Method for expanding activated lymphocytes with stable and controlled quality and its use in antitumor therapy - Patents.com

Abstract

The present invention relates to a method for producing a stock solution of activated and amplified lymphocytes, comprising the steps of: (1) culturing and activating lymphocytes by co-culturing peripheral blood mononuclear cells (PBMCs) obtained by centrifuging autologous peripheral whole blood with a lymphocyte activator in a serum-free medium; and (2) culturing and activating lymphocytes so that the initial density of the peripheral blood mononuclear cells in the serum-free medium is (0.2 to 1.6) × 10 6 (1) a serum-free medium having a concentration of (0.5 to 5) × 10 cells / ml, the serum-free medium being any one selected from KBM 581, GT-T551 H3, and a combination thereof; and (2) culturing the cultured activated lymphocytes produced in the step (1) in a concentration of (0.5 to 5) × 10 cells / ml. 6 The method includes the steps of: (1) producing a lymphocyte activation culture by subculturing the lymphocytes in a serum-free medium at a cell density of 100000000 / ml, the subculturing temperature being 37.0°C±1.0°C and the activation culture environment containing 7.5%±1.0% CO2; (2) adding 5-10 times the volume of serum-free medium to the lymphocyte activation culture obtained by the subculturing in step (2) to perform amplification culture, the number of generations of the amplification culture being 1-5; and (3) centrifuging, washing, and collecting the activated and amplified lymphocytes to obtain a stock solution of activated and amplified lymphocytes. The activated and amplified lymphocytes produced by the present invention have advantages such as high cell expansion efficiency, high cell survival rate, stable cell activity, long effective time, safety and effectiveness, and small side effects, and effectively solve the important problems in tumor treatment technology using individualized immune cells.

Description

[Technical field]

[0001] The present invention relates to the field of biomedicine, specifically to a method for expanding activated lymphocytes with a stable and controlled quality and its use in anti-tumor. [Background technology]

[0002] Malignant tumors are one of the major diseases that threaten human health and life, and their incidence rate shows a trend of increasing year by year. According to the data of "World Cancer Statistics 2015", the number of new cases of cancer in the world reached about 14.1 million in 2015, and the number of deaths reached 8.2 million. In China, the number of new cases of cancer reached 4.29 million in 2015, and the number of deaths reached 2.81 million. Surgery, radiotherapy, and chemotherapy have become the three common means of treating tumors, but all of them have the disadvantage of insufficient specificity, and while removing tumor cells, they also cause different degrees of damage to normal tissues and organs. In the 1980s, Rosenberg SA et al. recognized that when animals suffering from non-immune tumors were treated with recombinant IL-2, the animal's lymphocytes could cause tumor regression and metastasis. Since then, immune cell therapy has rapidly developed in clinical application and has become the fourth means of comprehensive tumor treatment. In 1988, Rosenberg SA et al. summarized the combined treatment of 214 tumor patients with IL-2 cells and LAK cells, and found that metastatic lesions were completely eliminated in 16 patients, and tumors in 26 patients shrank by more than 50%. This therapy has shown some therapeutic effect on patients with metastatic renal cell carcinoma, melanoma, colon cancer, and non-Hodgkin's lymphoma. LAK cells have low killing power, and large amounts must be infused for clinical application. Since their amplification ability is limited, high doses of IL-2 (single dose 1 × 10) are used together with cell infusion. 5 IU / Kg or [1-6] x 10 6 IU / m 2) is required, but during treatment with high doses of IL-2, obvious toxic reactions such as severe ulcers and renal failure may occur that are intolerable to patients. This therapy has been used mainly for a small number of tumors, such as renal cancer and melanoma, and has gradually been abandoned.

[0003] In 1991, the Stanford University School of Medicine Bone Marrow Transplant Research Center reported that cells obtained by stimulating and inducing peripheral blood mononuclear cells using IFN-γ, IL-2, anti-CD3 monoclonal antibody, and IL-1 have a strong killing effect on lymphoma cells but have little effect on normal hematopoietic stem cells. The cells obtained in this way are called cytokine-induced killer (CIK) cells, and the effector cells that exert antitumor effects are mainly CD3+CD56+ NKT cells, which combine the antitumor activity of T lymphocytes with the MHC-unrestricted characteristics of NK cells. Studies have shown that the amplification efficiency, tumor cell killing ability, and in vivo activity of CIK cells are significantly higher than that of LAK cells. Clinical studies on CIK cell therapy for metastatic renal cancer, colon cancer, non-small cell lung cancer, and lymphoma have been reported.

[0004] So far, non-specific immune cell therapy has been used in combination with tumor treatments such as surgery, radiation therapy, chemotherapy, targeted therapy, and immune activator therapy. It has been clinically observed that many non-specific immune cell therapies have brought therapeutic benefits to patients, such as preventing tumor recurrence, extending patient survival time, synergizing with chemotherapy, and significantly improving patients' quality of life. This allows non-specific immune cells to have long-term vitality in tumor treatment, which has attracted widespread attention and led to their widespread use.

[0005] In 2000, Yamazaki T et al. reported in "The Lancet" the results of a randomized controlled clinical trial in which autologous lymphocytes were expanded and activated in an in vitro culture system to prevent tumor recurrence after liver cancer surgery. A total of 150 liver cancer patients who had undergone curative surgical resection were enrolled in this study, with the treatment group (76 cases) receiving cellular immunotherapy and the control group (74 cases) not receiving any adjuvant therapy. Patients in the treatment group received a total of 370 cell infusions over the first 6 months, and the average number of lymphocytes infused per patient was 7.1 × 10 10 , of which CD3+ cells accounted for 78%. A total of 62 adverse reactions (including fever, headache, nausea, dizziness, dyskinesia, and tachycardia) occurred among 45 patients, and all adverse reactions were self-limiting. No patients had pulmonary or renal symptoms, signs of infection, decreased liver function, or autoimmune disorders. After a mean follow-up of 4.4 years (range, 0.2-6.7 years), the treatment group was found to have a significantly reduced risk of liver cancer recurrence of 41% (95% confidence interval, 12%-60%, p=0.01) compared with the control group. In the treatment group, the time to first recurrence was significantly slower than in the control group. The recurrence-free rate within 3 years was estimated to be 48% (95% confidence interval, 37%-59%) in the treatment group and 33% (95% confidence interval, 22%-43%) in the control group. The 5-year recurrence-free rate was estimated to be 38% (95% confidence interval: 22%-54%) in the treatment group and 22% (95% confidence interval: 11%-34%) in the control group. Patients in the treatment group had significantly higher recurrence-free survival (p=0.01) and disease-specific survival (p=0.04). The results of the study show that this nonspecific immunotherapy significantly extends the postoperative recurrence-free survival time of liver cancer patients. Japan has been using this technology in clinical tumor treatment since 2000, and by 2010, approximately 10,000 patients were treated annually. In 2007, South Korea approved the method to enter phase III clinical trials for the treatment of liver cancer and glioblastoma, and in 2012, it approved the commercialization of the nonspecific immune cell product Immuncell-LC, which significantly reduced the postoperative recurrence rate of liver cancer, and a single reinjection dose of cells was 1×10 9 ~2×10 10 Limited to individuals.

