A method for stable and controllable expansion of activated lymphocytes and its application in the prevention and treatment of neurological diseases.

Abstract

This invention relates to a method for preparing a stock solution for activating and expanding lymphocytes, comprising the following steps: (1) peripheral blood mononuclear cells (PBMCs) obtained by centrifugation of autologous peripheral whole blood and lymphocyte activators are co-cultured in a serum-free medium to complete the culture and activation of lymphocytes, wherein the serum-free medium is selected from any one of KBM 581, GT-T551H3 or a combination thereof; (2) the cultured and activated lymphocytes obtained in step (1) are placed in a serum-free medium and passaged to obtain a lymphocyte activated culture; (3) 5-10 times the volume of serum-free medium is added to the lymphocyte activated culture obtained in step (2) for expansion culture, the expansion culture number being 1-5 generations; (4) centrifugation, washing, and collection of activated and expanded lymphocytes are obtained. The activated and expanded lymphocytes prepared in this invention can be used to prevent and treat patients with neurological diseases. They can improve and enhance patients' physical control and make them more mentally alert. They can significantly reduce symptoms such as dizziness and difficulty in movement in patients with cerebellar ataxia, significantly improve patients' quality of life and accelerate their recovery, significantly improve the targeting and precision of immune cell therapy, and significantly improve patients' compliance.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a method for efficiently expanding and activating lymphocytes and its use in the prevention and treatment of neurological diseases. Background Technology

[0002] Cerebral stroke is an acute cerebrovascular disease caused by the sudden rupture or blockage of blood vessels in the brain, resulting in ischemic damage to brain tissue. It is characterized by high morbidity, high mortality, and high disability rates. Occlusion and stenosis of the internal carotid artery and vertebral artery can cause ischemic stroke, which can lead to death in severe cases.

[0003] Traumatic brain injury is mostly caused by external objects striking the head, and often results in varying degrees of permanent functional impairment. Damage to different areas of the brain can cause focal symptoms, including abnormalities in motor, sensory, speech, vision, and hearing areas. Diffuse brain damage can even affect memory, sleep, or lead to confusion and coma.

[0004] Neurodegenerative diseases are chronic, progressive degeneration of the central nervous system caused by the loss of neurons or glial cells in the brain or spinal cord. These include Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis.

[0005] Ataxia refers to clumsiness and incoordination of movement caused by dysfunction of the cerebellum, proprioception, and vestibular system. When the trunk, limbs, and pharyngeal muscles are involved, it can cause disorders of balance, posture, gait, and speech. Clinically, it is classified into cerebellar ataxia, cerebral ataxia, sensory ataxia, and vestibular ataxia. Ataxia is a complex manifestation of multiple diseases, requiring further investigation into its underlying etiology. Cerebellar ataxia is a common manifestation in neurological patients, with diverse causes. Cerebellar degeneration may have a long and slow progression, but acute cerebellar swelling caused by infarction, edema, or hemorrhage can have rapid and severe adverse effects, constituting a neurological emergency. The etiology of ataxia is complex. The development of molecular, genetic, and imaging diagnostic techniques provides more evidence for the accurate diagnosis of various types of ataxia. However, due to limitations in the timing of diagnosis and the availability of diagnostic tools, the pathogenesis of some patients remains unclear. Due to individual patient differences, clinical drug treatments lack sufficient evidence-based medicine, and their efficacy is uncertain. Research and development of safer and more effective drugs for the treatment of ataxia to meet unmet clinical needs.

[0006] Peripheral nerve injury is a common clinical condition mainly caused by factors such as trauma, tumors, and metabolic diseases. It often leads to partial or complete loss of motor, sensory, and autonomic functions in the affected segment, as well as intractable neuralgia, which severely impacts patients' quality of life and places a heavy burden on families and society. Currently, various strategies have been developed to promote the repair and regeneration of peripheral nerves after injury. However, in cases of significant cell loss, such as severe injury, complete cell replacement may be necessary. Cell therapy, where implanted cells can produce neurotrophic factors, structural and adhesive extracellular matrix molecules, etc., creates a favorable microenvironment for peripheral nerve axonal regeneration and functional circuit reconstruction, thereby stimulating and supporting axonal regeneration at the proximal stump. This is a promising therapeutic strategy for repairing long-segment peripheral nerve injuries. Cell types used for peripheral nerve injury repair mainly include mature somatic cells such as Schwann cells and olfactory ensheathing cells, and stem cells such as embryonic stem cells, neural stem cells, mesenchymal stem cells, myoblasts, and induced pluripotent stem cells.

[0007] According to the World Health Organization's 2017 Global Diabetes Report, the number of people with diabetes is increasing globally. Furthermore, rising living standards and increasing social competition are contributing to the rising incidence rate and its trend towards affecting younger people. In my country, there are 129 million people with diabetes, over 90% of whom have type 2 diabetes. Type 2 diabetes has a complex pathogenesis, and its various complications lead to serious consequences such as disability and death. Diabetic patients often have weakened immune systems, making them susceptible to various diseases: Disordered glucose metabolism, high blood sugar, and increased intracellular glucose and pyruvate accumulation create a favorable environment for pathogen growth and reproduction; impaired lipid metabolism and increased triglycerides provide abundant nutrients for pathogens; reduced albumin formation leads to decreased antibody and complement levels, weakened immunity, increased blood viscosity, and microcirculatory disturbances, affecting oxygen exchange between blood and tissues and further reducing the body's resistance. Metabolic disorders in diabetic patients cause liver damage and decreased vitamin A conversion, resulting in impaired respiratory mucosal epithelial integrity, reduced defense function, and increased susceptibility to pathogen infections. Diabetic patients or their relatives often have other autoimmune diseases, such as diffuse goiter with hyperthyroidism, chronic lymphocytic thyroiditis, myasthenia gravis, and pernicious anemia. In elderly patients with type II diabetes, the incidence of malignant tumors is significantly increased. Insulin resistance in these patients can lead to hyperinsulinemia, and insulin can promote the growth of malignant tumors.

[0008] The nasal cavity is easily invaded by bacteria, viruses, inhaled pollutants, dust, and harmful substances, triggering allergic rhinitis. Allergic rhinitis (AR) is a non-infectious inflammatory disease of the nasal mucosa. Allergens come into close contact with the skin, mucous membranes, and nasal mucosa, bridging IGE receptors on the surface of mast cells and causing mast cell degranulation. This releases inflammatory factors such as histamine, leukotrienes, and bradykinin into the local skin, mucous membranes, or blood vessels, leading to local capillary dilation, increased vascular permeability, smooth muscle contraction, and increased glandular secretion. Symptoms include sneezing, clear nasal discharge, nasal congestion, and nasal itching. It also easily leads to nasal flora imbalance and the growth of harmful bacteria. WHO survey data shows that the global prevalence of allergic rhinitis is as high as 11%, while the prevalence in the Chinese population is 10%-15%, with as many as 240 million people suffering from allergic rhinitis. Therefore, there is an urgent clinical need for safe and effective drug combinations to prevent and treat allergic rhinitis symptoms.

