A method for inducing and culturing gamma delta T cells in vitro

Abstract

The application discloses a method for inducing and culturing gamma delta T cells in vitro. The method provided by the application can induce gamma delta T (Vdelta2 type) cells with a purity of more than 80% (a conservative value) from peripheral blood of healthy adults in 19 days, realizes 400 (an average value) times (nearly 3 times of other culture methods) of total cell expansion in vitro under a serum-free condition, greatly saves the expansion cost of the gamma delta T cells and improves the expansion efficiency, and lays a foundation for the application of the gamma delta T cells in tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for in vitro induction and culture of γδT cells. Background Technology

[0002] γδT cells are a conserved population of innate lymphocytes with diverse structural and functional heterogeneity. They participate in various immune responses during tumor progression, performing effective immune surveillance through direct cytotoxicity, strong cytokine production, and indirect anti-tumor immune responses. This role makes γδT cells a promising cell therapy option for cancer treatment. Current in vitro induction and culture methods for γδT cells typically involve 20 days, often containing FBS (fetal bovine serum), posing safety risks for later cell application. Furthermore, the total cell expansion is usually less than 200-fold, increasing the cost of cell therapy and the waiting time for patient infusion. Therefore, a rapid induced expansion method for γδT cell culture needs to be developed. Summary of the Invention

[0003] To overcome the shortcomings of existing technologies, this invention provides a method for in vitro induction and culture of γδT cells.

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

[0005] A first aspect of the present invention provides a composition for inducing the culture of γδT cells, the composition comprising an activation medium, wherein a component of the activation medium comprises BTN3A1 protein.

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

[0007] Furthermore, the composition also includes an amplification culture medium, which includes basal culture medium, plasma, interleukins, 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] A second aspect of the present invention provides a method for inducing the culture of γδT cells, the method comprising culturing a cell population comprising γδT cells using the composition described in the first aspect of the present invention.

[0024] Furthermore, the method specifically includes:

[0025] 1) Activate a cell population including γδT cells using the activation culture medium described in the first aspect of the present invention;

[0026] 2) Amplify the cells obtained in 1) using the amplification culture medium described in the first aspect of the present invention.

[0027] Furthermore, the cell population including γδT cells is PBMC.

[0028] Furthermore, the inoculation density of the PBMCs is 1-5×10^6 / ml.

[0029] Furthermore, the inoculation density of the PBMCs is 2×10^6 / ml.

[0030] Furthermore, the activation time for step 1) is 1-3 days.

[0031] Furthermore, step 2) also includes a fluid replenishment step.

[0032] Furthermore, rehydration should be performed every 2-3 days.

[0033] Furthermore, the density of the γδT cells is 1-3×10^6 / ml.

[0034] Furthermore, the density of the γδT cells is 1×10^6 / ml.

[0035] The third aspect of the present invention provides the use of the composition described in the first aspect of the present invention or the method described in the second aspect of the present invention in the induced culture of γδT cells.

[0036] A fourth aspect of the present invention provides a γδT cell, said γδT cell being cultured from the composition described in the first aspect of the present invention or the method described in the second aspect of the present invention.

[0037] A fifth aspect of the present invention provides a pharmaceutical composition comprising the γδT cells described in the fourth aspect of the present invention.

[0038] 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 diseases, including cancer, infectious diseases, or autoimmune diseases.

[0039] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0040] Advantages and beneficial effects of the present invention:

[0041] The method provided in this application can induce γδT (Vδ2 type) cells with a purity of over 80% (conservative value) from peripheral blood of healthy adults within 19 days, and achieve a total cell count of 400 times (average value) in vitro under serum-free conditions (nearly 3 times that of other culture methods), which greatly saves the expansion cost of γδT cells and improves the expansion efficiency, laying the foundation for the application of γδT cells in the treatment of tumors. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of PBMCs isolated from peripheral blood;

[0043] Figure 2 This is a flow cytometry plot of the γδT proportion in donor batch 1 (the left plot shows the γδT proportion in PBMCs before induction on Day 0; the right plot shows the γδT proportion after induction on day 19).