[0006] The expanded activated lymphocyte system was produced from mononuclear cells in the patient's autologous peripheral blood by serum-free cell culture. The main component was expanded activated T lymphocytes (CD3+ cells), with CD8+ killer T lymphocytes as the main functional cells. The average number of CD8+ T cells reinfused into the patient per infusion was 4.70 ± 1.47 × 10 9 It has shown good tumor treatment activity in clinical practice. The prior art discloses a method for amplifying and activating lymphocytes by serum-free culture, but in order to improve the amplification rate, cell viability, and biological activity of lymphocytes produced by this method and meet the clinical needs for stable and controllable cell quality and safe and effective treatment, it is necessary to optimize the extraction and separation of peripheral blood mononuclear cells (PBMCs), the type and composition ratio of lymphocyte activators, serum-free medium, etc. Summary of the Invention

[0007] The object of the present invention is to provide a method for producing an activated and expanded lymphocyte stock solution, (1) A step of culturing and activating lymphocytes by co-culturing peripheral blood mononuclear cells (PBMCs) obtained by centrifuging autologous peripheral whole blood with a lymphocyte activator in a serum-free medium, wherein the initial density of the peripheral blood mononuclear cells in the serum-free medium for culture activation is (0.2 to 1.6) × 10 6 step (1), in which the concentration of lymphocytes in the culture medium is 100 / ml, the temperature of the cell activation culture is 37.0° C.±1.0° C., and the activation culture environment contains 7.5%±1.0% CO2, the lymphocyte activator is any one selected from anti-human CD2 antibody, anti-human CD3 antibody, anti-human CD28 antibody, and phytohemagglutinin (PHA), or a combination thereof, or an antibody-containing carrier having any one of these immobilized on a carrier, and the serum-free medium is any one selected from KBM 581 and GT-T551 H3, or a combination thereof; (2) The cultured activated lymphocytes produced in step (1) were diluted to (0.5–5) × 10 6(2) a step of producing a lymphocyte activation culture by subculturing the cells in a serum-free medium at a cell density of 1000000000 / ml, wherein the subculturing temperature is 37.0°C ± 1.0°C, the activation culture environment contains 7.5% ± 1.0% CO2, the serum-free medium is any one selected from KBM 581, GT-T551 H3, or a combination thereof, and the number of generations of subculturing is 1 to 5; (3) adding a serum-free medium having a volume 5 to 10 times that of the lymphocyte activation culture obtained by the subculture in step (2) to perform expansion culture, the temperature of the expansion culture is 37.0°C ± 1.0°C, the activation culture environment contains 7.5% ± 1.0% CO2, the serum-free medium is any one selected from KBM 581, GT-T551 H3, or a combination thereof, and the number of generations of the expansion culture is 1 to 5; and and (4) centrifuging, washing, and collecting the activated and expanded lymphocytes to obtain a stock solution of activated and expanded lymphocytes.

[0008] In a preferred technical solution of the present invention, the biological sample has an initial density of (0.2-1)×10 for culture activation in serum-free medium. 6 pieces / ml.

[0009] In a preferred technical solution of the present invention, the lymphocyte activator is an anti-human CD3 antibody, preferably 2.5 μg / ml-5 μg / ml and a volume of 8-15 ml, more preferably 2.5 μg / ml-3.8 μg / ml and a volume of 10-13 ml.

[0010] In a preferred technical solution of the present invention, the method for centrifugal extraction of peripheral blood mononuclear cells from whole blood includes the steps of adding a separation medium and a diluent to anticoagulated whole blood, stirring to homogenize the mixture, adding the mixture to a separation liquid, centrifuging at 1000-3000 rpm for 10-40 minutes, collecting the cell layer between the separation interface, adding a washing liquid, centrifuging to wash, and collecting the cells to obtain peripheral blood mononuclear cells, the separation medium is any one selected from hydroxyethyl starch 40 sodium chloride injection (HES), Percoll, Ficoll-Paque PLUS, or a combination thereof, the whole blood:diluent volume ratio is 1:1-2, the diluent is any one selected from sodium chloride injection, Hank's buffer, lactated Ringer's solution, Dulbecco's phosphate buffer, or a combination thereof, the separation liquid is hydroxyethyl starch 40 sodium chloride injection (HES), Ficoll, Lymphoprep, Lymphocyte Separation Media, Cell Separation Media, the osmotic pressure of the separation solution is 300 mOsmol / kg to 360 mOsmol / kg, and the washing solution is any one selected from 0.1% human albumin sodium chloride injection, Dulbecco's phosphate buffer, and sodium chloride injection, or a combination thereof.

[0011] In a preferred technical solution of the present invention, the centrifugation conditions are (1500-2500 rpm) x (15-30 min), preferably (2000-2500 rpm) x (20-25 min).

[0012] In a preferred technical solution of the present invention, the washing is centrifugal washing, the washing conditions are (500-2000 rpm) x (5-20 min), and the centrifugal washing is performed 1-5 times, preferably 2-3 times at (1000-1800 rpm) x (10-15 min).

[0013] In a preferred technical solution of the present invention, in step (2), the cell density of the subculture is (1-4) × 10 6The concentration of cells / ml is 7.00 to 7.80 after the addition of serum-free medium to the subculture system.

[0014] In a preferred technical solution of the present invention, in step (2), the cell density of the subculture is (2-3) × 10 6 The concentration of cells / ml is 7.02 to 7.76 after the addition of serum-free medium to the subculture system.

[0015] In a preferred technical solution of the present invention, the osmotic pressure of the separation liquid is 310-350 mOsmol / kg, preferably 316-347 mOsmol / kg.

[0016] In a preferred technical solution of the present invention, the number of generations of the subculture is 2 to 3.

[0017] In a preferred technical solution of the present invention, in step (3), the expansion culture is started when the cell density of the subculture increases to 1 to 10 times the culture activation cell density, and the lymphocyte culture obtained by the subculture in step (2) is added to a serum-free medium having a total volume of 6 to 8 times that of the lymphocyte culture, and the pH of the expansion culture system is 6.80 to 7.80.

[0018] In a preferred technical solution of the present invention, in step (3), the amplification culture is started when the cell density of the subculture increases to 1.2 to 3 times the culture activation cell density, and the pH of the amplification culture system is 6.88 to 7.70.

[0019] In a preferred technical solution of the present invention, the number of generations of the amplification culture is 2 to 3.

[0020] In a preferred technical solution of the present invention, the centrifugation conditions in step (4) are (1000-3000 rpm) x (1-10 min), preferably (1500-2800 rpm) x (2-8 min), and more preferably (2000-2500 rpm) x (5-6 min).

[0021] In a preferred technical solution of the present invention, the serum-free medium is optionally supplemented with the cytokine IL-2 at 300-600 IU / ml, preferably 400-500 IU / ml.

[0022] In a preferred technical solution of the present invention, the pH of the serum-free medium is 6.9-7.9, preferably 7.2-7.4.

[0023] In a preferred technical solution of the present invention, the culture device is any one selected from an incubator, a shaker, and a bioreactor.

[0024] In the preferred technical solution of the present invention, the amplification fold of the activated amplified lymphocytes is 900 times or more, preferably 1000 times or more, more preferably 1100 times or more.

[0025] In the preferred technical solution of the present invention, the survival rate of the activated expanded lymphocytes is more than 95%, preferably more than 98%.

[0026] In the preferred technical solution of the present invention, the number of CD8+ T cells in the activated expanded lymphocytes is 1×10 9 10 or more, preferably 1×10 9 ~2×10 10 pcs, more preferably 4 to 9.5 × 10 9 There are 10.

[0027] In a preferred technical solution of the present invention, the biological activity of lymphocytes after activation and amplification is 50 is 8.5 or less, preferably 4 or less, and more preferably 0.7141 or less.

[0028] Another object of the present invention is to provide a pharmaceutical composition containing a stock solution of activated and expanded lymphocytes, the pharmaceutical composition comprising (1 to 20) × 10 7 The kit consists of activated expanded lymphocytes at 100 / ml, 0.5-2% human albumin and injectable saline. The survival rate of the activated expanded lymphocytes is 95% or more, and the number of CD8+ T cells in the activated expanded lymphocytes is (1-20) x 107 / ml, and the pharmaceutical composition is free of preservatives and antibiotics.