[0009] Eczema is a common allergic disease that is prone to recurrence and affects people of all ages, genders, and regions. The causes of eczema may be related to internal factors, external environmental factors, and psychosocial factors. When the body's immune system is imbalanced and in a state of "immune activation," allergic reactions can occur. Some immune cells in the body become overactive, indiscriminately unleashing antibodies, complement, and other unconventional weapons to attack their own cells.

[0010] Urticaria, commonly known as hives, is a localized edema reaction caused by dilation and increased permeability of small blood vessels in the skin and mucous membranes. It usually subsides within 2 to 24 hours, but new rashes may recur. The course of the disease can last from several days to several months. It is quite common clinically.

[0011] Menopausal syndrome in women is a transitional period in which ovarian function gradually declines until it ceases completely. It is characterized by a series of clinical symptoms arising from physiological and psychological changes, commonly including hot flashes and sweating, irritability, palpitations, insomnia, or depression and forgetfulness. Based on clinical manifestations, this condition falls under the category of "pre- and post-menopausal symptoms" in Traditional Chinese Medicine. Stem cells can differentiate into sufficient oocytes, replenishing the number of oocytes, stimulating estrogen secretion, and maintaining the normal morphology and function of the ovaries. Applying stem cell therapy to ovarian aging can delay the onset of menopause, postpone the menopausal period, improve psychological age, target specific hormones, stimulate the secretion of estrogen, increase estrogen levels, improve many menopausal problems, and ultimately postpone the onset of menopause.

[0012] To date, numerous clinical observations have revealed cases where patients have benefited from immune cell therapy, or from its combination with other treatments such as surgery, radiotherapy, chemotherapy, targeted therapy, and immune activator therapy. This has demonstrated the longevity of immune cells in immunotherapy, leading to their widespread attention and application.

[0013] Serum-free cell culture expanded and activated lymphocytes were prepared from mononuclear cells in the patient's own peripheral blood. The main component was expanded and activated T lymphocytes (CD3+ cells), with CD8+ cytotoxic T lymphocytes as the primary functional cells. The average number of CD8+ T cells in a single infusion was 4.70 ± 1.47 × 10⁻⁶. 9 The results showed good therapeutic activity in clinical practice. Existing technologies disclose methods for serum-free culture to expand and activate lymphocytes, but optimization is needed in the extraction and separation of peripheral blood mononuclear cells (PBMCs), the type and ratio of lymphocyte activators, and serum-free culture media to improve the lymphocyte expansion fold, cell viability, and biological activity obtained by this method, thus meeting the clinical needs for stable and controllable cell quality and safe and effective treatment. Summary of the Invention

[0014] The purpose of this invention is to provide a method for preparing a stock solution for activating and expanding lymphocytes, comprising the following steps:

[0015] (1) Peripheral blood mononuclear cells (PBMCs) obtained by centrifugation of autologous peripheral whole blood were co-cultured with a lymphocyte activator in serum-free medium to complete the culture and activation of lymphocytes. The initial density of the peripheral blood mononuclear cells for activation in serum-free medium was (0.2-1.6)×10⁻⁶. 6 The cell count / ml was controlled at a cell activation culture temperature of 37.0℃±1.0℃, and the activation culture environment contained 7.5%±1.0% CO2. The lymphocyte activator was selected from any one or a combination of anti-human CD2 antibody, anti-human CD3 antibody, anti-human CD28 antibody, and phytohemagglutinin (PHA), or an antibody-containing carrier immobilized on a carrier. The serum-free culture medium was selected from any one or a combination of KBM 581 and GT-T551 H3.

[0016] (2) The cultured and activated lymphocytes obtained in step (1) are cultured at a cell density of (0.5-5)×10⁻⁶ cells / mL. 6Lymphocytes / ml were placed in serum-free culture medium and passaged to obtain activated lymphocyte culture. The passage temperature was 37.0℃±1.0℃, and the activation culture environment contained 7.5%±1.0% CO2. The serum-free culture medium was selected from any one or a combination of KBM 581, GT-T551H3, and the passage number was 1-5 generations.

[0017] (3) Add 5-10 times the volume of serum-free medium to the lymphocyte activated culture obtained in step (2) for amplification culture. The amplification culture temperature is 37.0℃±1.0℃, and the activation culture environment contains 7.5%±1.0% CO2. The serum-free medium is selected from any one or a combination of KBM 581, GT-T551 H3, and the amplification culture passage number is 1-5.

[0018] (4) Centrifuge, wash, and collect the activated and expanded lymphocytes to obtain the final product.

[0019] In a preferred embodiment of the present invention, the initial density of the biological sample for activation in serum-free culture medium is (0.2-1)×10⁻⁶. 6 per ml.

[0020] In a preferred embodiment of the present invention, the lymphocyte activator is an anti-human CD3 antibody, preferably 2.5 μg / ml-5 μg / ml, with a volume of 8-15 ml, more preferably 2.5 μg / ml-3.8 μg / ml, with a volume of 10-13 ml.

[0021] In a preferred embodiment of the present invention, the method for extracting peripheral blood mononuclear cells by centrifugation of whole blood includes the following steps: adding separation medium and diluent to anticoagulated whole blood, stirring and mixing, adding the mixture to the separation solution, centrifuging at 1000-3000 rpm for 10-40 min, collecting the cell layer at the interface, adding washing solution, centrifuging, washing, and collecting the cells to obtain the product. The separation medium is selected from any one or a combination of hydroxyethyl starch 40 sodium chloride injection (HES), Percol, Ficol-PaquePLUS, and the volume ratio of whole blood to diluent is 1:1-2. The diluent is selected from any one or a combination of sodium chloride injection, Hank's buffer, Lactated Ringer's solution, and Dulbecco's phosphate buffer. The separation solution is selected from hydroxyethyl starch 40 sodium chloride injection (HES), Ficol, Lymphoprep, Lymphocyte Separation Media, and Cel Separation Media. The separation solution has an osmotic pressure of 300 mOsmol / kg to 360 mOsmol / kg, and the washing solution is selected from any one or a combination of 0.1% human serum albumin sodium chloride injection, Dulbecco's phosphate buffer, and sodium chloride injection.

[0022] In the preferred embodiment of the present invention, the centrifugation conditions are (1500-2500 rpm)*(15-30 min), preferably (2000-2500 rpm)*(20-25 min).

[0023] In the preferred embodiment of the present invention, the washing is centrifugal washing, and the washing conditions are (500-2000 rpm) * (5-20 min), centrifugal washing 1-5 times, preferably (1000-1800 rpm) * (10-15 min), centrifugal washing 2-3 times.

[0024] In a preferred embodiment of the present invention, the cell density for passage culture in step (2) is (1-4)×10⁻⁶. 6 Cells / ml, and the pH of the passaged culture system after adding serum-free medium is 7.00-7.80.

