WO2023125696A2 - 一种质量稳定可控的扩增活化淋巴细胞的方法及其用于抗肿瘤用途 - Google Patents

一种质量稳定可控的扩增活化淋巴细胞的方法及其用于抗肿瘤用途 Download PDF

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WO2023125696A2
WO2023125696A2 PCT/CN2022/142940 CN2022142940W WO2023125696A2 WO 2023125696 A2 WO2023125696 A2 WO 2023125696A2 CN 2022142940 W CN2022142940 W CN 2022142940W WO 2023125696 A2 WO2023125696 A2 WO 2023125696A2
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activated
lymphocytes
cell
culture
serum
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WO2023125696A3 (zh
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王歈
孙磊
孙雪南
李雪姣
张永华
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北京永泰生物制品有限公司
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    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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Definitions

  • the invention relates to the field of biomedicine, in particular to a method for expanding and activating lymphocytes with stable and controllable quality and its anti-tumor application.
  • CIK cells have been reported in clinical research in the treatment of metastatic renal cancer, colon cancer, non-small cell lung cancer and lymphoma.
  • non-specific immune cell therapy has been used in combination with tumor treatments such as surgery, radiotherapy, chemotherapy, targeted therapy, and immune activator therapy.
  • tumor treatments such as surgery, radiotherapy, chemotherapy, targeted therapy, and immune activator therapy.
  • a large number of clinical cases have been observed in which non-specific immune cell therapy benefits patients, including the prevention of tumors.
  • Recurrence, prolongation of patient survival, synergistic effect with chemotherapy, and significant improvement in the quality of life of patients have enabled non-specific immune cells to gain long-term vitality in tumor treatment, which has attracted extensive attention and has been widely used.
  • Yamazaki T et al. reported the results of a randomized controlled clinical trial in "The Lancet" in 2000 using an in vitro culture system to expand and activate autologous lymphocytes to prevent tumor recurrence after liver cancer surgery.
  • a total of 150 patients with liver cancer who underwent radical surgical resection were enrolled in the study.
  • the treatment group (76 cases) received cellular immunotherapy, and the control group (74 cases) did not receive any adjuvant therapy.
  • Patients in the treatment group received a total of 370 cell reinfusions in the first 6 months, and the average number of lymphocytes reinfused in each patient was 7.1 ⁇ 10 10 , of which CD3+ cells accounted for 78%.
  • 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.
  • South Korea approved this method in 2007 to enter phase III clinical trials for the treatment of liver cancer and glioblastoma.
  • the quantity is defined as 1 ⁇ 10 9 -2 ⁇ 10 10 pieces.
  • the expanded activated lymphocytes (Expanded Activated Lymphocytes) in serum-free cell culture are prepared from mononuclear cells in the patient's own peripheral blood. Lymphocytes are the main functional cells, and the average number of CD8+ T cells in a single reinfusion of patients is 4.70 ⁇ 1.47 ⁇ 10 9 , which has shown good tumor therapeutic activity in clinical practice.
  • the prior art discloses methods for culturing and expanding activated lymphocytes without serum, but it is necessary to optimize the extraction and separation of peripheral blood mononuclear cells (PBMC), the types and ratios of lymphocyte activators, and serum-free medium to improve this method.
  • PBMC peripheral blood mononuclear cells
  • the lymphocyte expansion multiple, cell viability and biological activity obtained by the method meet the clinical needs of stable and controllable cell quality and safe and effective treatment.
  • the object of the present invention is to provide a method for preparing a stock solution of activated and expanded lymphocytes, comprising the following steps:
  • PBMC peripheral blood mononuclear cells
  • the lymphocyte activator is selected from any one of anti-human CD2 antibody, anti-human CD3 antibody, anti-human CD28 antibody, phytohemagglutinin (PHA) or a combination thereof or an antibody-containing carrier immobilized on the carrier, said
  • the serum-free medium is selected from any one of KBM 581, GT-T551H3 or a combination thereof;
  • step (2) Place the cultured activated lymphocytes prepared in step (1) in a serum-free medium according to the cell density of (0.5-5) ⁇ 106 /ml, and carry out subculture to obtain an activated lymphocyte culture,
  • the subculture temperature is 37.0°C ⁇ 1.0°C
  • the activation culture environment contains 7.5% ⁇ 1.0% CO 2
  • the serum-free medium is selected from any one of KBM 581, GT-T551H3 or a combination thereof, and the number of subculture generations For 1-5 generations;
  • step (3) Add serum-free medium 5-10 times its volume to the activated lymphocyte culture obtained in step (2) for subculture, and carry out expansion culture, wherein, the expansion culture temperature is 37.0 ° C ⁇ 1.0 ° C, the activation
  • the culture environment contains 7.5% ⁇ 1.0% CO 2
  • the serum-free medium is selected from any one of KBM 581, GT-T551 H3 or a combination thereof, and the number of expansion cultures is 1-5;
  • the initial density of the biological samples cultured and activated in the serum-free medium is (0.2-1) ⁇ 10 6 samples/ml.