[0029] In a preferred technical solution of the present invention, the pharmaceutical composition has a concentration of (2-18)×10 9 The combination of activated expanded lymphocytes (cells / ml), human albumin (concentration: 1-1.5%), and injectable saline solution is used. The survival rate of the activated expanded lymphocytes is 98% or higher, and the number of CD8+ T cells in the activated expanded lymphocytes is 1×10 9 ~2×10 10 There are 10 pieces.

[0030] In the preferred technical solution of the present invention, the cell amplification rate of the biological sample is 900 times or more, preferably 1000 times or more, more preferably 1100 times or more.

[0031] In the preferred technical solution of the present invention, the biological activity of activated amplified lymphocytes is 50 is 8.5 or less, preferably 4 or less, and more preferably 0.7141 or less.

[0032] In the preferred technical solution of the present invention, the total number of activated amplified lymphocytes reinjected into the patient at one time is 2×10 10 The number of cells is less than 100, and the cell viability during the valid period is 85% or more.

[0033] In a preferred technical solution of the present invention, the survival rate of the activated amplified lymphocytes is 85% or more when stored at 15 to 25°C for 12 hours.

[0034] Another object of the present invention is to provide a method for producing a pharmaceutical composition containing an activated and expanded lymphocyte stock solution, which comprises resuspending the activated and expanded lymphocyte stock solution in an injectable physiological saline solution containing human albumin.

[0035] Another object of the present invention is to provide a method for using the activated expanded lymphocytes of the present invention in immunotherapy, (1) obtaining a biological sample containing lymphocytes from an individual; (2) activating, passage and amplifying the lymphocytes using the production method of the present invention to obtain activated and amplified lymphocytes; (3) reinjecting the activated amplified lymphocytes or a pharmaceutical composition thereof into the individual.

[0036] Another object of the present invention is to provide an administration scheme for the use of the activated expanded lymphocytes of the present invention in immunotherapy, (1) In the first cycle, subjects will receive intravenous infusions once a week for 3-6 sessions. (2) In the second cycle, subjects will receive intravenous infusions once every two weeks for 3 to 6 sessions. (3) In the third cycle, subjects will receive intravenous infusions once every three weeks for three to six sessions. (4) In the fourth cycle, subjects will receive 7 to 10 intravenous infusions once every four weeks.

[0037] In a preferred technical solution of the present invention, (1) In the first cycle, subjects will receive intravenous infusions once a week for 4-5 sessions. (2) In the second cycle, subjects will receive intravenous infusions once every two weeks for 4-5 sessions. (3) In the third cycle, subjects will receive intravenous infusions once every three weeks for four to five sessions. (4) In the fourth cycle, subjects will receive 8 to 9 intravenous infusions once every 4 weeks.

[0038] In the preferred technical solution of the present invention, the number of activated amplified lymphocytes per intravenous infusion is 2×10 8 More than 10 lymphocytes, preferably (2-10) x 10 9 lymphocytes.

[0039] Another object of the present invention is to provide a use of the activated expanded lymphocytes of the present invention in the manufacture of an anti-tumor immunotherapeutic drug.

[0040] In a preferred technical solution of the present invention, the tumor is lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, renal cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancies, brain tumor, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine tumor, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer. , small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, and environmentally induced cancer, or a combination thereof.

[0041] In a preferred technical solution of the present invention, the lung cancer is any one selected from small cell lung cancer, non-small cell lung cancer, or a combination thereof.

[0042] In a preferred technical solution of the present invention, the leukemia is selected from acute lymphocytic leukemia, acute myeloid leukemia, B-cell chronic lymphocytic leukemia, chronic myeloid leukemia, or any combination thereof.

[0043] In a preferred technical solution of the present invention, the acute myeloid leukemia is selected from the following: acute myelocytic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute basophilic leukemia, acute eosinophilic leukemia, acute megakaryocytic leukemia, acute erythrocytic leukemia, acute panmyelopsis with myelofibrosis, or any combination thereof.

[0044] In a preferred technical solution of the present invention, the immunotherapy is selected from anti-tumor immunotherapy or post-tumor curative surgery immunotherapy, or a combination thereof.

[0045] In a preferred technical solution of the present invention, said immunotherapy is selected from primary hepatocellular carcinoma post-curative surgery immunotherapy.

[0046] In a preferred technical solution of the present invention, said drug is used in adult or pediatric patients.

[0047] Another object of the present invention is to provide a use of the activated expanded lymphocytes of the present invention in combination with any one of surgery, radiotherapy, and chemotherapy in anti-tumor or immunotherapy thereof.

[0048] Another object of the present invention is to provide a use of the activated expanded lymphocytes in the manufacture of an antiviral capacity enhancing drug.

[0049] Another object of the present invention is to provide a use of the activated expanded lymphocytes in the manufacture of an enhancing drug for the treatment of an autoimmune disease.

[0050] In a preferred technical solution of the present invention, the autoimmune disease is any one selected from systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, juvenile diabetes mellitus, essential thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, and skin diseases, or a complication thereof.

[0051] Unless otherwise specified, the present invention uses the biological activity of expanded activated lymphocytes (RTCA, e.g., effector-target ratio is 40:1) to represent therapeutic activity by activated expanded lymphocytes. For digested target cells (e.g., HepG2, etc.), the density is 2 × 10 5 Target cells were loaded at 100 μl per well at 100 cells / ml, and the effector-target ratio parameters (e.g., effector-target ratio of 40:1) were set to perform baseline measurements. After 16–24 h of loading detection, the target cells were loaded at a specific density according to the effector-target ratio (density of 1.6 × 10 in 50 μl per well). 7After adding effector cells (i.e., amplified activated lymphocytes) at 1000μg / ml, data such as cell morphology and proliferation / differentiation, i.e., changes in electrical impedance due to contact between live cells and the microelectrodes in the detection plate, were tracked in real time, dynamically, and quantitatively. When effector cells were added to wells containing adherent cells, the cells that adhered and grew on the surface of the microelectrodes caused a change in adhesion electrode interface impedance, which positively correlated with the change in cell index. The instrument's dedicated software converted the resistance signal into a specific cell index, and the generated cell index curve was monitored in real time to indicate the state of cell adhesion, spreading, growth, death, etc. at different times. By analyzing the changes in the cell index, the killing efficiency of effector cells against target cells (e.g., HepG2 cells) could be calculated, and the killing efficiency curve was used to calculate the KT. 50 (i.e., the time it takes for effector cells to kill 50% of target cells) was calculated and the KT 50 The KT value is used as an index to evaluate the biological activity of effector cells (i.e., activated and amplified lymphocytes). 50 A smaller value indicates a higher killing efficiency and a higher biological activity, whereas a smaller value indicates a lower biological activity.