[0025] In the preferred embodiment of the present invention, in step (2), the cell density for passage culture is (2-3)×10⁻⁶. 6 Cells / ml, pH 7.02-7.76 after adding serum-free medium to the passaged culture system.

[0026] In a preferred embodiment of the present invention, the osmotic pressure of the separation liquid is 310-350 mOsmol l / kg, preferably 316-347 mOsmol l / kg.

[0027] In a preferred embodiment of the present invention, the number of generations of subculture is 2-3.

[0028] In the preferred embodiment of the present invention, in step (3), when the density of passaged cells increases to 1-10 times the density of activated cells, expansion culture is started. The lymphocyte culture obtained from passaged culture in step (2) is added to a serum-free culture medium with a total volume of 6-8 times its volume for expansion culture. The pH of the expansion culture system is 6.80-7.80.

[0029] In a preferred embodiment of the present invention, in step (3), when the density of passaged cells increases to 1.2-3 times the density of activated cells, expansion culture begins, and the pH of the expansion culture system is 6.88-7.70.

[0030] In a preferred embodiment of the present invention, the amplification culture number is 2-3 generations.

[0031] In the preferred embodiment of the present invention, the centrifugation conditions in step (4) are (1000-3000rpm)*(1-10min), preferably (1500-2800rpm)*(2-8min), and more preferably (2000-2500rpm)*(5-6min).

[0032] In a preferred embodiment of the present invention, cytokine IL-2 300-600 I U / ml, preferably 400-500 I U / ml, is optionally added to the serum-free culture medium.

[0033] In a preferred embodiment of the present invention, the pH of the serum-free culture medium is 6.9-7.9, preferably 7.2-7.4.

[0034] In a preferred embodiment of the present invention, the culture equipment is selected from any one of an incubator, a shaker, or a bioreactor.

[0035] In the preferred embodiment of the present invention, the activation and amplification of lymphocytes is ≥900 times, preferably ≥1000 times, and more preferably ≥1100 times.

[0036] In the preferred embodiment of the present invention, the activation and expansion of lymphocytes has a viability rate of ≥95%, preferably ≥98%.

[0037] In a preferred embodiment of the present invention, the number of CD8+ T cells in the activated and expanded lymphocytes is ≥1×10⁻⁶. 9 1×10 9-2×10 10 More preferably 4-9.5×10 9 indivual.

[0038] In a preferred embodiment of the present invention, the activated and expanded lymphocytes exhibit biological activity KT 50 ≤8.5, preferably KT 50 ≤4, more preferably KT 50 ≤0.7141.

[0039] Another object of the present invention is to provide a pharmaceutical composition containing a stock solution of activated and expanded lymphocytes, said composition comprising (1-20)×10 7 The composition consists of activated and expanded lymphocytes per ml, 0.5-2% human serum albumin, and physiological saline for injection. The viability of the activated and expanded lymphocytes is ≥95%, and the CD8+ T cells among the activated and expanded lymphocytes are (1-20)×10⁶. 7 The pharmaceutical composition contains no preservatives or antibiotics.

[0040] In a preferred embodiment of the present invention, the composition comprises (2-18)×10 9 The concentration of activated and expanded lymphocytes per ml was ≥98%, with a concentration of 1-1.5% human serum albumin and physiological saline for injection. The activation and expanded lymphocyte viability was ≥98%, and the CD8+ T cell count among the activated and expanded lymphocytes was 1×10⁶. 9 -2×10 10 indivual.

[0041] In a preferred embodiment of the present invention, the cell amplification factor of the biological sample is ≥900 times, preferably ≥1000 times, and more preferably ≥1100 times.

[0042] In the preferred embodiment of the present invention, the biological activity of activated and expanded lymphocytes is KT50≤8.5, preferably KT50≤4, and more preferably KT50≤0.7141.

[0043] In a preferred embodiment of the present invention, the total number of activated and expanded lymphocytes in a single infusion by the patient is ≤2×10⁻⁶. 10 Each cell has a viability of ≥85% within its shelf life.

[0044] In a preferred embodiment of the present invention, the viability of activated and expanded lymphocytes obtained by storing the composition at 15-25°C for 12 hours is ≥85%.

[0045] Another object of the present invention is to provide a method for preparing a pharmaceutical composition containing activated and expanded lymphocyte stock solution, wherein the activated and expanded lymphocyte stock solution is resuspended in physiological saline for injection containing human serum albumin.

[0046] Another object of the present invention is to provide a method for using activated and expanded lymphocytes for immunotherapy, comprising the following steps:

[0047] (1) Obtain biological samples containing lymphocytes from individuals;

[0048] (2) The lymphocytes are activated, passaged, and expanded using the preparation method of the present invention to obtain activated and expanded lymphocytes;

[0049] (3) The activated and expanded lymphocytes or their pharmaceutical composition are reinfused into the individual.

[0050] Another object of the present invention is to provide a dosing regimen for the activated and expanded lymphocytes of the present invention for immunotherapy, comprising the following regimens:

[0051] (1) In the first course of treatment, the patient receives 1-5 intravenous infusions, once every two weeks;

[0052] (2) In the second course of treatment, the patient receives 1-5 intravenous infusions, once every three weeks;

[0053] If necessary, the treatment course can be increased, and the infusion interval can be extended.

[0054] The preferred technical solution of the present invention includes the following:

[0055] (1) In the first course of treatment, the patient receives 2-4 intravenous infusions, once every two weeks;

[0056] (2) In the second course of treatment, the patient receives 2-4 intravenous infusions, once every three weeks;

[0057] (3) In the third course of treatment, the patient receives 2-4 intravenous infusions, once every four weeks.

[0058] In a preferred embodiment of the present invention, the number of activated and expanded lymphocytes in each intravenous infusion is ≥5 x 102 8 Lymphocytes, preferably ≥(1-20)x10 9 One lymphocyte.

[0059] Another object of the present invention is to provide the application of activated and expanded lymphocytes in the preparation of medicaments for the prevention and treatment of patients with neurological diseases who require enhanced immunity, nerve repair and / or neurotrophic effects to repair and / or nourish nerve damage caused by neurological trauma or ischemia-hypoxia.

[0060] In a preferred embodiment of the present invention, the neurological disease is selected from any one of ataxia, Alzheimer's disease, epilepsy, stroke, sequelae of stroke, Parkinson's disease, neurodegenerative disease, hand tremor, peripheral nerve and muscle diseases, headache, sleep disorder, dizziness, and inflammation of the nervous system or their complications.

[0061] In a preferred embodiment of the present invention, the ataxia is selected from any one or a combination of cerebellar ataxia, cerebral ataxia, sensory ataxia, and vestibular ataxia.

[0062] In a preferred embodiment of the present invention, the ataxia is selected from any one of acquired ataxia, hereditary ataxia, and non-hereditary degenerative ataxia.

[0063] Another objective of this invention is to provide an application of activated and expanded lymphocytes combined with rehabilitation training for the prevention and treatment of neurological diseases.

