow cytometric graph of the γδT percentage of donors in batch 3 (the left figure is the γδT percentage in PBMC before induction on Day 0; the right figure is the γδT percentage after induction on Day 19). DETAILED DESCRIPTION

[0040] The following provides definitions of some terms used in this specification. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0041] The present invention provides a composition for inducing and culturing γδT cells. The composition comprises an activation culture medium, and components of the activation culture medium comprise BTN3A1 protein.

[0042] In a specific embodiment, the BTN3A1 protein (CD277 protein) is a recombinant human BTN3A1 protein.

[0043] In some embodiments, γδT cells are a subpopulation of T cells that provide a link between innate and adaptive immune responses. These cells undergo V-(D)-J segment rearrangement to produce antigen-specific γδT cell receptors (γδTCRs) and γδT cells, and can be directly activated by recognizing antigens through γδTCRs or other non-TCR proteins, thereby activating γδT cell effector functions independently or together. γδT cells represent a small part of the overall T cell population in mammals, approximately 1%-5% of T cells in peripheral blood and lymphoid organs, and they appear to be primarily present in epithelial cell-rich compartments, like the skin, liver, digestive tract, respiratory tract, and reproductive tract. Unlike αβTCRs that recognize antigens bound to major histocompatibility complex molecules (MHC), γδTCRs can directly recognize bacterial antigens, viral antigens, stress antigens expressed by diseased cells, and tumor antigens in the form of intact proteins or non-peptide compounds.

[0044] The components of the activation medium also include basal medium, plasma, and interleukin.

[0045] The composition also includes an expansion medium, which includes a basal medium, plasma, interleukin, antibody, and TCR.

[0046] In some embodiments, the basic culture medium includes but is not limited to GT-T551 medium, Optivitro medium, X-VIVO15 medium, cellgrowSCGM medium, and AIM-V medium.

[0047] In a specific embodiment, the basal culture medium is selected from GT-T551 medium.

[0048] In some embodiments, the plasma can be autologous plasma, or plasma from any other source.

[0049] In a specific embodiment, said plasma is selected from autologous plasma.

[0050] In some embodiments, the interleukins include but are not limited to IL-2 (interleukin 2), IL-7 (interleukin 7), IL-12 (interleukin 12), IL-15 (interleukin 15), IL-18 (interleukin 18), and IL-21 (interleukin 21).

[0051] In a specific embodiment, the interleukin is selected from IL-2 (interleukin 2).

[0052] In some embodiments, the antibodies include but are not limited to CD3, CD28, CD46, CD30, CD70, CD80, CD86, CD122, CD16, CD161, and γδTCR.

[0053] In a specific embodiment, the antibody is selected from CD3, CD28 and γδ TCR.

[0054] In some embodiments, the present application does not exclude the use of other culture media, or the addition of some conventional nutritional supplements, such as salts, antibiotics, vitamins and amino acids, etc., in the culture media of the present application. Amino acid supplements may include any amino acids, including glycine, alanine, valine, leucine, isoleucine, arginine, lysine, aspartic acid, cysteine, cysteine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, asparagine, glutamine, histidine and serine. Antibiotics such as gentamicin, penicillin, streptomycin, ampicillin, kanamycin, etc.

[0055] The medium of the present application may also contain a buffer, and its type is not specifically limited and can be a type well known to those skilled in the art. A buffer is a solution that resists pH changes through the action of its acid-base conjugate component. It can be based on, for example, the desired pH of the buffer (as well as microbial growth and metabolic characteristics, microbial culture culture system, pH control and culture medium used). In some embodiments, the buffer has a pH in the range of about 2 to about 9, or about 3 to about 8, or about 4 to about 7, or about 5 to about 7. Non-limiting examples of buffers that will control pH within this range include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate and ammonium buffers, and combinations of these. In some embodiments, the buffer includes 3-(N-morpholino) propanesulfonic acid (MOPS) free acid, 3-(N-morpholino) propanesulfonic acid (MOPS) Na, hydroxyethylpiperazineethanesulfonic acid (HEPES) and sodium bicarbonate.

[0056] The present invention provides a method for inducing and culturing γδT cells, the method specifically comprising: 1) activating a cell population including γδT cells using the above activation medium; 2) The cells obtained in 1) are expanded using the above-mentioned expansion medium.