[0052] Unless otherwise stated, in the present invention, when it concerns percentages between liquids and liquids, said percentages are volume / volume percentages, when it concerns percentages between liquids and solids, said percentages are volume / weight percentages, when it concerns percentages between solids and liquids, said percentages are weight / volume percentages and the rest are weight / weight percentages. Effect of the Invention

[0053] Compared with the prior art, the present invention has the following beneficial technical effects:

[0054] 1. The activated amplified lymphocytes prepared by the present invention have the advantages of high cell amplification efficiency, high cell viability, stable cell activity, long effective time, safety and effectiveness, and small side effects, effectively solving the important problems of tumor treatment technology using individualized immune cells. First, a serum-free culture system with stable cell culture efficiency is used to continuously culture for 12 days, maintaining the killing activity and amplification ability of cells, and avoiding pathogenic microbial contamination and other unfavorable factors for cell growth caused by the use of serum. Second, the cell amplification efficiency is high, and there is no need to use a mononuclear cell recovery machine, and the cells can be efficiently amplified by more than 1000 times. Conventional immune cell therapy requires the use of a mononuclear cell recovery machine to recover a large amount of starting peripheral blood mononuclear cells, which avoids the destructive effect on the patient's entire immune cell system. Third, the cells are highly active and stable, and the therapeutic effect is not affected even if they are stored for 12 hours. Fourth, a standardized quality control system for personalized products will be established, solving the technical challenges of large-scale production and achieving the technology to obtain cell products with relatively uniform quality, thereby significantly improving the targeting of treatment, treatment accuracy, and patient compliance.

[0055] 2. When the activated amplified lymphocytes of the present invention are used in the treatment of tumor patients, they can prevent post-surgery recurrence of tumors, prolong the post-surgery recurrence period, significantly prolong the survival time of tumor patients, and significantly improve the quality of life of patients; and during the treatment process, they can reduce the amount of chemotherapy drugs used, the dose of radiation therapy, and adverse reactions such as nausea, vomiting, hair loss, and insomnia caused by chemotherapy and radiation therapy, eliminate serious adverse reactions such as deterioration of blood test results and damage to liver and kidney function, relieve pain caused by tumors, and greatly improve the quality of life of patients.

[0056] 3. The lymphocyte amplification method of the present invention has the advantages of being easy to operate, having stable and controllable quality, and being suitable for industrial production. [Brief description of the drawings]

[0057] [Figure 1] 1 shows the growth state of EAL cells in the first subculture in Example 1 (observed under a microscope at 250x magnification). [Diagram 2] 1 shows the growth state of EAL cells in the first amplification culture in Example 1 (observed under a microscope at 250x magnification). [Diagram 3] 1 shows the growth state of EAL cells in the second amplification culture in Example 1 (observed under a microscope at 250x magnification). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] The present invention will be further described below with reference to examples, however, the present invention is not limited to the scope of the described examples.

[0059] Example 1: Preparation of lymphocyte activation and amplification stock solution (1) Isolation, inoculation, and activation culture of peripheral blood mononuclear cells (PBMCs) (Day 0) Patient 1: Liver cancer patient, Age: 55 years old, Gender: Male The serum-free cell culture medium: GT-T551 H3 serum-free cell culture medium (IL-2 concentration in the medium is 500 IU / ml, pH is 7.2 to 7.4) 37ml of whole blood from the patient was placed in a new 250ml centrifuge tube, and the same volume of 0.9% sodium chloride injection as the blood sample was added and mixed uniformly. Then, it was gently added to a 50ml centrifuge tube (two centrifuge tubes) containing 15ml of Ficoll to clarify the interface, and centrifuged at 2000rpm for 20min, with the acceleration of the centrifuge set to 7 and the deceleration to 0. The cell layer between the interface was aspirated into a new 50ml centrifuge tube, mixed uniformly with 0.9% sodium chloride injection at a ratio of 1:1, and centrifuged at 1200rpm for 10min. After centrifugation was completed, the supernatant was discarded, the cell precipitate was shaken to disperse, and then resuspended in 50ml of 0.9% sodium chloride injection and centrifuged at 1200rpm for 10min. After centrifugation was completed, the supernatant was discarded, the cell precipitate was shaken to disperse, and then 5ml of serum-free cell culture medium was added and mixed uniformly. After that, 10μl of the cell suspension was taken and the cells were counted. Cells were cultured in serum-free cell culture medium at 0.8 × 10 6 The cells were resuspended to a concentration of 100 / ml and then transferred to a 225cm plate coated with anti-human CD3 antibody. 2Cell culture flasks were inoculated, samples were taken, sterility checked and marked, and then placed in a cell incubator for incubation. During the preparation of lymphocyte activation and amplification stock solution, the culture conditions in the incubator are 37° C. and 7.5% CO 2 .

[0060] (2) First subculture (day 3) When the color of the medium became light yellow (3 days after inoculation culture, assuming the day of blood collection as day 0), the cell culture flask was removed from the incubator. The morphology of the cells was observed under a microscope, and the growth condition was good (see Figure 1). 50 ml of serum-free cell culture medium was added to the cell culture flask, and the cell culture flask was returned to the cell incubator for culture.

[0061] (3) Second subculture (day 4) When the color of the medium became light yellow, the cell culture flask was removed from the incubator. The morphology of the cells was observed under a microscope, and the growth condition was good. 140 ml of serum-free cell culture medium was added, and the flask was returned to the cell incubator for culture.

[0062] (4) First amplification culture (day 5) The cell culture flask was removed from the incubator and the cell morphology was observed under a microscope, which indicated good growth conditions (see Figure 2). A sample was taken and the cells were counted, and the viable cell density was 1.0 × 10 6 When the number of cells / ml or more was reached, 760 ml of the cell culture solution and serum-free cell culture medium were poured into the cell culture bag, and the cell culture bag was returned to the cell incubator for culture.

[0063] (5) Second amplification culture (day 8) When the color of the culture medium became yellowish, a new cell culture bag was placed in a biological safety cabinet, 1000 ml of serum-free cell medium (bag A) was poured in by a syringe sleeve, and the opening of the tube was sealed. The cell culture bag (bag B) containing the cells being cultured was taken out of the incubator, and the two bags were connected by a sterile connection device. First, all the liquid in bag A was poured into bag B, and after uniform mixing, 1000 ml was returned to bag A, so that the amount of liquid in both bags was 1000 ml. The opening of the tube was sealed, and a sample was taken for sterility testing. The morphology of the cells was observed under a microscope (see Figure 3), and the two bags containing the cells were returned to the incubator for culture.

[0064] (6) Cell purification, centrifugation, concentration, collection, and preparation of cell stock solution (Day 12) On the fourth day after the two cell culture bags, one cell culture bag was taken out of the incubator and placed in a biological safety cabinet, the cell suspension was divided equally into four 250ml centrifuge tubes, and centrifuged at 2000rpm for 5 minutes. The supernatant was discarded, and the precipitate was dispersed by shaking. Then, another cell culture bag of the same batch was taken out of the incubator and placed in a biological safety cabinet, the cell suspension was divided equally into the original four 250ml centrifuge tubes, and centrifuged at 2000rpm for 5 minutes. The supernatant was discarded, the precipitate was dispersed by shaking, and the cells in one tube were rinsed three times with 250ml of 0.9% sodium chloride injection containing 0.1% human albumin, and they were combined into another tube, and the four tubes were combined into two tubes, and centrifuged at 2000rpm for 5 minutes. The supernatant was discarded, the precipitate was shaken to disperse, and the cells in the two tubes were rinsed three times with 200 ml of 0.1% human albumin sodium chloride injection, combined into one tube, and mixed uniformly. Then, a sample was taken to count the number of cells, centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, and the activated amplified lymphocyte stock solution was obtained, with 2.30 × 10 10 Activated amplified lymphocytes were obtained.

[0065] (7) Preparation of the composition The concentration of activated amplified lymphocytes is 8×107 The cells were obtained by resuspending the cell stock solution in physiological saline for injection containing 1% human albumin to a concentration of 10 cells / ml.