  • the lymphocyte activator is an anti-human CD3 antibody, preferably 2.5 ⁇ g/ml-5 ⁇ g/ml, with a volume of 8-15 ml, more preferably 2.5 ⁇ g/ml-3.8 ⁇ g/ml, The volume is 10-13ml.
  • the method for centrifuging whole blood to extract peripheral blood mononuclear cells comprises the following steps: adding a separation medium and a diluent to the anticoagulated whole blood, stirring, mixing, adding it to In the separation liquid, centrifuge at 1000-3000rpm*10-40min, collect the cell layer between the interface, add washing liquid, centrifuge, wash, collect the cells, and obtain that, wherein the separation medium is selected from hydroxyethyl starch 40 sodium chloride Any one of injection (HES), Percoll, Ficoll-Paque PLUS or a combination thereof, the volume ratio of whole blood: diluent is 1:1-2, and the diluent is selected from sodium chloride injection, Hank's buffer , Lactated Ringer's solution, any one of Dulbecco's phosphate buffer or a combination thereof, the separating liquid is selected from Hetastarch 40 Sodium Chloride Injection (HES), Ficoll, Lymphoprep,
  • HES Sodium Chloride In
  • the centrifugal condition is (1500-2500rpm)*(15-30min), preferably (2000-2500rpm)*(20-25min).
  • washing is centrifugal washing, and washing condition is (500-2000rpm)*(5-20min), centrifugal washing 1-5 time, is preferably (1000-1800rpm)*(10-15min), centrifugal Wash 2-3 times.
  • the cell density of the subculture in step (2) is (1-4) ⁇ 10 6 cells/ml
  • the pH of the subculture system after adding serum-free medium is 7.00-7.80.
  • step (2) the cell density of the subculture is (2-3) ⁇ 10 6 cells/ml, and the pH of the subculture system after adding serum-free medium is 7.02-7.76.
  • the osmotic pressure of the separation liquid is 310-350 mOsmol/kg, preferably 316-347 mOsmol/kg.
  • the number of subcultures is 2-3 generations.
  • step (3) when the subculture cell density increases to 1-10 times of the culture activated cell density, the expansion culture is started, and the lymphocyte culture obtained in step (2) subculture is added to Its volume is 6-8 times in total in serum-free medium for expansion culture, and the pH of the expansion culture system is 6.80-7.80.
  • step (3) when the subcultured cell density increases to 1.2-3 times of the cultured activated cell density, the expanded culture is started, and the pH of the expanded culture system is 6.88-7.70.
  • the number of expansion culture generations is 2-3 generations.
  • the centrifugal condition in step (4) is (1000-3000rpm)*(1-10min), preferably (1500-2800rpm)*(2-8min), more preferably (2000-2500rpm )*(5-6min).
  • the cytokine IL-2 300-600IU/ml is optionally added in the serum-free medium, preferably 400-500IU/ml.
  • the pH in the serum-free medium is 6.9-7.9, preferably 7.2-7.4.
  • the culture equipment is selected from any one of incubators, shakers, and bioreactors.
  • the expansion factor of activated and expanded lymphocytes is ⁇ 900 times, preferably ⁇ 1000 times, more preferably ⁇ 1100 times.
  • the viability of activated and expanded lymphocytes is ⁇ 95%, preferably ⁇ 98%.
  • the number of CD8+ T cells in the activated and expanded lymphocytes is ⁇ 1 ⁇ 10 9 , preferably 1 ⁇ 10 9 -2 ⁇ 10 10 , more preferably 4-9.5 ⁇ 10 9 .