[0057] In some embodiments, the cell population comprising γδ T cells comprises a blood sample, an umbilical cord blood sample, a tumor, a stem cell precursor, a tissue, a lymph, or an epithelial site of a subject in direct contact with the external environment.

[0058] In a preferred embodiment, the cell population comprising γδ T cells is selected from a blood sample.

[0059] In a more preferred embodiment, the blood sample is selected from peripheral blood.

[0060] In a specific embodiment, the cell population comprising γδ T cells is PBMCs isolated from peripheral blood.

[0061] The present invention provides use of the above composition, the above method or the above γδT cells in preparing a pharmaceutical composition for treating a disease, wherein the disease includes cancer, an infectious disease or an autoimmune disease.

[0062] In some embodiments, non-limiting examples of cancer include adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, neuroblastoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors (such as cerebellar astrocytoma, cerebral astrocytoma / malignant glioma), ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, optic pathway and hypothalamic glioma, breast cancer, bronchial adenoma, Burkitt's lymphoma, cancer of unknown primary site, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumor, gallbladder cancer, stomach cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liposarcoma, liver cancer, lung cancer (such as non-small cell lung cancer), cellular and small cell lung cancer), lymphoma, leukemia, malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma, melanoma, mesothelioma, metastatic squamous cell carcinoma of unknown primary, oral cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myeloid leukemia, nasal and sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, epithelial ovarian cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic islet cell cancer, sinus and nasal cancer, parathyroid cancer, penile cancer, oropharyngeal cancer, pheochromocytoma, pineal gland Astrocytoma, pineal germ cell tumor, pituitary adenoma, pleuropulmonary blastoma, plasmacytoma, primary central nervous system lymphoma, prostate cancer, colorectal cancer, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, skin cancer merkel cell, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, T-cell lymphoma, pharyngeal cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (pregnancy), cancer of unknown primary, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms tumor.

[0063] In some embodiments, the infectious disease may be caused, for example, by pathogenic bacteria or by a virus. Non-limiting examples of pathogenic bacteria can be found in: a) Bordetella, such as species Bordetella pertussis; b) Borrelia, such as species Borrelia burgdorferi; c) Brucelia, such as species Brucella abortus, Brucella canis, Brucela meliterisis and / or Brucella suis; d) Campylobacter, such as species Campylobacter jejuni; e) Chlamydia and Chlamydophila, such as Chlamydia pneumonia, Chlamydia trachomatis and / or Chlamydophila psittaci. psittaci); f) Clostridium, such as Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani; g) Corynebacterium, such as Corynebacterium diphtheria; h) Enterococcus, such as Enterococcus faecalis and / or Enterococcus faecium; i) Escherichia, such as Escherichia coli; j) Francisella, such as Francisella tularensis; k) Haemophilus, such as Haemophilus influenzae. influenza) species; l) Helicobacter, such as Helicobacter pylori; m) Legionella, such as Legionella pneumophila;n) Leptospira, such as Leptospira interrogans; o) Listeria, such as Listeria monocytogenes; p) Mycobacterium, such as Mycobacterium leprae, Mycobacterium tuberculosis and / or Mycobacterium ulcerans; q) Mycoplasma, such as Mycoplasma pneumonia; r) Neisseria, such as Neisseria gonorrhoeae and / or Neisseria meningitidia; s) Pseudomonas, such as Pseudomonas aeruginosa. aeruginosa species; t) Rickettsia, such as Rickettsia rickettsii species; u) Salmonella, such as Salmonella typhi and / or Salmonella typhimurium species; v) Shigella, such as Shigella sonnei species; w) Staphylococcus, such as Staphylococcus aureus, Staphylococcus epidermidis and / or Staphylococcus saprophyticus species; x) Streptococcus, such as Streptococcus agalactiae, Streptococcus pneumoniae and / or Streptococcus pyogenes. pneumonia) and / or Streptococcus pyogenes species; y) Treponema, such as Treponema pallidum species; z) Vibrio, such as Vibrio cholera; and / or aa) Yersinia, such as Yersinia pestis species. ;