[0066] Example 2: Preparation of lymphocyte activation and amplification stock solution (1) Isolation, inoculation, and activation culture of PBMCs (Day 0) Patient 2: Breast cancer patient, Age: 50, Gender: Female The serum-free cell culture medium: KBM 581 serum-free cell culture medium (IL-2 concentration in the medium is 500 IU / ml, pH is 7.2 to 7.4) 63 ml of whole blood was placed in a new 250 ml centrifuge tube, and the same volume of sodium chloride injection as the blood sample was added, and after uniform mixing, it was gently added to a 50 ml centrifuge tube (four centrifuge tubes) into which 15 ml of Ficoll had been dispensed, to clarify the interface, and centrifuged at 2000 rpm for 20 min, with the centrifuge acceleration set to 7 and deceleration set to 0. The cell layer between the interface was aspirated into a new 50 ml centrifuge tube, uniformly mixed with 0.9% sodium chloride injection at 1:1, and centrifuged at 1200 rpm for 10 min. After centrifugation was completed, the supernatant was discarded, the cell precipitate was shaken to disperse, and then resuspended in 50 ml of sodium chloride injection, and centrifuged at 1200 rpm for 10 min. After centrifugation was completed, the supernatant was discarded, the cell precipitate was shaken to disperse, and then 5 ml of serum-free cell medium was added, and after uniform mixing, 10 μl of the cell suspension was taken and the cells were counted. The cells were then counted in serum-free cell medium at a concentration of 0.2 × 10 6 The cells were resuspended to a concentration of 100 / ml and then transferred to a 225cm plate coated with anti-human CD3 antibody. 2 Cell culture flasks were inoculated, samples were taken, sterility checked and marked, and then placed in a cell incubator for incubation. During the preparation of lymphocyte activation and amplification stock solution, the culture conditions in the incubator are 37° C. and 7.5% CO 2 .

[0067] (2) First subculture (day 4) When the color of the medium became light yellow (4 days after inoculation culture, assuming the day of blood collection as day 0), the cell culture flask was removed from the incubator. The morphology of the cells was observed under a microscope, and the growth condition was good. The cells were counted, and 50 ml of serum-free cell culture medium was added to the cell culture flask, and the cell culture flask was returned to the cell incubator for culture.

[0068] (3) Second subculture (day 5) When the medium color became light yellow, the cell culture flask was removed from the incubator. The morphology of the cells was observed under a microscope and the growth condition was good. The cells were counted, and 140 ml of serum-free cell culture medium was added to the cell culture flask, and the cell culture flask was returned to the cell incubator for culture.

[0069] (4) First amplification culture (day 6) The cell culture flask was removed from the incubator and the cell morphology was observed under a microscope, which indicated good growth. A sample was taken and the cells were counted, and the viable cell density was 1×10 6 When the number of cells / ml or more was reached, 750 ml of the cell culture solution and serum-free cell medium were poured into the cell culture bag, and the cell culture bag was returned to the cell incubator for culture.

[0070] (5) Second amplification culture (day 8) When the color of the culture medium turned yellowish, a new cell culture bag was placed in a biological safety cabinet, 1000 ml of serum-free cell culture medium (bag A) was poured in by a syringe sleeve, and the opening of the tube was sealed. The cell culture bag (bag B) containing the cells being cultured was taken out of the incubator, and the two bags were connected by a sterile connection device. First, all the liquid in bag A was poured into bag B, and after uniform mixing, 1000 ml was returned to bag A, so that the amount of liquid in both bags was 1000 ml. The opening of the tube was sealed, and a sample was taken for sterility testing. The morphology of the cells was observed under a microscope, and the two bags containing the cells were returned to the incubator for culture.

[0071] (6) Cell purification, centrifugation, concentration, collection, and preparation of cell stock solution (Day 12) On the fourth day, of the two cell culture bags, one cell culture bag was removed from the incubator and placed in a biological safety cabinet, and the cell suspension was divided equally into four 250 ml centrifuge tubes and centrifuged at 2000 rpm for 5 minutes. The supernatant was discarded and the precipitate was dispersed by shaking. Then, another cell culture bag of the same batch was removed from the incubator and placed in a biological safety cabinet, and the cell suspension was divided equally into the original four 250 ml centrifuge tubes and centrifuged at 2000 rpm for 5 minutes. The supernatant was discarded and the precipitate was dispersed by shaking. The cells in one tube were rinsed three times with 250 ml of 0.9% sodium chloride injection, and they were combined into another tube, and the four tubes were combined into two tubes and centrifuged at 2000 rpm for 5 minutes. The supernatant was discarded, the precipitate was shaken to disperse, and the cells in the two tubes were rinsed three times with 200 ml of sodium chloride injection, combined into one tube, and mixed uniformly. Then, a sample was taken to count the number of cells, centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, and the activated amplified lymphocyte stock solution was obtained, with a cell count of 2.84 × 10 10 Activated amplified lymphocytes were obtained.

[0072] (7) Preparation of the composition The solution of activated amplified lymphocytes was 6 × 10 7 The cells were obtained by resuspending the cell stock solution in physiological saline for injection containing 1.5% human albumin to a concentration of 1.5 cells / ml.

[0073] Example 3: Preparation of lymphocyte activation and amplification stock solution (1) Isolation, inoculation, and activation culture of PBMCs (Day 0) Patient 3: Non-small cell lung cancer patient, age: 53 years old, gender: male The serum-free cell culture medium: KBM 581 (CORNING) serum-free cell culture medium (IL-2 concentration in the medium is 400 IU / ml, pH is 7.2 to 7.4) 58 ml of the patient's whole blood was placed in a new 250 ml centrifuge tube, and the same volume of 0.9% sodium chloride injection as the blood sample was added and mixed uniformly. Then, it was gently added to a 50 ml centrifuge tube (four centrifuge tubes) containing 15 ml of Ficoll to clarify the interface, and centrifuged at 2000 rpm for 20 min, with the acceleration of the centrifuge set to 7 and the deceleration to 0. The cell layer between the interface was aspirated into a new 50 ml centrifuge tube, mixed uniformly with 0.9% sodium chloride injection at a ratio of 1:1, and centrifuged at 1200 rpm for 10 min. After centrifugation was completed, the supernatant was discarded, the cell precipitate was shaken to disperse, and then the precipitate was heavily suspended in 50 ml of sodium chloride injection and centrifuged at 1200 rpm for 10 min. After centrifugation was completed, the supernatant was discarded, the cell precipitate was shaken to disperse, 5 ml of serum-free cell culture medium was added, and after uniform mixing, 10 μl of the cell suspension was taken and the cells were counted. The cells were then cultured in serum-free cell culture medium at a ratio of 0.3 × 10 6 The cells were resuspended to a concentration of 100 / ml and then transferred to a 225cm plate coated with anti-human CD3 antibody. 2 Cell culture flasks were inoculated, samples were taken, sterility checked and marked, and then placed in a cell incubator for incubation. During the preparation of lymphocyte activation and amplification stock solution, the culture conditions in the incubator are 37° C. and 5% CO 2 .

[0074] (2) First subculture (day 3) When the color of the medium became light yellow (3 days after inoculation culture, assuming the day of blood collection as day 0), the cell culture flask was removed from the incubator. The morphology of the cells was observed under a microscope, and the growth condition was good. The cells were counted, and 50 ml of serum-free cell culture medium was added to the cell culture flask, and the cell culture flask was returned to the cell incubator for culture.

[0075] (3) Second subculture (day 4) When the medium color became light yellow, the cell culture flask was removed from the incubator. The morphology of the cells was observed under a microscope and the growth condition was good. The cells were counted, and 140 ml of serum-free cell culture medium was added to the cell culture flask, and the cell culture flask was returned to the cell incubator for culture.

[0076] (4) First amplification culture (day 6) The cell culture flask was removed from the incubator and the cell morphology was observed under a microscope, which indicated good growth. A sample was taken and the cells were counted, and the viable cell density was 1×10 6 When the number of cells / ml or more was reached, 750 ml of the cell culture solution and serum-free cell medium were poured into the cell culture bag, and the cell culture bag was returned to the cell incubator for culture.