  • the biological activity of activated and expanded lymphocytes is KT 50 ⁇ 8.5, preferably KT 50 ⁇ 4, more preferably KT 50 ⁇ 0.7141.
  • Another object of the present invention is to provide a pharmaceutical composition containing the stock solution of activated and expanded lymphocytes, the composition is composed of (1-20) ⁇ 10 7 activated and expanded lymphocytes/ml, Composed of human serum albumin and physiological saline for injection, the viability of the activated and expanded lymphocytes is ⁇ 95%, wherein the CD8+ T cells in the activated and expanded lymphocytes are (1-20) ⁇ 10 7 /ml, the The pharmaceutical composition does not contain preservatives and antibiotics.
  • the composition is composed of (2-18) ⁇ 10 9 /ml activated and expanded lymphocyte concentration, 1-1.5% human serum albumin concentration and physiological saline for injection, and the activated and expanded lymphocyte concentration is 1-1.5%.
  • the viability of increased lymphocytes is ⁇ 98%, and the number of CD8+T cells in the activated and expanded lymphocytes is 1 ⁇ 10 9 -2 ⁇ 10 10 .
  • the cell amplification factor of the biological sample is ⁇ 900 times, preferably ⁇ 1000 times, more preferably ⁇ 1100 times.
  • the biological activity of activated and expanded lymphocytes is KT50 ⁇ 8.5, preferably KT50 ⁇ 4, more preferably KT50 ⁇ 0.7141.
  • the total number of activated and expanded lymphocytes in a single reinfusion of the patient is ⁇ 2 ⁇ 10 10 , and the cell viability within the validity period is ⁇ 85%.
  • the activity rate of activated and expanded lymphocytes of the composition within 12 hours of storage at 15-25°C is ⁇ 85%.
  • Another object of the present invention is to provide a method for preparing a pharmaceutical composition containing a stock solution of activated and expanded lymphocytes.
  • the stock solution of activated and expanded lymphocytes is resuspended in physiological saline for injection containing human albumin.
  • Another object of the present invention is to provide the method for activating and expanding lymphocytes of the present invention for immunotherapy, comprising the following steps:
  • Another object of the present invention is to provide the dosage regimen of activated and expanded lymphocytes of the present invention for immunotherapy, including the following regimens:
  • the subjects received 7-10 intravenous infusions, once every four weeks.
  • the subject received 8-9 times of intravenous infusion, once every four weeks.
  • the number of activated and expanded lymphocytes in each intravenous infusion is ⁇ 2 ⁇ 10 8 lymphocytes, preferably ⁇ (2-10) ⁇ 10 9 lymphocytes.
  • Another object of the present invention is to provide the application of the activated and expanded lymphocytes of the present invention in the preparation of drugs for anti-tumor immunotherapy.
  • the tumor is selected from lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, malignant blood disease, brain tumor, head and neck cancer , Glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumor, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, uterine cancer, anal region cancer, testicular cancer, fallopian tube cancer, uterus Endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophagus, small intestine, endocrine system, thyroid, parathyroid, adrenal, soft tissue sarcoma, urethra, penis Carcinoma, chronic or acute leukemia, childhood solid tumors, lymphocytic lymphoma,
  • the lung cancer is selected from any one of small cell lung cancer, non-small cell lung cancer or their complications.
  • the leukemia is selected from any one of acute lymphoblastic leukemia, acute myelogenous leukemia, B-cell chronic lymphocytic leukemia, chronic myelogenous leukemia or their complications.
  • the acute myeloid leukemia is selected from acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute basophilic leukemia, acute eosinophilic leukemia , acute megakaryocytic leukemia, acute erythroleukemia, acute panmyeloid hyperplasia with myelofibrosis, or any combination thereof.
  • the immunotherapy is selected from any one or combination of anti-tumor immunotherapy or immunotherapy after radical tumor resection.
  • the immunotherapy is selected from immunotherapy after radical resection of primary hepatocellular carcinoma.
  • the drug is used for adult patients or children patients.
  • Another object of the present invention is to provide the application of the activated and expanded lymphocytes of the present invention in combination with any one of surgery, radiotherapy and chemotherapy in anti-tumor or immunotherapy.
  • Another object of the present invention is to provide the application of activated and expanded lymphocytes in the preparation of drugs for enhancing antiviral ability.