[0044] Figure 3 This is a line graph showing the amplification of total cells from donor Batch 1 after 19 days of culture;

[0045] Figure 4 This is a line graph showing the expansion of γδT cells from donor Batch 1 after 19 days of culture;

[0046] Figure 5 This is a flow cytometry plot of the γδT proportion in donor 2 of batch 2 (the left plot shows the γδT proportion in PBMCs before induction on Day 0; the right plot shows the γδT proportion after induction on day 19).

[0047] Figure 6 This is a flow cytometry plot of the γδT proportion in donor 3 of batch (the left plot is the γδT proportion in PBMC before induction on Day 0; the right plot is the γδT proportion after induction on Day 19). Detailed Implementation

[0048] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, 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 pertains.

[0049] The present invention provides a composition for inducing the culture of γδT cells, the composition comprising an activation medium, wherein the components of the activation medium include BTN3A1 protein.

[0050] In a specific implementation, the BTN3A1 protein (CD277 protein) is recombinant human BTN3A1 protein.

[0051] In some implementations, γδT cells are a subset 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 acting independently or in combination to activate γδT cell effector functions. γδT cells represent a small fraction 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 located in epithelial cell-rich compartments such as the skin, liver, digestive tract, respiratory tract, and reproductive tract. Unlike αβTCRs, which recognize antigens that bind to the major histocompatibility complex (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.

[0052] The components of the activation medium also include basal medium, plasma, and interleukin.

[0053] The composition further includes an amplification culture medium, which includes basal culture medium, plasma, interleukins, antibodies, and TCRs.

[0054] In some embodiments, the basal culture medium includes, but is not limited to, GT-T551 medium, Optivitro medium, X-VIVO15 medium, cellgrowSCGM medium, and AIM-V medium.

[0055] In a specific implementation, the basal culture medium is selected from GT-T551 culture medium.

[0056] In some embodiments, the plasma may be autologous plasma or plasma from any other source.

[0057] In a specific implementation, the plasma is selected from autologous plasma.

[0058] 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).

[0059] In a specific implementation, the interleukin is selected from IL-2 (interleukin 2).

[0060] In some embodiments, the antibodies include, but are not limited to, CD3, CD28, CD46, CD30, CD70, CD80, CD86, CD122, CD16, CD161, and γδTCR.

[0061] In a specific implementation, the antibody is selected from CD3, CD28, and γδTCR.

[0062] In some embodiments, this application does not exclude the use in combination with other culture media, or the addition of conventional nutritional supplements such as salts, antibiotics, vitamins, and amino acids to the culture media of this application. Amino acid supplements may include any amino acid, including glycine, alanine, valine, leucine, isoleucine, arginine, lysine, aspartic acid, cysteine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, asparagine, glutamine, histidine, and serine. Antibiotics include gentamicin, penicillin, streptomycin, ampicillin, kanamycin, etc.

[0063] The culture medium of this application may also contain a buffer, and its type is not specifically limited, but may be of 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 components. The desired pH of the buffer can be determined, for example, by the microbial growth and metabolic characteristics, the microbial culture system, pH control, and the 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 control the pH within this range include MES, MOPS, MOPSO, Tris, HEPES, phosphates, acetates, citrates, succinates, and ammonium buffers, as well as combinations thereof. In some embodiments, the buffer comprises 3-(N-morpholino)propanesulfonic acid (MOPS) free acid, 3-(N-morpholino)propanesulfonic acid (MOPS)Na, hydroxyethylpiperazine ethanesulfonic acid (HEPES), and sodium bicarbonate.

[0064] This invention provides a method for inducing and culturing γδT cells, the method specifically comprising:

[0065] 1) Activate a cell population including γδT cells using the above-mentioned activation medium;

[0066] 2) Amplify the cells obtained in 1) using the amplification medium described above.

[0067] In some embodiments, the cell population including γδT cells includes blood samples, umbilical cord blood samples, tumors, stem cell precursors, tissues, lymph nodes, or epithelial sites of subjects in direct contact with the external environment.