[0077] (5) Second amplification culture (day 8) When the color of the culture medium turned yellowish, a new cell culture bag was placed in a biological safety cabinet, 1000 ml of serum-free cell culture medium (bag A) was poured in by a syringe sleeve, and the opening of the tube was sealed. The cell culture bag (bag B) containing the cells being cultured was taken out of the incubator, and the two bags were connected by a sterile connection device. First, all the liquid in bag A was poured into bag B, and after uniform mixing, 1000 ml was returned to bag A, so that the amount of liquid in both bags was 1000 ml. The opening of the tube was sealed, and a sample was taken for sterility testing. After observing the cell morphology under a microscope, the two bags containing the cells were returned to the incubator for culture.

[0078] (6) Cell purification, centrifugation, concentration, collection, and preparation of cell stock solution (Day 12) On the fourth day, one of the two cell culture bags was removed from the incubator and placed in a biological safety cabinet, and the cell suspension was divided equally into four 250 ml centrifuge tubes and centrifuged at 2000 rpm for 5 minutes. The supernatant was discarded and the precipitate was dispersed by shaking. Then, another cell culture bag of the same batch was removed from the incubator and placed in a biological safety cabinet, and the cell suspension was divided equally into the original four 250 ml centrifuge tubes and centrifuged at 2000 rpm for 5 minutes. The supernatant was discarded and the precipitate was dispersed by shaking, and the cells in one tube were rinsed three times with 250 ml of 0.9% sodium chloride injection, and they were combined into another tube, and the four tubes were combined into two tubes and centrifuged at 2000 rpm for 5 minutes. The supernatant was discarded, the precipitate was shaken to disperse, and the cells in the two tubes were rinsed three times with 200 ml of sodium chloride injection, combined into one tube, and mixed uniformly. Then, a sample was taken to count the number of cells, centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, and the activated amplified lymphocyte stock solution was obtained, containing 2.23 × 10 10 Activated amplified lymphocytes were obtained.

[0079] (7) Preparation of the composition The concentration of activated amplified lymphocytes is 8×10 7 The cells were obtained by resuspending the cell stock solution in physiological saline for injection containing 1% human albumin to a concentration of 10 cells / ml. Example 4 Screening of serum-free media

[0080] Using the same activation amplification process as in Example 1, the effects of three serum-free media, KBM 581 (CORNING), GT-T551 H3 (TAKARA), and TexMACS GMP Medium (Miltentyi biotec), on the production of EAL cells were compared and studied in vitro.

[0081] Peripheral blood from three healthy subjects was collected to separate PBMCs, which were then subjected to cell counting and phenotypic detection. Each PBMC was divided into three equal parts and inoculated into cell culture flasks coated with activating antibodies using three types of serum-free media. Cells were counted every 4 to 6 days, and the corresponding cell media were replenished. After 12 days, the culture was terminated and cell counting, viability, phenotypic detection, and killing activity detection were performed. The cells were cultured using serum-free medium TexMACS GMP Medium. The cells expanded slowly after 6-8 days of culture, but could not meet the quality requirements for in vitro lymphocyte expansion.

[0082] [Table 1]

[0083] [Table 2]

[0084] [Table 3]

[0085] Example 5: Effect of expanding activated lymphocytes in patients (1) Peripheral blood was collected from a total of 50 patients with different genders, ages, and tumor types. Basic information on the patients is shown in Tables 4 to 6.

[0086] [Table 4]

[0087] [Table 5]

[0088] [Table 6]

[0089] For the preparation of lymphocyte expansion activation stock solution, see Example 1.

[0090] The cells were counted using a cell counting plate to measure the percentage of expansion. The viability of expanded cells was obtained by trypan blue staining and cell counting. The phenotype of expanded cells was detected by flow cytometry using FITC-labeled CD3 antibody and APC-labeled CD8 antibody.

[0091] [Table 7] TIFF2024545912000009.tif219164

[0092] (2) Using the above amplified cells, 787 clinical killing tests were performed to confirm the biological activity of the cells. 50 KT of less than 1 h was detected, i.e., the time to reach 50% killing; the shorter this time, the better the biological effectiveness. 50 The frequency is 2.29%, 1~2h KT 50 The frequency of the KT is 34.81%, 2~3h 50 The frequency of the KT is 31.77%, 3~4h 50 The frequency of KT is 18.3%, 4~5h 50 The frequency is 8.39%, 5~6h KT 50 The frequency is 2.92%, KT of 6~8h 50 The frequency of this incident was 1.52%.

[0093] Test Example 1: Study on activated and amplified lymphocytes according to the present invention for use in immunotherapy The selection criteria for the EAL therapy group were as follows: Patients were histologically or cytologically diagnosed with gastric cancer, lung cancer, etc., had a life expectancy of more than 12 weeks, and had an Eastern Cooperative Oncology Group (ECOG) performance status (PS) score of 0 to 2. Patients with an ECOG score of more than 2 were excluded. All patients agreed to the treatment regimen and signed an informed consent form.

[0094] EAL therapy group: 50 cases, patient age, sex, and disease information are shown in Example 5. Autologous amplified activated lymphocyte preparation was infused. Treatment regimen: In the first treatment course, subjects received intravenous infusion once a week for a total of four times. In the second treatment course, subjects received intravenous infusion once every two weeks for a total of four times. In the third treatment course, subjects received intravenous infusion once every three weeks for a total of four times. In the fourth treatment course, subjects received intravenous infusion once every four weeks for a total of eight times.

[0095] Control group: 20 cancer patients (5 each for gastric cancer, small cell lung cancer, liver cancer, and leukemia) who were hospitalized for treatment during the same period and received chemotherapy or radiotherapy with conventional treatment regimens. Patients with a history of cell therapy and ECOG>2 were excluded. Chemotherapy regimens of study subjects undergoing treatment included CE (etoposide + carboplatin), EP (etoposide + cisplatin), IP (irinotecan + cisplatin), PP (paclitaxel + cisplatin / nedaplatin), DP (docetaxel + cisplatin), CAV (cyclophosphamide + famomycin + vincristine), NP (nelzarabine + cisplatin), pemetrexed alone, docetaxel alone, temozolomide capsule alone, and etoposide capsule alone. Patients with incomplete treatment history or patients who were lost to follow-up were not included in the statistics. The efficacy and safety of immune cell therapy were analyzed.

[0096] (1) Evaluation of treatment efficacy The median OS time of the EEAL therapy group was significantly longer than that of the control group. EAL immunotherapy can extend the survival time of cancer patients by 1 to 7 years, improve side effects and adverse reactions such as nausea, vomiting, hair loss, diarrhea, loss of appetite, fever, chills, headache, itch, rash, tachycardia, and other adverse reactions in patients undergoing chemotherapy, and eliminate serious adverse reactions such as deterioration of blood picture results and damage to liver and kidney function, greatly improving the quality of life of patients.

[0097] (2) Safety evaluation EAL-treated group: Only self-limiting adverse events of grade 1 or 2 occurred in patients treated with EAL (see Table 8). No patients developed pulmonary or renal symptoms, infectious symptoms, deterioration of liver function, or autoimmune diseases. No treatment-related deaths were recorded.

[0098] Control group: Severe adverse reactions occurred, including deterioration of blood picture results and damage to liver and kidney function, as well as more severe adverse reactions such as nausea, vomiting, diarrhea, and hair loss.

[0099] [Table 8]

[0100] Test Example 2: Study on combined use with activated amplified lymphocytes of the present invention 1. Patient Selection A clinical treatment study was conducted on 100 children with low- or intermediate-risk acute myeloid leukemia (AML) and low levels of minimal residual disease (MRD).