[0064] In some embodiments, the infectious disease may be caused by a virus. Non-limiting examples of viruses can be found in the following virus families and are illustrated by exemplary species: a) Adenoviridae, such as adenovirus species; b) Herpesviridae, such as herpes simplex 1, herpes simplex 2, varicella-zoster virus, Epstein-barr virus, human cytomegalovirus, human herpesvirus 8 species; c) Papillomaviridae, such as human papillomavirus species; d) Polyomaviridae, such as BK virus, JC virus species; e) Poxviridae, such as leukemia virus, ... e) such as smallpox species; f) Hepadnaviridae such as hepatitis B virus species; g) Parvoviridae such as human bocavirus, parvovirus B19 species; h) Astroviridae such as human astrovirus species; i) Caliciviridae such as Norwalk virus species; j) Flaviviridae such as hepatitis C virus (HCV), yellow fever virus, dengue virus, West Nile virus species; k) Togaviridae such as rubella virus species; l) Hepaciviridae viridae, such as Hepatitis E virus species; m) Retroviridae, such as Human Immunodeficiency Virus (HIV) species; n) Orthomyxoviridae, such as Influenza virus species; o) Arenaviridae, such as Guanarito, Junin, Lassa, Machupo and / or Sabiá virus species; p) Bunyaviridae, such as Crimean-Congo virus species; Hemorrhagic fever virus species; q) Filoviridae, such as Ebola virus and / or Marburg virus species; Paramyxoviridae, such as measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, Hendra virus and / or Nipah virus species; r) Rhabdoviridae, such as rabies virus species; s) Reoviridae, such as rotavirus, orbivirus, Colorado tick fever virus and / or Banna virus species. In some examples, the virus is not assigned to a virus family, such as hepatitis D.

[0065] In some embodiments, examples of autoimmune diseases or disorders include: rheumatoid arthritis, rheumatic fever, multiple sclerosis, experimental autoimmune encephalomyelitis, psoriasis, uveitis, diabetes, systemic lupus erythematosus (SLE), lupus nephritis, eczema, scleroderma, polymyositis / scleroderma, polymyositis / dermatomyositis, ulcerative colitis, severe combined immunodeficiency (SCID), DiGeorge syndrome, ataxia-telangiectasia, seasonal allergies, perennial allergies, food allergies, Allergic reactions, mastocytosis, allergic rhinitis, atopic dermatitis, Parkinson's disease, Alzheimer's disease, hypersplenism, leukocyte adhesion defect, X-linked lymphoproliferative disease, X-linked agammaglobulinemia, selective immunoglobulin A deficiency, hyper-IgM syndrome, HIV, autoimmune lymphoproliferative syndrome, Wiskott-Aldrich syndrome, chronic granulomatous disease, common variant immune CVID, hyperimmunoglobulin E syndrome, Hashimoto's thyroiditis, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, polyglandular syndrome, bullous pemphigoid, Henoch-Schonlein purpura, poststreptococcal nephritis, erythema nodosum, erythema multiforme, gA nephropathy, Takayasu's arteritis, Addison's disease, sarcoidosis, ulcerative colitis inflammatory inflammatory disease, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangiitis obliterans, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, chronic active hepatitis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes tuberculosis, giant cell arteritis / polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, fibrosing alveolitis, and cancer.

[0066] The pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0067] In some embodiments, pharmaceutically acceptable adjuvants include but are not limited to diluents, solubilizing agents, emulsifiers, preservatives, preservatives and / or adjuvants. The adjuvant is preferably nontoxic or substantially nontoxic to the recipient at the dosage and concentration adopted. This type of adjuvant includes but is not limited to saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the pharmaceutical composition may contain a material for improving, maintaining or retaining, for example, pH, permeability, viscosity, clarity, color, isotonicity, smell, sterility, stability, dissolution or release rate, absorption or penetration of the composition. Determine the best pharmaceutical composition such as a biological preparation depending on the expected route of administration, delivery mode and required dosage.

[0068] Pharmaceutical compositions for in vivo administration are usually provided in the form of sterile preparations. Sterilization is achieved by filtering through a sterile filter membrane. When the composition is freeze-dried, this method can be used for sterilization before or after freeze-drying, reconstitution, dilution. The pharmaceutical composition of the present application can be selected for parenteral delivery. The composition for parenteral administration can be stored in a freeze-dried form or in a solution. For example, it is prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Parenteral compositions are usually placed in a container with a sterile access hole, such as an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle. Alternatively, the composition can be selected for inhalation or delivery through the digestive tract (such as oral). The preparation of pharmaceutical compositions is within the technical scope of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing antibodies in sustained or controlled release delivery formulations. Technologies for preparing a variety of other sustained or controlled delivery methods (such as liposome carriers, bio-erodible microparticles or porous beads and accumulated injections) are also known to those skilled in the art.