[0068] In a preferred embodiment, the cell population including γδT cells is selected from a blood sample.

[0069] In a more preferred embodiment, the blood sample is selected from peripheral blood.

[0070] In a specific implementation, the cell population including γδT cells is PBMC isolated from peripheral blood.

[0071] The present invention provides the use of the above-described composition, method, or γδT cells in the preparation of pharmaceutical compositions for treating diseases, including cancer, infectious diseases, or autoimmune diseases.

[0072] In some implementations, non-limiting examples of cancer include: adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal 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 lymphoma, cancer of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancer, chronic... Lymphoblastic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, fibroblastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, liposarcoma, liver cancer, lung cancer (such as non-small cell lung cancer). Small cell lung cancer, lymphoma, leukemia, malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma, melanoma, mesothelioma, metastatic squamous cell carcinoma of unknown primary origin in the neck, oral cancer, multiple endocrine gland tumor syndrome, myelodysplastic syndrome, myeloid leukemia, nasal cavity and sinus cancer, nasopharyngeal carcinoma, 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 tumors, pancreatic cancer, pancreatic cancer islet cells, sinus and nasal cavity cancer, parathyroid cancer, penile cancer, oropharyngeal cancer, pheochromocytoma, pineal gland Astrocytoma, pineal germ cell tumor, pituitary adenoma, pleural pulmonary blastoma, plasmacytoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, Merkel cell carcinoma of the skin, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, T-cell lymphoma, pharyngeal cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (pregnancy), cancer of unknown primary origin, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor.

[0073] In some implementations, infectious diseases may be caused, for example, by pathogenic bacteria or viruses. Non-limiting examples of pathogenic bacteria include: a) the genus *Bordetella*, such as *Bordetella pertussis*; b) the genus *Borrelia*, such as *Borrelia burgdorferi*; c) the genus *Brucelia*, such as *Brucella abortus*, *Brucella canis*, *Brucella meliteris*, and / or *Brucella suis*; d) the genus *Campylobacter*, such as *Campylobacter jejuni*; e) the genera *Chlamydia* and *Chlamydophila*, such as *Chlamydia pneumoniae*, *Chlamydia trachomatis*, and / or *Chlamydophila psittaci*. f) Clostridium species, such as *Clostridium botulinum*, *Clostridium difficile*, *Clostridium perfringens*, and *Clostridium tetani*; g) Corynebacterium species, such as *Corynebacterium diphtheria*; h) Enterococcus species, such as *Enterococcus faecalis* and / or *Enterococcus faecium*; i) Escherichia species, such as *Escherichia coli*; j) Francisella species, such as *Francisella tularensis*; k) Haemophilus species, such as *Haemophilus influenzae*. (l) *Helicobacter*, such as *Helicobacter pylori*; (m) *Legionella*, such as *Legionella pneumophila*.n) *Leptospira* species, such as *Leptospira interrogans*; o) *Listeria* species, such as *Listeria monocytogenes*; p) *Mycobacterium* species, such as *Mycobacterium leprae*, *Mycobacterium tuberculosis*, and / or *Mycobacterium ulcerans*; q) *Mycoplasma* species, such as *Mycoplasma pneumoniae*; r) *Neisseria* species, such as *Neisseriagonorrhoeae* and / or *Neisseria meningitidia*; s) *Pseudomonas* species, such as *Pseudomonas aeruginosa*. t) genus *Aeruginosa*, including species such as *Rickettsia rickettsii*; u) genus *Salmonella*, including species such as *Salmonella typhi* and / or *Salmonella typhimurium*; v) genus *Shigella*, including species such as *Shigella sonnei*; w) genus *Staphylococcus*, including species such as *Staphylococcus aureus*, *Staphylococcus epidermidis*, and / or *Staphylococcus saprophyticus*; x) genus *Streptococcus*, including species such as *Streptococcus agalactiae* and *Streptococcus pneumoniae*. y) *Streptococcus pyogenes* species, including *Treponema* species such as *Treponemapallidum*; z) *Vibrio* species, including *Vibrio cholera*; and / or aa) *Yersinia* species, including *Yersinia pestis*.