[0101] Low-level MRD was defined as a percentage of leukemic blasts <0.5% per bone marrow aspirate. Patients with high-risk AML, acute promyelocytic leukemia, disease relapse, low-risk disease who remained MRD-negative after induction chemotherapy, and patients with MRD >0.5% after 6 months of treatment were excluded. The 100 patients enrolled in this study were divided into two groups according to their parents' preference and financial situation. As shown in Table 9, the combination treatment group (n=50) received chemotherapy and EAL therapy, and the chemotherapy group (n=50) received chemotherapy alone. The parents or legal guardians of each patient provided written informed consent for chemotherapy and EAL therapy.

[0102] 2. Treatment regimen (1) Chemotherapy regimen Induction chemotherapy consisted of cytarabine 100-150mg / m 2 for 7 days, and 2 for 2 days) and etoposide 100-150 mg / m2 After two cycles of induction, consolidation chemotherapy was started. The consolidation chemotherapy consisted of three regimens: Regimen 1: High-dose cytarabine (Ara-c 3 g / m 2 , for 3 days) and anthracycline (idarubicin 8–10 mg / m 2 Regimen 2: Harringtonine 4-6 mg / m 2 cytarabine 100-150mg / m for 7 days 2 Regimen 3: Cytarabine 100-150 mg / m 2 for 7 days, and 2 for 2 days) and etoposide 100-150 mg / m 2 and combined for 3 days. The three regimens were alternated for a total of 12 to 18 months. During each cycle of low-dose Ara-c consolidation chemotherapy, intrathecal triple chemotherapy (methotrexate, cytarabine, and hydrocortisone) was administered for a total of 4 to 8 times to prevent central nervous system leukemia.

[0103] (2)EAL therapy EAL therapy was administered to low-risk and intermediate-risk AML patients with persistently positive MRD status (<0.5%) and those whose MRD status became positive or whose MRD burden increased by one copy. In this study, patients in the combination treatment group received a combination of EAL therapy and chemotherapy. EAL was performed by drawing 10-80 mL of peripheral blood from each patient (determined according to the absolute number of peripheral blood lymphocytes). The PBMC suspension was then placed in a flask coated with immobilized anti-CD3 antibody and subjected to activation, passage, and amplification culture according to the manufacturing method of Example 1. The lymphocytes were then collected, filtered through a membrane with a pore size of 100 μm, and resuspended in 100 mL of saline containing 1% human serum albumin for intravenous infusion to individual patients. The final product was divided into 1-3 infusion bags depending on the total number of cells after culture, with each bag containing 1 × 10 9 ~2×10 10 CD3+CD8+ T cells were included.

[0104] EAL was stored in a thermostat at 15–25°C and transfused to patients within 12 hours to ensure that cell viability was always 90% or higher. Each transfusion contained 3 × 10 9 The study protocol was approved by the Institutional Review Board. Informed consent was obtained from the parents or legal guardians of all patients.

[0105] 3. Monitoring and follow-up Monitoring measures: MRD in core-binding factor AML (CBF AML) was monitored by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) assay. For non-CBF AML patients, MRD was detected by flow cytometry according to leukemia-associated immunophenotype (LAIP).

[0106] Monitoring and follow-up times: Bone marrow samples were collected at the time of diagnosis, before each cycle of chemotherapy, and then every 3 months to 3 years, and every 6 months to 5 years.

[0107] 4.Results Among the 100 children enrolled in the study, no significant differences were observed between groups in terms of sex, age, genetic characteristics, risk stratification, MRD status 8 months after chemotherapy, or chemotherapy intensity.

[0108] The combined treatment group included 26 men and 24 women, with a median age of 10.5 years (range 1-15 years). Of these, 42 cases (84.00%) were in the low-risk group and 8 cases were in the intermediate-risk group.

[0109] The mean white blood cell count at onset was 27.9 × 10 9 (2.1~100×10 9 The combined treatment group received an average of four EAL treatments (range 1-12). In this study, EAL therapy was initiated 6-12 months (mean 8 months) after chemotherapy initiation. Two patients had central nervous system leukemia and one patient had a large mandibular mass. The combined treatment group received an average of four EAL treatments (range 1-12). In this study, EAL therapy was initiated 6-12 months (mean 8 months) after chemotherapy initiation.

[0110] The 50 children in the combination therapy group received a total of 220 EAL transfusions (mean 4 transfusions per patient). Analysis of cell phenotype and cell count from these 220 EAL transfusions revealed a mean cell count of 7.5 ± 0.5 × 10 9 / L, and mean cell viability was 98.9%. T cell phenotype distribution was CD3+ 98.95% and CD3+CD8+ 87.26%.

[0111] The control group included 25 males and 25 females, with a median age of 10.6 years (range 1-15 years). Of these, 42 cases (84.00%) were in the low-risk group and 8 cases were in the intermediate-risk group. At the time of presentation, one patient had central nervous system leukemia and one patient had extensive thoracic and lumbar spine involvement.

[0112] [Table 9]

[0113] (2) MRD negativity rate in both groups during consolidation therapy EAL therapy was initiated when pre-EAL MRD was <0.5% and the mean MRD burden was 0.055%. After EAL and chemotherapy, 49 of 50 patients (98.00%) had a negative MRD status, while one patient's MRD status remained positive after combination therapy. In the combination group, the mean time that MRD status remained negative during consolidation chemotherapy was 7.9 months.

[0114] In the control group, 30 patients (60.00%) achieved negative MRD status. During consolidation chemotherapy, MRD status remained negative for a mean of 4.8 months.

[0115] 98.00% of patients in the combination therapy group achieved negative MRD status during consolidation chemotherapy, which was significantly higher than that of patients in the control group. Furthermore, the duration of time that the MRD status remained negative in the combination therapy group was 7.9 months, which was significantly longer than that of the chemotherapy group.

[0116] (3) Comparison of survival rates between the two groups Median EFS was 81 months in the combination treatment group and 50 months in the control group.

[0117] The mean 5-year EFS rate was 88.9% in the combination treatment group and 69.7% in the control group.

[0118] In the combination treatment group, four patients (8.00%) experienced disease recurrence: three patients developed bone marrow relapse and one patient developed bone marrow relapse after CNS relapse.

[0119] In the control group, 12 patients (24.0%) had disease recurrence, all of which were bone marrow relapses. The median time to recurrence was 15.3 months (range 6-34 months). In the combination therapy group, the median time to recurrence was 35.8 months, which was significantly longer than in the chemotherapy group.

[0120] (4) Adverse reactions Although occasional fevers and chills occurred in the combination treatment group, no severe inflammatory cytokine cascades causing organ damage were observed during treatment, and no cell therapy-related deaths were observed.

[0121] The above description of the specific embodiments of the present invention does not limit the present invention, and those skilled in the art may make various modifications or variations based on the present invention, and all of them fall within the scope of protection of the claims of the present invention as long as they do not deviate from the spirit of the present invention.