[0064] Another object of the present invention is to provide the use of activated and expanded lymphocytes in the preparation of medicaments for treating patients with allergic diseases.

[0065] In a preferred embodiment of the present invention, the allergic disease includes any one of rhinitis, eczema, and urticaria.

[0066] Another object of the present invention is to provide the use of activated and expanded lymphocytes in the preparation of medicaments for treating patients with menopausal syndrome.

[0067] Another object of the present invention is to provide the use of activated and expanded lymphocytes in the preparation of medicaments for treating diabetes or its complications.

[0068] Unless otherwise stated, this invention uses the biological activity of expanded and activated lymphocytes (RTCA, with an effector-to-target ratio of 40:1 as an example) to characterize the therapeutic activity of activated and expanded lymphocytes. Digested target cells (such as HepG2) are added at a density of 2 × 10⁻¹¹ μL per well. 5 Target cells were pre-coated using cells / ml and the effector-to-target ratio was set (e.g., 40:1) for baseline measurement. 16-24 hours after target cell coagulation, a specific density (50 μl per well at a density of 1.6 × 10⁻⁶ cells / ml) was added according to the set effector-to-target ratio. 7After adding effector cells (i.e., expanded and activated lymphocytes) at a density of (cells / ml), the instrument tracks cell morphology, proliferation, and differentiation data in real-time, dynamically, and quantitatively. This involves monitoring the change in electrical impedance caused by the contact between live cells and microelectrodes in the detection plate. When effector cells are added to wells containing adherent cells, the adherent growth of these cells on the microelectrode surface alters the interfacial impedance of the adherent electrode. This alteration is positively correlated with changes in the cell index (Cell Index). The instrument's dedicated software converts the electrical resistance signal into a specific cell index. The software monitors the generated cell index curve in real-time to display the cell adhesion, extension, growth, and death states at different time points. By analyzing changes in the cell index, the killing efficiency of effector cells against target cells (such as HepG2 cells) can be calculated. The KT value is then calculated from the killing efficiency curve. 50 (i.e., the time it takes for effector cells to kill 50% of target cells), and in KT 50 The KT value serves as an indicator for determining the biological activity of effector cells (i.e., activated and expanded lymphocytes). 50 The smaller the value, the higher the killing efficiency and the higher the biological activity. Conversely, the larger the value, the lower the biological activity.

[0069] Unless otherwise stated, when this invention relates to percentages between liquids, the percentage is volume / volume percentage; when this invention relates to percentages between liquids and solids, the percentage is volume / weight percentage; when this invention relates to percentages between solids and liquids, the percentage is weight / volume percentage; the remainder is weight / weight percentage.

[0070] Compared with the prior art, the present invention has the following beneficial technical effects:

[0071] 1. The activated and expanded lymphocytes obtained by this invention have advantages such as high cell expansion efficiency, high cell viability, stable cell activity, long shelf life, safety and effectiveness, and few side effects. They effectively solve key problems in personalized immunotherapy. First, they use a serum-free culture system with stable cell culture efficiency, continuously culturing for 12 days while maintaining cell killing activity and expansion capacity, avoiding potential pathogenic microbial contamination and other adverse factors on cell growth that may occur with serum. Second, they have high cell expansion efficiency, eliminating the need for a mononuclear cell collection machine, achieving more than 1000-fold expansion, avoiding the destructive effect on the patient's overall immune cell system caused by the traditional immunotherapy method of using apheresis to collect large numbers of initial peripheral blood mononuclear cells. Third, they have high cell activity and good stability, and can be stored for 12 hours without affecting their therapeutic effect. Fourth, they solve the technical problems of standardized quality control systems and large-scale production for personalized products, obtaining relatively uniform cell products, significantly improving treatment targeting and precision, and patient compliance.

[0072] 2. The activated and expanded lymphocytes prepared in this invention are used to prevent and treat patients with neurological diseases. These patients need enhanced immunity, nerve repair, and / or nerve nutrition to repair and / or nourish nerve damage caused by neurological trauma and ischemia-hypoxia. They significantly improve nerve conduction, joint flexibility, and motor coordination, which helps improve and enhance the patient's body control and makes them more mentally alert. They significantly reduce symptoms such as dizziness and difficulty in movement in patients with cerebellar ataxia, significantly improve the patient's quality of life, and accelerate the patient's recovery. They have the advantages of high cell expansion efficiency, high cell viability, good cell stability, significant curative effect, fast recovery, safety and effectiveness, and few side effects. They significantly improve the targeting and precision of immune cell therapy and significantly improve patient compliance.

[0073] 3. The lymphocyte amplification method of the present invention has the advantages of simple operation, controllable quality, and suitability for industrial production. Attached Figure Description

[0074] Figure 1 Example 1: Growth status of EAL cells during the first passage culture (observed under a 250× microscope);

[0075] Figure 2 Example 1: EAL cell growth during the first expansion culture (observed under a 250× microscope);

[0076] Figure 3 Example 1: EAL cell growth during the second expansion culture (observed under a 250× microscope). Detailed Implementation

[0077] The present invention will be further described below with reference to the embodiments, but the invention is not limited to the scope of the described embodiments.

[0078] Example 1 Preparation of Lymphocyte Activation and Expansion Stock Solution

[0079] (1) Isolate peripheral blood mononuclear cells (PBMCs) and inoculate them for activation culture (day 0).

[0080] Patient 1: Cerebellar ataxia, age: 52, gender: male.

[0081] The serum-free cell culture medium is: GT-T551 H3 serum-free cell culture medium (IL-2 concentration in the medium is 500 IU / ml, pH 7.2-7.4).

[0082] 37 ml of whole blood was drawn from the patient and placed in a new 250 ml centrifuge tube. An equal volume of 0.9% sodium chloride injection solution was added and mixed well. This mixture was then slowly added to two 50 ml centrifuge tubes (each containing 15 ml of Ficol.) to ensure a clear interface. The tubes were centrifuged at 2000 rpm for 20 minutes with the centrifuge set to speed 7 and deceleration 0. The cell layer from the interface was then transferred to a new 50 ml centrifuge tube and mixed with 0.9% sodium chloride injection solution at a 1:1 ratio. The tubes were centrifuged at 1200 rpm for 10 minutes. After centrifugation, the supernatant was discarded, the cell pellet was shaken to separate, and the cells were resuspended in 50 ml of 0.9% sodium chloride injection solution. The cells were centrifuged at 1200 rpm for 10 minutes. After centrifugation, the supernatant was discarded, the cell pellet was shaken to separate, and 5 ml of serum-free cell culture medium was added. 10 μl of the cell suspension was taken for cell counting. The cell concentration after resuspending in serum-free cell culture medium was 0.8 × 10⁻⁶ cells / mL. 6 After inoculating at a rate of 100 cells / ml, the cells were inoculated into 225 cm² cells coated with anti-human CD3 antibodies. 2 Samples are taken from cell culture flasks, subjected to aseptic testing and labeled, and then placed in a cell culture incubator for culture.