[0074] In some implementations, infectious diseases may be caused by viruses. Non-limiting examples of viruses can be found in the following viral families, and are illustrated by exemplary species: a) Adenoviridae, such as adenovirus species; b) Herpesviridae, such as herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, Epstein-Barr virus, human cytomegalovirus, and human herpesvirus type 8; c) Papillomaviridae, such as human papillomavirus species; d) Polyomaviridae, such as BK virus and JC virus species; e) Poxviridae... e) such as smallpox; f) Hepatoviridae, such as hepatitis B virus; g) Parvoviridae, such as human bocavirus and parvovirus B19; h) Astroviridae, such as human astrovirus; i) Caliciviridae, such as Norwalk virus; j) Flaviviridae, such as hepatitis C virus (HCV), yellow fever virus, dengue virus, and West Nile virus; k) Togaviridae, such as rubella virus; l) Hepatitis Viridae (Hepe (m) Viral family, such as hepatitis E virus species; (n) Retroviridae family, such as human immunodeficiency virus (HIV) species; (o) Orthomyxoviridaw family, such as influenza virus species; (p) Arenaviridae family, such as Guanarito virus, Junin virus, Lassa virus, Machupo virus, and / or Sabiá virus species; (v) Bunyaviridae family, such as Crimean-Congo virus species. Hemorrhagic fever viruses; q) Filoviridae, such as Ebola virus and / or Marburg virus; Paramyxoviridae, such as measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, Hendra virus and / or Nipah virus; r) Rhabdoviridae, such as rabies virus; s) Reoviridae, such as rotavirus, circovirus, Colorado tick fever virus and / or Banna virus. In some instances, viruses are not classified into a virology family, such as hepatitis D.

[0075] In some implementations, examples of autoimmune diseases or conditions 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, etc. Allergic reactions, mast cell hyperplasia, allergic rhinitis, atopic dermatitis, Parkinson's disease, Alzheimer's disease, hypersplenism, leukocyte adhesion defect, X-linked lymphoproliferative disorders, X-linked agammaglobulinemia, selective immunoglobulin A deficiency, hyper-IgM syndrome, HIV, autoimmune lymphoproliferative syndrome, Wiskott-Aldrich syndrome, chronic granulomatous disease, common variant immunodeficiency syndromes. CVID (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, allergic purpura, post-streptococcal nephritis, erythema nodosum, erythema multiforme, gA nephropathy, Takayasu arteritis, Addison's disease, sarcoidosis, ulcerative colitis Inflammation, polyarteritis nodosa, ankylosing spondylitis, Goodpasture 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 dorsalis, giant cell arteritis / polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, fibrotic alveolitis, and cancer.

[0076] The pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0077] In some embodiments, pharmaceutically acceptable excipients include, but are not limited to, diluents, solubilizers, emulsifiers, preservatives, and / or adjuvants. The excipients are preferably non-toxic or substantially non-toxic to the recipient at the dosage and concentration used. Such excipients include, but are not limited to, saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. In some embodiments, the pharmaceutical composition may contain substances for improving, maintaining, or retaining, for example, the composition's pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeation. The optimal pharmaceutical composition, such as a biologic, is determined based on the intended route of administration, delivery method, and required dosage.

[0078] Pharmaceutical compositions intended for internal administration are typically provided in sterile formulations. Sterilization is achieved by filtration through a sterile filter membrane. This method can be used to sterilize the composition before or after lyophilization, reconstitution, or dilution. The pharmaceutical compositions of this application may be used for parenteral delivery. Compositions intended for parenteral delivery may be lyophilized or stored in solution. They may be prepared, for example, using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. Parenteral compositions are typically placed in containers with sterile access openings, such as intravenous solution bags or vials with stoppers that can be punctured by a hypodermic needle. Alternatively, the composition may be intended for inhalation or delivery via the digestive tract (e.g., orally). The preparation of pharmaceutical compositions is within the 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. Techniques for formulating a variety of other sustained or controlled delivery methods, such as liposome carriers, bioeasily perishable microparticles or porous beads, and accumulation injection, are also known to those skilled in the art.