Claims

1. A method for producing an activated and expanded lymphocyte stock solution, comprising the steps of: (1) A step of culturing and activating lymphocytes by co-culturing peripheral blood mononuclear cells (PBMCs) obtained by centrifuging autologous peripheral whole blood with a lymphocyte activator in a serum-free medium, wherein the initial density of the peripheral blood mononuclear cells in the serum-free medium for the culture activation is (0.2 to 1.6) x 10 6 The temperature of the cell activation culture is 37.0° C.±1.0° C., and the activation culture environment is CO 2 a step (1) in which the serum-free medium contains 7.5%±1.0% of the above, the lymphocyte activator being any one selected from anti-human CD2 antibody, anti-human CD3 antibody, anti-human CD28 antibody, and phytohemagglutinin (PHA), or a combination thereof, or an antibody-containing carrier having any one of these immobilized on a carrier, and the serum-free medium being any one selected from KBM 581 and GT-T551 H3, or a combination thereof; (2) The cultured activated lymphocytes produced in step (1) are diluted with (0.5-5) x 10 6 and subculturing the cells in a serum-free medium at a cell density of 1000 / ml to produce a lymphocyte activation culture, the subculturing temperature is 37.0° C.±1.0° C., and CO is added to the activation culture environment. 2 (2) containing 7.5%±1.0% of α-aminobutyric acid; wherein the serum-free medium is any one selected from KBM 581, GT-T551 H3, or a combination thereof, and the number of generations of subculture is 1 to 5; (3) A step of adding a serum-free medium in an amount 5 to 10 times the volume of the lymphocyte activation culture obtained by the subculture in step (2) to perform expansion culture, the temperature of the expansion culture being 37.0° C.±1.0° C., and the activation culture environment being free of CO 2 Step (3), wherein the serum-free medium is any one selected from KBM 581, GT-T551 H3, or a combination thereof, and the number of generations of the amplification culture is 1 to 5; and (4) centrifuging, washing, and collecting the activated and amplified lymphocytes to obtain an activated and amplified lymphocyte stock solution.

2. The method for centrifugal extraction of peripheral blood mononuclear cells from whole blood is as follows: The method includes the steps of adding a separation medium and a diluent to the anticoagulated whole blood, stirring the mixture to obtain a homogeneous mixture, adding the mixture to a separation solution, centrifuging the mixture at 1000-3000 rpm for 10-40 minutes, collecting the cell layer between the separation interface, adding a washing solution, washing the cells by centrifugation, and collecting the cells to obtain peripheral blood mononuclear cells; The separation medium is any one selected from hydroxyethyl starch 40 sodium chloride injection (HES), Percoll, Ficoll-Paque PLUS, or a combination thereof, the volume ratio of whole blood to diluent is 1:1 to 2, the diluent is any one selected from sodium chloride injection, Hank's buffer, lactated Ringer's solution, Dulbecco's phosphate buffer, or a combination thereof, and the separation solution is hydroxyethyl starch 40 sodium chloride injection (HES), Ficoll, Lymphoprep, Lymphocyte Separation Media, Cell ... The method according to claim 1, characterized in that the separation solution is any one selected from the group consisting of 0.1% human albumin sodium chloride injection, Dulbecco's phosphate buffer, and sodium chloride injection, or a combination thereof, and the osmotic pressure of the separation solution is 300 mOsmol / kg to 360 mOsmol / kg.

3. The method according to any one of claims 1 to 2, characterized in that the centrifugation conditions are (1500-2500 rpm) x (15-30 min), preferably (2000-2500 rpm) x (20-25 min), the washing is centrifugal washing, the washing conditions are (500-2000 rpm) x (5-20 min), and the centrifugal washing is performed 1 to 5 times, preferably 2 to 3 times at (1000-1800 rpm) x (10-15 min).

4. The method according to any one of claims 1 to 3, characterized in that the serum-free medium is optionally supplemented with the cytokine IL-2 at 300-600 IU / ml, preferably 400-500 IU / ml.

5. The method according to any one of claims 1 to 4, wherein the amplification fold of the activated amplified lymphocytes is 900-fold or more, preferably 1000-fold or more, and more preferably 1100-fold or more.

6. 6. The method according to any one of claims 1 to 5, wherein the survival rate of the activated and amplified lymphocytes is 95% or more, preferably 98% or more.

7. The number of CD8+ T cells in the activated and amplified lymphocytes was 1 x 10 9 10 or more, preferably 1×10 9 ~2×10 10 pcs, more preferably 4 to 9.5 × 10 9 The method according to any one of claims 1 to 6, characterized in that

8. Biological activity of lymphocytes after activation and amplification 50 The method according to any one of claims 1 to 7, characterized in that is equal to or less than 8.5, preferably equal to or less than 4, more preferably equal to or less than 0.7141.

9. A pharmaceutical composition comprising an activated amplified lymphocyte stock solution produced by the production method according to any one of claims 1 to 8, The pharmaceutical composition is (1 to 20) x 10 7 The composition comprises activated expanded lymphocytes at 100 / ml, 0.5-2% human albumin and injectable physiological saline, the survival rate of the activated expanded lymphocytes being 95% or more, and the number of CD8+ T cells in the activated expanded lymphocytes being (1-20) x 10 7 5. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition contains no preservatives and no antibiotics.

10. The pharmaceutical composition according to claim 9, characterized in that the cell amplification rate of the biological sample is 900 times or more, preferably 1000 times or more, and more preferably 1100 times or more.

11. Biological activity of activated amplified lymphocytes KT 50 The pharmaceutical composition according to any one of claims 9 to 10, wherein the β-amino acid value is not more than 8.5, preferably not more than 4, more preferably not more than 0.7141.

12. The total number of activated amplified lymphocytes reinjected into the patient was 2 × 10 10 The pharmaceutical composition according to any one of claims 9 to 11, characterized in that the number of cells is not more than 100, and the cell viability during the effective period is 85% or more.

13. The pharmaceutical composition according to any one of claims 9 to 12, characterized in that the survival rate of activated amplified lymphocytes is 85% or more when stored at 15 to 25°C for 12 hours.

14. A method for producing a pharmaceutical composition containing the activated amplified lymphocyte stock solution according to any one of claims 9 to 13, characterized in that the activated amplified lymphocyte stock solution is resuspended in an injectable physiological saline solution containing human albumin.

15. A method for using activated and amplified lymphocytes produced by the method according to any one of claims 1 to 8 in immunotherapy, comprising: (1) obtaining a biological sample containing lymphocytes from an individual; (2) activating, passage and amplifying the lymphocytes using the production method of the present invention to obtain activated and amplified lymphocytes; (3) re-injecting the activated expanded lymphocytes or a pharmaceutical composition thereof into the individual.

16. An administration scheme for using activated and amplified lymphocytes produced by the production method according to any one of claims 1 to 8 in immunotherapy, comprising: (1) In the first cycle, subjects will receive intravenous infusions once a week for 3 to 6 sessions. (2) In the second cycle, subjects will receive intravenous infusions once every two weeks for three to six sessions. (3) In the third cycle, subjects will receive intravenous infusions once every three weeks for three to six sessions. (4) In a fourth cycle, the subject receives 7 to 10 intravenous infusions once every four weeks.

17. (1) In the first course, subjects will receive intravenous infusions once a week for 4-5 sessions. (2) In the second cycle, subjects will receive intravenous infusions once every two weeks for 4-5 sessions. (3) In the third cycle, subjects will receive intravenous infusions once every three weeks for four to five sessions. (4) In a fourth course, the subject receives 8 to 9 intravenous infusions once every 4 weeks.

18. The number of activated amplified lymphocytes per intravenous infusion was 2 × 10 8 lymphocytes or more, preferably (2-10) x 10 9 18. The administration scheme according to any one of claims 15 to 17, characterized in that the lymphocytes are more than one.

19. 10. Use of activated and amplified lymphocytes produced by the production method according to any one of claims 1 to 8 in the production of any one of an antitumor immunotherapy drug, an antiviral ability enhancing drug, and an autoimmune disease treatment enhancing drug.

20. 9. Use of the activated and amplified lymphocytes produced by the method according to any one of claims 1 to 8 in combination with surgery, radiotherapy, or chemotherapy in antitumor therapy or immunotherapy.

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