[0083] During the preparation of the lymphocyte activation and expansion stock solution, the incubator conditions were: 37℃, 7.5% CO2.

[0084] (2) First passaging (day 3)

[0085] When the culture medium turns lighter or yellower (day 0 is counted as the day of blood collection, day 3 after inoculation), remove the cell culture flask from the incubator; observe the cell morphology under a microscope: the growth is good (see...). Figure 1 Add 50ml of serum-free cell culture medium to the cell culture flask and place it back into the cell culture incubator for further culture.

[0086] (3) Second subculture (day 4)

[0087] When the culture medium turns light yellow, remove the cell culture flask from the incubator; observe the cell morphology under a microscope: the growth is good. Add 140 ml of serum-free cell culture medium and return it to the cell culture incubator for further culture.

[0088] (4) First amplification culture (day 5)

[0089] Remove the cell culture flask from the incubator and observe the cell morphology under a microscope: the cells are growing well (see...). Figure 2 Sampling was performed for cell counting, and the viable cell density was not less than 1.0 × 10⁻⁶. 6 When the cell culture volume is 760 ml, pour the cell culture medium and 760 ml of serum-free cell culture medium into the cell culture bag and place it back into the cell culture incubator for culture.

[0090] (5) Second amplification culture (day 8)

[0091] When the culture medium turns yellowish, take a new cell culture bag to the biosafety cabinet and pour 1000ml of serum-free cell culture medium (bag A) into it through a syringe sleeve, then seal the tube. Remove the cell culture bag containing the cultured cells from the incubator (bag B), and connect the two bags using an aseptic joining machine. First, allow all the liquid in bag A to flow into bag B, mix well, and then return 1000ml to bag A, so that both bags contain 1000ml of liquid. Seal the tube and extract a sample for sterility testing. Observe the cell morphology under a microscope (see...). Figure 3 Then, place the two bags of cells back into the incubator for further culture.

[0092] (6) Purify, centrifuge, concentrate, and collect cells to prepare cell stock solution (Day 12)

[0093] On day 4 after culturing two cell culture bags, remove one cell culture bag from the incubator and transfer it to the biosafety cabinet. Divide the cell suspension into four 250ml centrifuge tubes and centrifuge at 2000rpm for 5 minutes. Discard the supernatant and disperse the precipitate. Then, remove another cell culture bag from the same batch from the incubator and transfer it to the biosafety cabinet. Divide the cell suspension into the original four 250ml centrifuge tubes and centrifuge at 2000rpm for 5 minutes. Discard the supernatant and disperse the precipitate. Use 250ml of 0.9% sodium chloride injection containing 0.1% human serum albumin to wash the cells in one tube three times and combine them into another tube. Combine the four tubes into two tubes and centrifuge at 2000rpm for 5 minutes. Discard the supernatant, disperse the precipitate, and then use 200ml of 0.1% human serum albumin sodium chloride injection to wash the cells in the two tubes three times and combine them into one tube. After mixing, take a sample for cell counting, centrifuge at 2000rpm for 5 minutes, and discard the supernatant. This is the stock solution for activating and expanding lymphocytes.

[0094] (7) Preparation of the composition

[0095] Activated and expanded lymphocytes at a concentration of 8 × 10 7 The cell stock solution is resuspended at 1 / ml in physiological saline for injection containing 1% human serum albumin.

[0096] Example 2 Preparation of Lymphocyte Activation and Expansion Stock Solution

[0097] (1) Isolate PBMCs and inoculate them for activation culture (day 0)

[0098] Patient 2: Cerebellar ataxia, age: 65, gender: male.

[0099] The serum-free cell culture medium is: KBM 581 serum-free cell culture medium (I L-2 concentration in the medium is 500 IU / ml, pH 7.2-7.4).

[0100] Transfer 63 ml of whole blood to a new 250 ml centrifuge tube. Add an equal volume of sodium chloride injection solution to the blood sample, mix well, and slowly add to four 50 ml centrifuge tubes (each containing 15 ml of Ficol. I) to ensure a clear interface. Centrifuge at 2000 rpm for 20 min with the centrifuge set to speed 7 and deceleration 0. Transfer the cell layer from the interface to a new 50 ml centrifuge tube, mix with 0.9% sodium chloride injection solution at a 1:1 ratio, and centrifuge at 1200 rpm for 10 min. After centrifugation, discard the supernatant, shake the cell pellet, resuspend in 50 ml of sodium chloride injection solution, and centrifuge at 1200 rpm for 10 min. After centrifugation, discard the supernatant, shake the cell pellet, add 5 ml of serum-free cell culture medium, mix well, and take 10 μl of the cell suspension for cell counting. Resuspend the cells in serum-free cell culture medium to a concentration of 0.2 × 10⁻⁶ cells / mL. 6 After inoculating at a rate of 100 cells / ml, the cells were inoculated into 225 cm² cells coated with anti-human CD3 antibodies. 2 In cell culture flasks, samples are taken, sterility is checked and labeled, and then placed in a cell culture incubator for incubation.

[0101] During the preparation of the lymphocyte activation and expansion stock solution, the incubator conditions were: 37℃, 7.5% CO2.

[0102] (2) First passaging (day 4)

[0103] When the culture medium turns lighter or yellow (day 0 is counted as the day of blood collection, day 4 after inoculation), remove the cell culture flask from the incubator; observe the cell morphology under a microscope: the growth is good. Perform cell counting, add 50 ml of serum-free cell culture medium to the cell culture flask, and return it to the cell culture incubator for further culture.

[0104] (3) Second passaging (day 5)

[0105] When the culture medium turns lighter in color (to a pale yellow), remove the cell culture flask from the incubator. Observe the cell morphology under a microscope: the growth is good. Perform cell counting, add 140 ml of serum-free cell culture medium to the cell culture flask, and return it to the cell culture incubator for further culture.

[0106] (4) First amplification culture (day 6)

[0107] Remove the cell culture flask from the incubator and observe the cell morphology under a microscope: The cells are growing well. Take samples for cell counting; when the viable cell density is not less than 1 × 10⁻⁶... 6 When the cell count is 750 ml, pour the cell culture medium and 750 ml of serum-free cell culture medium into the cell culture bag and place it back into the cell culture incubator for culture.

[0108] (5) Second amplification culture (day 8)

[0109] When the culture medium turns yellowish, take a new cell culture bag to the biosafety cabinet and pour 1000ml of serum-free cell culture medium (bag A) into it using a syringe sleeve, then seal the tube. Remove the cell culture bag containing the cultured cells from the incubator (bag B), and connect the two bags using an aseptic connection machine. First, allow all the liquid in bag A to flow into bag B, mix well, and then return 1000ml to bag A, so that both bags contain 1000ml of liquid. Seal the tube and take a sample for sterility testing. Observe the cell morphology under a microscope, and then return both bags of cells to the incubator for further culture.