[0079] Once formulated, the pharmaceutical composition is stored in sterile vials in the form of a solution, suspension, gel, emulsion, solid, crystal, or lyophilized powder. The formulation may be stored in a ready-to-use form or in a reconstituted form prior to administration (e.g., lyophilized).

[0080] In some implementations, prevention and similar terms, such as prevent, indicate methods for preventing, suppressing, or reducing the likelihood of the occurrence or recurrence of a condition. As used in this application, prevention and similar terms also include reducing the intensity, effect, symptoms, and / or burden of a disease or condition before its onset or recurrence.

[0081] In some implementations, treatment is directed at the subject using the methods described in this application to achieve at least one positive therapeutic effect (e.g., a reduction in the number of cancer cells, a reduction in tumor volume, a decrease in the rate of cancer cell infiltration into surrounding organs, or a decrease in the rate of tumor metastasis or tumor growth). Effective treatment methods for patients can vary depending on various factors, such as the patient's disease state, age, weight, and the ability of the therapy to elicit an anti-cancer response in the subject.

[0082] The treatment described in this application includes any beneficial or desired effects associated with the treatment. Treatment does not necessarily indicate the complete eradication or cure of the disease or condition, or its associated symptoms.

[0083] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0084] Example

[0085] Example 1

[0086] 1. Experimental Materials and Methods

[0087] Table 1 Experimental Materials

[0088]

[0089] Experimental method for the experimental group:

[0090] Day 0: Isolation of PBMCs from peripheral blood:

[0091] 1) Collect 15ml of fresh peripheral blood using a heparin sodium anticoagulant tube. Add 15ml of PBS to the 15ml of peripheral blood and mix well. Then slowly add the mixture to the upper layer of 15ml of Ficoll (sucrose-diatrizoate meglumine) lymphocyte separation medium. Centrifuge at 1200g for 20min, with the centrifuge setting at 1 step up and 1 step down.

[0092] 2) After centrifugation, carefully collect PBMCs ( Figure 1Add PBS to a 50ml centrifuge tube to a final volume of 45ml. Centrifuge at 500g for 10min with the centrifuge set to 7°C (upper 7, lower 7). Discard the supernatant, loosen the cell pellet, and resuspend the cells in 45ml of PBS. Centrifuge at 500g for 10min with the centrifuge set to 7°C (upper 7, lower 7). Discard the supernatant, resuspend the cells in basal medium, mix well, count the cells, and adjust the cell density to 2×10^6 / ml. Resuspend the cells in complete medium (GT-T551 + 10% autologous plasma + 500U / ml IL-2 + 3μg / ml Recombinant Human CD277 / BTN3A1 Protein) and culture in a T25 flask.

[0093] Day 3: Cell observation showed small clonal clusters 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, 5ml of additional culture medium was added.

[0094] Day 5: Cell observation showed that the small clonal cluster at the bottom had become significantly 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.

[0095] Day 7: Cell observation showed a large number of cell clones 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 additional culture medium was added.

[0096] Day 9: Cell observation. Many clone clusters were visible at the bottom. Cells were 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) returned to room temperature, the medium was replenished and the cell density was adjusted to 1×10^6 / ml.

[0097] Day 11: Cell observation. Many clone clusters were visible at the bottom. Cells were 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) returned to room temperature, the cell density was adjusted to 1×10^6 / ml.

[0098] Day 13: Cell observation. Many clone clusters were visible at the bottom. Cells were mixed and counted. After the culture medium (GT-T551 + 500U / ml IL-2, 50ng / ml CD3, 10ng / ml CD28, 3μg / ml anti-human γδTCR) returned to room temperature, the cell density was adjusted to 1×10^6 / ml.