[0069] Once formulated, the pharmaceutical composition is stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid, crystal or lyophilized powder. The formulation can be stored in a ready-to-use form or in a form that is reconstituted (e.g., lyophilized) prior to administration.

[0070] In some embodiments, prevention and similar words, such as prevention, etc., indicate methods for preventing, inhibiting or reducing the likelihood of occurrence or recurrence of a condition. As used herein, prevention and similar words also include reducing the intensity, effects, symptoms and / or burden of a disease or condition before onset or recurrence.

[0071] In some embodiments, treatment is directed to a subject, using the treatment methods described herein to achieve at least one positive treatment effect (e.g., a decrease in the number of cancer cells, a decrease in tumor volume, a decrease in the rate of cancer cell infiltration into peripheral organs, or a decrease in the rate of tumor metastasis or tumor growth). The treatment method that effectively treats a patient may vary according to a variety of factors (e.g., the patient's disease state, age, weight, and the ability of the therapy to stimulate an anti-cancer response in the subject).

[0072] Treatment, as used herein, includes any beneficial or desired effects associated with treatment. Treatment does not necessarily indicate complete eradication or cure of a disease or condition, or its associated symptoms.

[0073] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0074] Example Example 1

[0075] 1. Experimental Materials and Methods Table 1 Experimental materials

[0076] Experimental group experimental method: Day 0: Isolation of PBMC from peripheral blood: 1) Collect 15 ml of fresh peripheral blood using a sodium heparin anticoagulant tube. Add 15 ml of PBS to the 15 ml peripheral blood and mix well. Slowly add the mixture to the upper layer of 15 ml Ficoll (polysucrose-diatrizoate) lymphocyte separation solution. Set the centrifuge to 1 / 1, 1200 g for 20 min.

[0077] 2) After centrifugation, carefully collect PBMCs ( Figure 1 ) in a 50ml centrifuge tube, add PBS to 45ml, set the centrifuge to 7 / 7, centrifuge at 500g for 10min, discard the supernatant, loosen the cell pellet, resuspend it with 45ml PBS again, set the centrifuge to 7 / 7, centrifuge at 500g for 10min, discard the supernatant, resuspend it with basal culture medium, mix and count, adjust the cell density to 2×10^6 / ml, resuspend it with complete culture medium (GT-T551+10% autologous plasma+500U / ml IL-2 +3μg / mlRecombinant Human CD277 / BTN3A1 Protein), and culture it in a T25 flask.

[0078] Day 3: Cell observation, small clones can be seen at the bottom. After the culture medium (GT-T551+10% autologous plasma+500U / ml IL-2, 50ng / ml CD3, 10ng / ml CD28, 3μg / ml anti human γδTCR) returns to room temperature, 5ml of fluid is replenished.

[0079] Day 5: Cell observation showed that the small clones at the bottom had obviously grown larger. After the culture medium (GT-T551 + 10% autologous plasma + 500U / ml IL-2, 50ng / ml CD3, 10ng / ml CD28, 3μg / ml anti human γδTCR) returned to room temperature, 10ml of fluid was added.

[0080] Day7: Cell observation: a large number of cell clones were visible at the bottom. After the culture medium (GT-T551+10% autologous plasma+500U / ml IL-2, 50ng / ml CD3, 10ng / ml CD28, 3μg / ml anti human γδTCR) returned to room temperature, 20ml of fluid was replenished.

[0081] Day9: Cell observation. Many clones can be seen at the bottom. The cells are mixed and counted. After the culture medium (GT-T551+10% autologous plasma+500U / ml IL-2, 50ng / ml CD3, 10ng / ml CD28, 3μg / ml anti human γδTCR) returns to room temperature, the fluid is replenished and the cell density is adjusted to 1×10^6 / ml.

[0082] Day 11: Cell observation. Many clones can be seen at the bottom. The cells are mixed and counted. After the culture medium (GT-T551+10% autologous plasma+500U / ml IL-2, 50ng / ml CD3, 10ng / ml CD28, 3μg / ml anti human γδTCR) returns to room temperature, the cell density is adjusted to 1×10^6 / ml.

[0083] Day 13: Cell observation. Many clones can be seen at the bottom. The cells are mixed and counted. After the culture medium (GT-T551+500U / mlIL-2, 50ng / ml CD3, 10ng / ml CD28, 3μg / ml anti human γδTCR) returns to room temperature, the cell density is adjusted to 1×10^6 / ml.