  • Another object of the present invention is to provide the application of activated and expanded lymphocytes for the preparation of drugs for enhanced treatment of autoimmune diseases.
  • the autoimmune disease is selected from systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, juvenile diabetes, primary thrombocytopenia, autoimmune hemolytic anemia , ulcerative colitis, any of skin diseases or their complications.
  • the present invention uses the biological activity of expanded and activated lymphocytes (RTCA, taking the effect-to-target ratio of 40:1 as an example) to characterize the therapeutic activity of activated and expanded lymphocytes.
  • RTCA biological activity of expanded and activated lymphocytes
  • effector cells i.e., expanded and activated lymphocytes
  • a specific density 50 ⁇ l per well with a density of 1.6 ⁇ 10 7 cell/ml
  • real-time , dynamic and quantitative tracking of cell morphology, proliferation and differentiation data that is, the change of electrical impedance produced by the contact of living cells with the microelectrodes in the detection plate.
  • the instrument-specific software converts the resistance signal into a specific cell index, and the software monitors the generated cell index curve in real time to show the state of cell adhesion, stretching, growth, death, etc. in different time periods.
  • target cells such as HepG2 cells
  • KT 50 i.e., the time for effector cells to kill 50% of target cells
  • KT 50 value i.e. activated Expanded lymphocytes
  • said percentage is volume/volume percentage;
  • said percentage is volume/weight percentage;
  • said percentages are weight/volume percentages; the remainder are weight/weight percentages.
  • the present invention has the following beneficial technical effects:
  • the activated and expanded lymphocytes prepared by the present invention have the advantages of high cell expansion efficiency, high cell viability, stable cell activity, long validity period, safety and effectiveness, and small side effects, effectively solving the problem of individualized immune cell therapy.
  • the key issues of tumor technology are: first, use a serum-free culture system with stable cell culture efficiency, maintain the killing activity and expansion ability of the cells through continuous culture for 12 days, and avoid the possible contamination of pathogenic microorganisms and other adverse effects caused by the use of serum.
  • the cell expansion efficiency is high, no need to use a mononuclear cell collection machine, and the high-efficiency amplification is more than 1000 times, avoiding the need for a large number of initial peripheral blood mononuclear cells to be collected by an apheresis machine for traditional immune cell therapy
  • the destructive effect on the patient's overall immune cell system is that the cells have high activity and good stability, and the cells can be stored for 12 hours without affecting their therapeutic effect;
  • the fourth is to solve the technical problems of the standardized quality control system and large-scale production of individualized products , the technological process of obtaining cell products with relatively uniform quality, which can significantly improve the targeting and precision of treatment and the compliance of patients.
  • the activated and amplified lymphocytes of the present invention can prevent postoperative tumor recurrence, prolong the postoperative recurrence period of tumor, significantly prolong the survival period of tumor patients and significantly improve the quality of life of patients, and during the treatment process, Reduce the dose of chemotherapy drugs, radiotherapy dose, and adverse reactions such as nausea, vomiting, hair loss, insomnia during radiotherapy and chemotherapy, no serious adverse reactions such as decreased blood count, liver and kidney function damage, reduce pain caused by tumors, and significantly improve the life of patients quality.
  • the lymphocyte expansion method of the present invention has the advantages of simple operation, stable and controllable quality, and is suitable for industrial production.
  • Figure 1 The growth status of EAL cells during the first subculture in Example 1 (observed under a microscope at 250 ⁇ )
  • Figure 2 The growth status of EAL cells in the first expansion culture in Example 1 (observed under a microscope at 250 ⁇ )
  • Figure 3 Example 1 Growth status of EAL cells during the second expansion culture (observed under 250 ⁇ microscope)
  • PBMC peripheral blood mononuclear cells
  • Patient 1 liver cancer patient, age: 55 years old, sex: male.
  • the serum-free cell culture medium is: GT-T551 H3 serum-free cell culture medium (the concentration of IL-2 in the culture medium is 500IU/ml, pH 7.2-7.4)
  • the supernatant was poured off, the cell pellet was shaken off, resuspended with 50ml of 0.9% sodium chloride injection, and centrifuged at 1200rpm for 10min. After the centrifugation is completed, pour off the supernatant, shake off the cell pellet, add 5ml of serum-free cell culture medium, mix well, take 10 ⁇ l of cell suspension for cell counting. After the cells were resuspended in serum-free cell culture medium at a concentration of 0.8 ⁇ 106 cells/ml, they were inoculated into 225 cm 2 cell culture flasks coated with anti-human CD3 antibody, samples were taken, sterility checked and labeled, and the Place in a cell culture incubator for cultivation.