[0099] Day 16: Cell observation. Many clone clusters were visible at the bottom. Cells were mixed and counted. After the culture medium (GT-T551 + 500U / ml IL-2, 50ng / ml CD3, 10ng / ml CD28, 3μg / ml anti-human γδTCR) returned to room temperature, the cell density was adjusted to 1×10^6 / ml.

[0100] Day 19: Cell observation. Many clone clusters were visible at the bottom. Cells were mixed and counted. Cells were taken for flow cytometry analysis.

[0101] Control group experimental method:

[0102] The control group's amplification medium consisted of X-VIVO15 + 500 U / ml IL-2 + 5% serum substitute; the activation medium consisted of X-VIVO15 + 5% autologous plasma + 500 U / ml IL-2, 50 ng / ml CD3, and 10 ng / ml CD28. The remaining experimental procedures were the same as those for the experimental group.

[0103] 2. Experimental Results

[0104] After 19 days of culture, the purity of γδT cells from donor batch 1 was 92.52%. Figure 2 The total number of cells increased more than 850 times. Figure 3 (Table 2) γδT cells expanded more than 14,000 times ( Figure 4 (Table 3).

[0105] Table 2 Total Cell Expansion Fold

[0106]

[0107] Table 3. Folding of γδT cell expansion

[0108]

[0109] Example 2

[0110] 1. Experimental Materials and Methods

[0111] Same as Example 1.

[0112] 2. Experimental Results

[0113] After 19 days of culture, the purity of γδT cells from donor batch 2 was 82.35%. Figure 5 The total number of cells increased by 276 times (Table 4), and the number of γδT cells increased by 5361 times (Table 5).

[0114] Table 4 Total Cell Expansion Fold

[0115]

[0116] Table 5. Folding of γδT cell expansion

[0117]

[0118] Example 3

[0119] 1. Experimental Materials and Methods

[0120] Same as Example 1.

[0121] 2. Experimental Results

[0122] After 19 days of culture, the purity of γδT cells from batch 3 donors was 86.98%. Figure 6 The total number of cells increased by 230-fold (Table 6), and the number of γδT cells increased by 3448-fold (Table 7).

[0123] Table 6 Total Cell Expansion Folds

[0124]

[0125] Table 7. Folding of γδT cells

[0126]

[0127] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A composition for inducing culture of γδ T cells, characterized by, The composition comprises an activation medium, components of the activation medium comprise BTN3A1 protein; components of the activation medium further comprise basal medium, plasma, interleukin; The composition further comprises an expansion medium, the expansion medium comprises basal medium, plasma, interleukin, antibody; The interleukin is IL-2; The antibody is selected from CD3 antibody, CD28 antibody and γδTCR antibody; The concentration of the BTN3A1 protein is 1-5 μg / ml; The concentration of the IL-2 is 100-500 U / ml; The concentration of the CD3 antibody is 30-100 ng / ml; The concentration of the CD28 antibody is 5-10 ng / ml; The concentration of the γδTCR antibody is 1-5 μg / ml; The basal medium is selected from GT-T551; The plasma is autologous plasma; the concentration of the autologous plasma is 10%.

2. A method of inducing culture of γδ T cells, characterized by, The method comprises culturing a cell population comprising γδT cells using the composition of claim 1; the cell population comprising γδT cells is PBMC.

3. The method of claim 2, wherein, The method specifically comprises: Step 1) activating a cell population comprising γδT cells using the activation medium of claim 1; Step 2) expanding the obtained cells in step 1) using the expansion medium of claim 1.

4. The method of claim 2, wherein, The seeding density of the PBMC is 2×10^6 / ml.

5. The method of claim 3, wherein, The activation time of step 1) is 1-3 days.

6. The method of claim 3, wherein, Step 2) further comprises a step of liquid supplementing.

7. The method of claim 6, wherein, Liquid supplementing is performed every 2-3 days.

8. The method of claim 3, wherein, The density of the γδT cells is 1×10^6 / ml.

9. Use of the composition of claim 1 or the method of any one of claims 2-8 in inducing culture of γδT cells.

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