[0084] Day 16: Cell observation. Many clones can be seen at the bottom. The cells are mixed and counted. After the culture medium (GT-T551+500U / mlIL-2, 50ng / ml CD3, 10ng / ml CD28, 3μg / ml anti human γδTCR) returns to room temperature, the cell density is adjusted to 1×10^6 / ml.

[0085] Day 19: Cell observation. Many clones can be seen at the bottom. The cells are mixed and counted, and the cells are taken for flow cytometry analysis.

[0086] Control group experimental method: The expansion medium of the control group was X-VIVO15+500U / ml IL-2+5% serum replacement; the activation medium was X-VIVO15+5% autologous plasma+500U / ml IL-2, 50ng / ml CD3, 10ng / ml CD28. The rest of the experimental process was the same as the experimental group.

[0087] 2. Experimental results After 19 days of culture, the purity of γδT cells from batch 1 donors was 92.52% ( Figure 2 ), the total cell expansion was more than 850 times ( Figure 3 , Table 2 ), γδT cells expanded more than 14,000-fold ( Figure 4 , Table 3).

[0088] Table 2 Total cell expansion times

[0089] Table 3 γδT cell expansion fold

[0090] Example 2 1. Experimental Materials and Methods Same as Example 1.

[0091] 2. Experimental results After 19 days of culture, the purity of γδT cells from batch 2 donors was 82.35% ( Figure 5 ), total cells expanded 276-fold (Table 4), and γδT cells expanded 5361-fold (Table 5).

[0092] Table 4 Total cell expansion times

[0093] Table 5 γδT cell expansion fold

[0094] Example 3 1. Experimental Materials and Methods Same as Example 1.

[0095] 2. Experimental results After 19 days of culture, the purity of γδT cells from batch 3 donors was 86.98% ( Figure 6 ), total cells expanded 230-fold (Table 6), and γδT cells expanded 3448-fold (Table 7).

[0096] Table 6 Total cell expansion times

[0097] Table 7 γδT cell expansion times

[0098] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A composition for inducing and culturing γδT cells, characterized in that: The composition comprises an activation medium, and the components of the activation medium comprise BTN3A1 protein; Preferably, the components of the activation medium further include basal medium, plasma, and interleukin; Preferably, the composition further comprises an expansion medium, wherein the expansion medium comprises a basal medium, plasma, interleukin, and antibody; Preferably, the basal medium is selected from GT-T551; Preferably, the plasma is autologous plasma; Preferably, the interleukin is IL-2; Preferably, the antibody is selected from one or more of CD3, CD28 or γδTCR.

2. The composition according to claim 1, characterized in that The concentration of the BTN3A1 protein is 1-5 μg / ml; Preferably, the concentration of the autologous plasma is 10%; Preferably, the concentration of IL-2 is 100-500 U / ml; Preferably, the concentration of CD3 is 30-100 ng / ml; Preferably, the concentration of CD28 is 5-10 ng / ml; Preferably, the concentration of the γδTCR is 1-5 μg / ml.

3. A method for inducing and culturing γδT cells, characterized in that: The method comprises culturing a cell population comprising γδ T cells using the composition of claim 1 or 2.

4. The method according to claim 3, characterized in that: The method is specifically as follows: 1) activating a cell population including γδT cells using the activation medium according to claim 1; 2) Proliferating the cells obtained in 1) using the proliferation medium according to claim 2.

5. The method according to claim 4, characterized in that The cell population including γδT cells is PBMC; Preferably, the seeding density of the PBMC is 2×10^6 / ml.

6. The method according to claim 4, characterized in that The activation time for step 1) is 1-3 days; Preferably, step 2) further comprises the step of replenishing fluid; Preferably, rehydration is performed every 2-3 days; Preferably, the density of the γδT cells is 1×10^6 / ml.

7. Use of the composition according to any one of claims 1 to 2 or the method according to any one of claims 3 to 6 in inducing and culturing γδT cells.

8. A γδT cell, characterized in that: The γδT cells are obtained by culturing the composition according to any one of claims 1 to 2 or the method according to any one of claims 3 to 6.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the γδT cells according to claim 8.

10. Use of the composition according to any one of claims 1 to 2, the method according to any one of claims 3 to 6, or the γδT cells according to claim 8 in preparing a pharmaceutical composition for treating a disease, wherein the disease comprises cancer, an infectious disease, or an autoimmune disease; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

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