  • the culture conditions in the incubator are: 37° C., 7.5% CO 2 .
  • the growth condition is good (see Figure 2), take a sample and carry out cell counting, when the viable cell density is not lower than 1.0 ⁇ 106 /ml, mix the cell culture medium with 760ml Serum-free cell culture medium, poured into the cell culture bag, and put it back into the cell culture incubator for cultivation.
  • Activated and expanded lymphocytes are obtained by resuspending the cell stock solution at a concentration of 8 ⁇ 10 7 /ml in physiological saline for injection containing 1% human serum albumin.
  • Patient 2 breast cancer patient, age: 50, sex: female.
  • the serum-free cell culture medium is: KBM 581 serum-free cell culture medium (the concentration of IL-2 in the culture medium is 500IU/ml, pH 7.2-7.4)
  • the supernatant was poured off, the cell pellet was shaken off, resuspended with 50ml of sodium chloride injection, and centrifuged at 1200rpm for 10min. After the centrifugation was completed, the supernatant was poured off, the cell pellet was shaken off, and 5ml of serum-free cell culture medium was added, after mixing, 10 ⁇ l of the cell suspension was taken for cell counting. After the cells were resuspended in serum-free cell culture medium at a concentration of 0.2 ⁇ 10 6 cells/ml, they were inoculated into 225 cm 2 cell culture flasks coated with anti-human CD3 antibody. A sample is taken, checked for sterility and labeled, and placed in a cell incubator for incubation.
  • the culture conditions in the incubator are: 37° C., 7.5% CO 2 .
  • the day of blood collection is considered as day 0, and the day after inoculation and culture is the 4th day
  • Perform cell counting add 50ml of serum-free cell culture medium into the cell culture flask, and put it back into the cell culture incubator for culturing.
  • the growth condition is good, take a sample for cell counting, when the viable cell density is not lower than 1 ⁇ 106 cells/ml, mix the cell culture medium with 750ml serum-free cell culture medium Pour into a cell culture bag and place it back in the cell culture incubator for cultivation.
  • Activated and expanded lymphocytes are obtained by resuspending the original cell solution at a concentration of 6 ⁇ 10 7 cells/ml in physiological saline for injection containing 1.5% human serum albumin.
  • Patient 3 A patient with non-small cell lung cancer, age: 53, sex: male.
  • the serum-free cell culture medium is: KBM 581 (CORNING) serum-free cell culture medium (the concentration of IL-2 in the culture medium is 400IU/ml, pH7.2-7.4)
  • the supernatant was poured off, the cell pellet was shaken off, resuspended with 50ml of sodium chloride injection, and centrifuged at 1200rpm for 10min. After the centrifugation was completed, the supernatant was poured off, the cell pellet was shaken off, and 5ml of serum-free cell culture medium was added, after mixing, 10 ⁇ l of the cell suspension was taken for cell counting. After the cells were resuspended in serum-free cell culture medium at a concentration of 0.3 ⁇ 10 6 cells/ml, they were inoculated into 225 cm 2 cell culture flasks coated with anti-human CD3 antibody. A sample is taken, checked for sterility and labeled, and placed in a cell incubator for incubation.
  • the culture conditions in the incubator are: 37° C., 5% CO 2 .
  • the cell culture bottle is taken out from the incubator; the cell morphology is observed under a microscope: the growth condition is good. Perform cell counting, add 50ml of serum-free cell culture medium into the cell culture flask, and put it back into the cell culture incubator for culturing.
  • the growth condition is good, take a sample for cell counting, when the viable cell density is not lower than 1 ⁇ 106 cells/ml, mix the cell culture medium with 750ml serum-free cell culture medium Pour into a cell culture bag and place it back in the cell culture incubator for cultivation.
  • Activated and expanded lymphocytes are obtained by resuspending the cell stock solution at a concentration of 8 ⁇ 10 7 /ml in physiological saline for injection containing 1% human serum albumin.