[0110] (6) Purify, centrifuge, concentrate, and collect cells to prepare cell stock solution (Day 12)

[0111] On day 4 after culturing two cell culture bags, remove one cell culture bag from the incubator and transfer it to the biosafety cabinet. Divide the cell suspension into four 250ml centrifuge tubes and centrifuge at 2000rpm for 5 minutes. Discard the supernatant and disperse the precipitate. Then, remove another cell culture bag from the same batch from the incubator and transfer it to the biosafety cabinet. Divide the cell suspension into the original four 250ml centrifuge tubes and centrifuge at 2000rpm for 5 minutes. Discard the supernatant and disperse the precipitate. Wash the cells from one tube three times with 250ml of 0.9% sodium chloride injection and combine them into another tube. Combine the four tubes into two tubes and centrifuge at 2000rpm for 5 minutes. Discard the supernatant and disperse the precipitate. Then, wash the cells from the two tubes three times with 200ml of sodium chloride injection and combine them into one tube. After mixing, take a sample for cell counting, centrifuge at 2000rpm for 5 minutes, and discard the supernatant. This is the stock solution for activating and expanding lymphocytes.

[0112] (7) Preparation of the composition

[0113] Activated and expanded lymphocytes at a concentration of 6 × 10 7 The cell stock solution is resuspended at 1 / ml in physiological saline for injection containing 1.5% human serum albumin.

[0114] Example 3 Preparation of Lymphocyte Activation and Expansion Stock Solution

[0115] (1) Isolate PBMCs and inoculate them for activation culture (day 0)

[0116] Patient 3: Stroke patient, age: 62, gender: male.

[0117] The serum-free cell culture medium was KBM 581 (CORN 1 NG) serum-free cell culture medium (IL-2 concentration in the medium was 400 I U / ml, pH 7.2-7.4).

[0118] 58 ml of whole blood was drawn from the patient and placed into a new 250 ml centrifuge tube. An equal volume of 0.9% sodium chloride injection solution was added, mixed well, and then slowly added to four 50 ml centrifuge tubes (each containing 15 ml of Fico Ill solution), ensuring a clear interface. The tubes were centrifuged at 2000 rpm for 20 minutes with the centrifuge set to speed 7 and deceleration 0. The cell layer between the cells was aspirated into a new 50 ml centrifuge tube, mixed with 0.9% sodium chloride injection solution at a 1:1 ratio, and centrifuged at 1200 rpm for 10 minutes. After centrifugation, the supernatant was discarded, the cell pellet was shaken apart, and the cells were resuspended in 50 ml of sodium chloride injection solution. The cells were centrifuged at 1200 rpm for 10 minutes. After centrifugation, the supernatant was discarded, the cell pellet was shaken apart, and 5 ml of serum-free cell culture medium was added. 10 μl of the cell suspension was taken for cell counting. The cell concentration after resuspending in serum-free cell culture medium was 0.3 × 10⁻⁶ cells / mL. 6 After inoculating at a rate of 100 cells / ml, the cells were inoculated into 225 cm² cells coated with anti-human CD3 antibodies. 2 In cell culture flasks, samples are taken, sterility is checked and labeled, and then placed in a cell culture incubator for incubation.

[0119] During the preparation of the lymphocyte activation and expansion stock solution, the incubator conditions were: 37℃, 5% CO2.

[0120] (2) First passaging (day 3)

[0121] When the culture medium turns lighter or yellow (day 0 is counted as the day of blood collection, day 3 after inoculation), remove the cell culture flask from the incubator; observe the cell morphology under a microscope: the growth is good. Perform cell counting, add 50 ml of serum-free cell culture medium to the cell culture flask, and return it to the cell culture incubator for further culture.

[0122] (3) Second subculture (day 4)

[0123] When the culture medium turns lighter in color (to a pale yellow), remove the cell culture flask from the incubator. Observe the cell morphology under a microscope: the growth is good. Perform cell counting, add 140 ml of serum-free cell culture medium to the cell culture flask, and return it to the cell culture incubator for further culture.

[0124] (4) First amplification culture (day 6)

[0125] Remove the cell culture flask from the incubator and observe the cell morphology under a microscope: The cells are growing well. Take samples for cell counting; when the viable cell density is not less than 1 × 10⁻⁶... 6 When the cell count is 750 ml, pour the cell culture medium and 750 ml of serum-free cell culture medium into the cell culture bag and place it back into the cell culture incubator for culture.

[0126] (5) Second amplification culture (day 8)

[0127] When the culture medium turns yellowish, take a new cell culture bag to the biosafety cabinet and pour 1000ml of serum-free cell culture medium (bag A) into it using a syringe sleeve, then seal the tube. Remove the cell culture bag containing the cultured cells from the incubator (bag B), and connect the two bags using an aseptic connection machine. First, allow all the liquid in bag A to flow into bag B, mix well, and then return 1000ml to bag A, so that both bags contain 1000ml of liquid. Seal the tube and take a sample for sterility testing. Observe the cell morphology under a microscope, and then return both bags of cells to the incubator for further culture.

[0128] (6) Purify, centrifuge, concentrate, and collect cells to prepare cell stock solution (Day 12)

[0129] On day 4 after culturing two cell culture bags, remove one cell culture bag from the incubator and transfer it to the biosafety cabinet. Divide the cell suspension into four 250ml centrifuge tubes and centrifuge at 2000rpm for 5 minutes. Discard the supernatant and disperse the precipitate. Then, remove another cell culture bag from the same batch from the incubator and transfer it to the biosafety cabinet. Divide the cell suspension into the original four 250ml centrifuge tubes and centrifuge at 2000rpm for 5 minutes. Discard the supernatant and disperse the precipitate. Use 250ml of 0.9% sodium chloride injection to wash the cells in one tube three times and combine them into another tube. Combine the four tubes into two tubes and centrifuge at 2000rpm for 5 minutes. Discard the supernatant, disperse the precipitate, and then use 200ml of sodium chloride injection to wash the cells in the two tubes three times and combine them into one tube. After mixing, take a sample for cell counting, centrifuge at 2000rpm for 5 minutes, and discard the supernatant. This is the stock solution for expanding and activating lymphocytes.

[0130] (7) Preparation of the composition

[0131] Activated and expanded lymphocytes at a concentration of 8 × 10 7 The cell stock solution is resuspended at 1 / ml in physiological saline for injection containing 1% human serum albumin.

[0132] Example 4: Screening of serum-free culture medium

[0133] Using the same activation and amplification process as in Example 1, the effects of three serum-free culture media—KBM 581 (CORNING), GT-T551 H3 (TAKARA), and TexMACS GMP Medium (Miltenybiotec)—on EAL cell preparation were compared in vitro.

[0134] Peripheral blood cells (PBMCs) were collected from three normal individuals for cell counting and phenotypic analysis. Each PBMC was trisected and cultured in three different serum-free culture media. Cell counts were performed every 4-6 days, and the corresponding culture medium was replenished. After 12 days, the culture was terminated, and cell counts, viability, phenotypic analysis, and cytotoxic activity were performed.

[0135] Cells were cultured in serum-free TexMACS GMP Medium, but cell proliferation was slow after 6–8 days of culture, failing to meet the quality requirements for in vitro lymphocyte expansion.

[0136] Table 1

[0137]

[0138]

[0139] Table 2

[0140]

[0141] Table 3

[0142]

[0143] Example 5 Preparation of activated and expanded lymphocyte stock solution

[0144] (1) Peripheral blood was collected from a total of 18 patients, representing different genders, ages, and types. Basic patient information is shown in Table 4. The preparation of the lymphocyte expansion and activation stock solution followed Example 1. Cells were counted using a cell counting chamber to determine the expansion ratio. Cell viability was obtained by trypan blue staining and cell counting. Phenotypic detection of the expanded cells was performed using FITC-labeled CD3 antibody and APC-labeled CD8 antibody, and the results are shown in Table 4.

[0145] Table 4

[0146]

[0147] Experimental Case 1: Treatment Study of Cerebellar Ataxia

[0148] The sample was prepared according to Example 1 and intravenously injected back into cerebellar ataxia patient 1 in Table 4. The treatment plan was as follows: First course of treatment: the patient received 2 intravenous infusions every two weeks; Second course of treatment: the patient received 2 intravenous infusions every three weeks; Third course of treatment: the patient received 3 intravenous infusions every four weeks.

[0149] Before treatment: dizziness; inability to fully manage daily life activities (such as holding objects, dressing, eating, sitting up, etc.), and bilateral limb imbalance, inability to stand or walk.

[0150] Post-treatment follow-up results:

[0151] After the first treatment: The patient felt significant improvement in all aspects of their body, and there was a trend of accelerated recovery. The improvement in walking was particularly obvious: dizziness was significantly reduced, joints were more flexible, walking was more natural, and gait was easier.

[0152] After the second treatment: The patient no longer felt dizzy at rest, and the dizziness during walking was significantly reduced. While walking, the patient felt a gradual return to balance in the strength of both feet, and the hip, knee, and ankle joints became more flexible and elastic. Self-control improved, and walking became more controlled. The patient felt significant progress.

[0153] After the third treatment: the physical rehabilitation progress reached a breakthrough new level: first, the dizziness was less severe, the mind was clearer, and the body control was enhanced; second, the whole body was more coordinated; third, the hip joints, knee joints and other joints were much more flexible, the lower limbs were significantly lighter, the walking progress was very significant, and walking was already more comfortable and the steps were consciously flexible and easy.

[0154] After the sixth treatment: the physical rehabilitation progress has reached a breakthrough new level: first, the mind is clearer and the body control is enhanced; second, the whole body is more coordinated; third, the hip joints, knee joints and other joints are more flexible, the lower limbs are significantly lighter, the self-control of walking is good, walking is more comfortable and the gait is consciously flexible and easy.

[0155] Two other patients with cerebellar ataxia showed significant improvement after treatment.

[0156] Experimental Case 2: Stroke Treatment Study

[0157] Three stroke patients, all of whom had suffered major strokes more than two years prior to treatment, underwent peripheral blood lymphocyte expansion and culture according to the protocol in Example 1. The resulting compound was then intravenously reinfused. The treatment regimen was as follows: First cycle: two intravenous infusions every two weeks; Second cycle: two intravenous infusions every three weeks; Third cycle: three intravenous infusions every four weeks. After two cycles of treatment, all three patients showed significant improvements in cognitive function, gait speed, and Barthel index performance.

[0158] Experimental Case 3: Eczema Treatment Study

[0159] Three patients with eczema presented with skin eczema, poor sleep, and decreased energy. Multiple treatments had failed to cure the condition, and eczema recurred repeatedly. Their MISS immune score was -5, and they exhibited a severe decrease and imbalance in lymphocyte subsets, with compensatory activation at the CD4+ T cell level.

[0160] Peripheral blood was collected according to the protocol of Example 1 for lymphocyte expansion culture to prepare a composition, which was then intravenously reinfused into the patient. The treatment regimen was as follows: First course of treatment: the patient received two intravenous infusions every two weeks; Second course of treatment: the patient received two intravenous infusions every three weeks; Third course of treatment: the patient received three intravenous infusions every four weeks. After one month of treatment, the eczema completely disappeared, and after three months, the skin was completely restored.

[0161] Case 4: Rhinitis Treatment Study

[0162] Three patients with rhinitis, suffering from seasonal allergic rhinitis, experienced attacks every autumn, each lasting 1-2 months, accompanied by symptoms such as nasal congestion, runny nose, tearing, and dizziness.

[0163] Peripheral blood was collected according to the protocol of Example 1 for lymphocyte amplification culture to prepare a composition, which was then intravenously injected back into the patient. The treatment regimen was as follows: First course of treatment: the patient received 2 intravenous infusions every two weeks; Second course of treatment: the patient received 2 intravenous infusions every three weeks; Third course of treatment: the patient received 3 intravenous infusions every four weeks.

[0164] Follow-up results after two courses of treatment: The patient reported significant improvement in all aspects of their health, and the symptoms of nasal congestion and runny nose were reduced.

[0165] Case 5: Study on the Treatment of Urticaria

[0166] Three patients with chronic urticaria experienced no improvement despite taking loratadine, levocetirizine (an antihistamine), and topical medications. They had numerous red wheals on their faces that persisted for a long time, causing intense itching, sleepless nights, poor mental state, and severely impacting their daily lives and social interactions.

[0167] Peripheral blood was collected according to the protocol of Example 1 for lymphocyte expansion culture to prepare a composition, which was then intravenously reinfused into the patient. The treatment regimen was as follows: First course of treatment: the patient received two intravenous infusions every two weeks; Second course of treatment: the patient received two intravenous infusions every three weeks; Third course of treatment: the patient received three intravenous infusions every four weeks. After three courses of treatment, the patient's condition was basically cured.

[0168] Trial Case 6: Treatment Study of Menopausal Syndrome

[0169] Three patients with menopausal syndrome, who had gone through menopause six months prior, experienced insomnia and fatigue, and were diagnosed with menopausal symptoms at the hospital.

[0170] Peripheral blood was collected according to the protocol of Example 1 for lymphocyte expansion culture to prepare a composition, which was then intravenously reinfused into the patient. The treatment regimen was as follows: First course of treatment: the patient received two intravenous infusions every two weeks; Second course of treatment: the patient received two intravenous infusions every three weeks; Third course of treatment: the patient received three intravenous infusions every four weeks. After four courses of treatment, the discomfort symptoms disappeared.

[0171] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a stock solution for activating and expanding lymphocytes, comprising the following steps: (1) Peripheral blood mononuclear cells (PBMCs) obtained by centrifugation of autologous peripheral whole blood were placed in serum-free culture medium with a lymphocyte activator and then cultured at 37.0℃±1.0℃ and 7.5%±1.0%CO2 to obtain activated lymphocytes. The preparation of mononuclear cells includes the following steps: Add separation medium and diluent to anticoagulated whole blood, stir and mix well, add to the separation solution, centrifuge at 1000-3000 rpm for 10-40 min, collect the cell layer at the interface, add washing buffer, centrifuge, wash, and collect the cells. The separation medium is selected from any one or a combination of hydroxyethyl starch 40 sodium chloride injection, Percoll, Ficoll-Paque PLUS, etc., with a whole blood:diluent volume ratio of 1:1-2. The diluent is selected from any one or a combination of sodium chloride injection, Hank's buffer, Lactated Ringer's solution, Dulbecco's phosphate buffer, etc. The separation solution is selected from hydroxyethyl starch 40 sodium chloride injection, Ficoll, Lymphoprep, Lymphocyte Separation Media, Cell Separation... The media are any one or a combination thereof, wherein the osmotic pressure of the separation solution is 300 mOsmol / kg-360 mOsmol / kg, and the washing solution is selected from any one or a combination thereof, including 0.1% human serum albumin sodium chloride injection, Dulbecco's phosphate buffer, and sodium chloride injection; the initial density of mononuclear cells in serum-free culture medium is (0.2-1.6) × 10⁻⁶. 6 The lymphocyte activator is selected from any one of anti-human CD3 antibody or antibody-containing carrier immobilized on a carrier; the serum-free culture medium is selected from any one of KBM 581, GT-T551 H3 or a combination thereof, and the serum-free culture medium contains 300-600 IU / ml of cytokine IL-2. (2) Resuspend the activated lymphocytes obtained in step (1) in serum-free medium until the cell density is (0.5-5)×10⁻⁶. 6 Cells / ml were then passaged for 1-5 generations at 37.0℃±1.0℃ and 7.5%±1.0%CO2 to obtain activated passaged lymphocytes. The serum-free culture medium was selected from any one or a combination of KBM 581, GT-T551 H3. (3) Add serum-free culture medium at 5-10 times its volume to the activated passaged lymphocytes obtained in step (2), and then place them in amplification culture at 37.0℃±1.0℃ and 7.5%±1.0%CO2 for 1-5 generations to obtain activated and expanded lymphocytes. The serum-free culture medium is selected from any one or a combination of KBM 581, GT-T551 H3. (4) Centrifuge, wash, and collect the activated and expanded lymphocytes to obtain the final product.

2. The method as described in claim 1, wherein the initial density of mononuclear cells in serum-free culture medium in step (1) is (0.2-1)×10⁻⁶. 6 per ml.

3. The method according to claim 1, wherein the lymphocyte activator is an anti-human CD3 antibody, at a concentration of 2.5 μg / ml-5 μg / ml, and a volume of 8-15 ml.

4. The method according to claim 3, wherein the lymphocyte activator is an anti-human CD3 antibody, at a concentration of 2.5 μg / ml-3.8 μg / ml, and a volume of 10-13 ml.

5. The method as described in claim 1, wherein the centrifugation conditions are (1500-2500 rpm) * (15-30 min).

6. The method as described in claim 5, wherein the centrifugation conditions are (2000-2500 rpm) * (20-25 min).

7. The method as described in claim 1, wherein the washing is centrifugal washing, and the centrifugal washing conditions are (500-2000 rpm) * (5-20 min), and the centrifugal washing is performed 1-5 times.

8. The method as described in claim 7, wherein the centrifugal washing conditions are (1000-1800 rpm) * (10-15 min), and the centrifugal washing is performed 2-3 times.

9. The method as described in claim 1, wherein in step (2), the cell density for subculture is (1-4)×10⁻⁶. 6 The culture volume was 10 cells / ml, and the pH of the subculture system after adding serum-free medium was 7.00-7.

80.

10. The method of claim 9, wherein the cell density for subculture is (2-3) × 10⁻⁶. 6 The culture volume was 10 cells / ml, and the pH of the subculture system after adding serum-free medium was 7.02-7.

76.

11. The method according to claim 1, wherein the osmotic pressure of the separation liquid is 310-350 mOsmol / kg.

12. The method of claim 11, wherein the osmotic pressure of the separation liquid is 316-347 mOsmol / kg.

13. The method as described in claim 1, wherein the number of subculture generations is 2-3 generations.

14. The method according to claim 1, in step (3), when the density of passaged cells increases to 1-10 times the density of activated cells, expansion culture is started, and serum-free culture medium with a total volume of 6-8 times is added to the activated passaged lymphocyte culture system prepared in step (2) for expansion culture, wherein the pH of the expansion culture system is 6.80-7.

80.

15. The method of claim 14, wherein expansion culture begins when the density of passaged cells increases to 1.2-3 times the density of activated cells, wherein the expansion culture system has a pH of 6.88-7.

70.

16. The method as described in claim 1, wherein the amplification culture generation is 2-3 generations.

17. The method as described in claim 1, wherein the centrifugation conditions in step (4) are (1000-3000 rpm) * (1-10 min).

18. The method of claim 17, wherein the centrifugation conditions in step (4) are (1500-2800 rpm) * (2-8 min).

19. The method of claim 18, wherein the centrifugation conditions in step (4) are (2000-2500 rpm) * (5-6 min).

20. The method of claim 1, wherein the serum-free culture medium contains 400-500 IU / ml of the cytokine IL-2.

21. The method of claim 1, wherein the culture equipment is selected from any one of an incubator, a shaker, or a bioreactor.

22. The method according to any one of claims 1-21, wherein the activation and amplification fold of the lymphocytes is ≥900-fold.

23. The method of claim 22, wherein the activation and amplification fold of the lymphocytes is ≥1000-fold.

24. The method of claim 23, wherein the activation and amplification fold of the lymphocytes is ≥1100-fold.

25. The method according to any one of claims 1-21, wherein the cell viability of the activated and expanded lymphocytes is ≥95%.

26. The method of claim 25, wherein the cell viability of the activated and expanded lymphocytes is ≥98%.

27. The method according to any one of claims 1-21, wherein the number of CD8+ T cells in the activated and expanded lymphocytes is ≥1×10⁻⁶. 9 per mL.

28. The method of claim 27, wherein the number of CD8+ T cells in the activated and expanded lymphocytes is 1 × 10⁻⁶. 9 -2×10 10 per mL.

29. The method of claim 28, wherein the number of CD8+ T cells in the activated and expanded lymphocytes is 4 × 10⁻⁶. 9 -9.5×10 9 per mL.

30. The method according to any one of claims 1-21, wherein the biological activity of activated and expanded lymphocytes is KT 50 ≤8.

5.

31. The method of claim 30, wherein the biological activity of activated and expanded lymphocytes is KT 50 ≤4.

32. The method of claim 31, wherein the biological activity of activated and expanded lymphocytes is KT 50 ≤0.7141.

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