  • each PBMC is divided into three equal parts, and then inoculated in 3 kinds of serum-free medium respectively, cultured in the cell culture bottle coated with activated antibody, every 4- Cell counting was carried out every 6 days and the corresponding cell culture medium was supplemented. After 12 days, the culture was terminated and cell counting, viability, phenotype detection, and killing activity detection were performed.
  • the serum-free medium TexMACS GMP Medium was used to culture the cells. After 6-8 days of culture, the cell expansion was slow, which could not meet the quality requirements of lymphocyte expansion in vitro.
  • Embodiment 5 patient's activated lymphocyte expansion effect
  • Example 1 for the preparation of the lymphocyte expansion and activation stock solution.
  • Expanded cell viability was obtained by trypan blue staining and cell counting. Phenotypic detection of expanded cells by flow cytometry was determined using FITC-labeled CD3 antibody, APC-labeled CD8 antibody.
  • Experimental Example 1 The present invention activates and expands lymphocytes for immunotherapy research
  • EAL treatment group The inclusion criteria for the EAL treatment group are as follows: patients diagnosed with gastric cancer, lung cancer, etc. by histology or cytology, life expectancy > 12 weeks, Eastern Cooperative Oncology Group (ECOG) performance status (PS) score 0-2; ECOG Patients with a score >2 were excluded. All patients agreed to their treatment plan and signed informed consent.
  • ECOG Eastern Cooperative Oncology Group
  • PS performance status
  • EAL treatment group 50 cases, the patient's age, gender, and disease information are shown in Example 5, autologous input of expanded and activated lymphocyte preparations.
  • Treatment plan In the first course of treatment, the subject received 4 times of intravenous infusion, once a week; in the second course of treatment, the subject received 4 times of intravenous infusion, once every two weeks; in the third course of treatment, the subject received 4 times of intravenous infusion Infusion, once every three weeks; in the fourth course of treatment, subjects received 8 intravenous infusions, once every four weeks.
  • Control group 20 cases, cancer patients who entered the hospital for treatment at the same time (5 cases each for gastric cancer, small cell lung cancer, liver cancer, and leukemia), and experienced chemotherapy or radiotherapy, conventional treatment plan; excluded those with a history of cell therapy and ECOG>2 patient.
  • the chemotherapy regimens of the research subjects included: CE (etoposide + carboplatin), EP (etoposide + cisplatin), IP (irinotecan + cisplatin), PP (paclitaxel + cisplatin/neda Platinum) and DP (docetaxel + cisplatin), CAV (cyclophosphamide + famomycin + vincristine), NP (navirbine + cisplatin), pemetrexed alone, doxe alone Paclitaxel, temozolomide capsules alone, etoposide capsules alone. Patients with incomplete medical history or lost follow-up were not included in the statistics. To analyze the efficacy and safety of immune cell therapy.
  • the median OS time of the EEAL treatment group was significantly longer than that of the control group.
  • EAL immunotherapy prolongs the survival of cancer patients by 1-7 years; improves side effects and adverse reactions such as nausea, vomiting, hair loss, diarrhea, loss of appetite, fever, chills, headache, itching, rash, tachycardia, etc. There were no serious adverse reactions such as decreased blood picture, liver and kidney function damage, etc., and the quality of life of patients was significantly improved and improved.
  • EAL treatment group only grade 1 or 2 self-limited adverse events occurred in EAL treated patients (see Table 8); no patients experienced pulmonary or renal symptoms, infection symptoms, worsening liver function or autoimmune disease; no treatment-related death record.
  • Control group There were serious adverse reactions such as decreased blood picture, damage to liver and kidney function, and severe nausea, vomiting, diarrhea, hair loss and other adverse reactions.
  • a clinical treatment study was conducted in 100 low- or intermediate-risk pediatric acute myeloid leukemia (AML) patients with low levels of minimal residual disease (MRD).
  • AML acute myeloid leukemia
  • MRD minimal residual disease
  • Induction chemotherapy included cytarabine 100-150 mg/m 2 for 7 days, combined with anthracyclines (idarubicin 8-10 mg/m 2 for 2 days) and etoposide 100-150 mg/m 2 for 3 days. Consolidation chemotherapy was started after 2 cycles of induction.
  • Consolidation consists of three regimens: regimen 1, high-dose cytarabine (Ara-c 3g/m 2 , 3 days) and anthracycline (8-10mg/m 2 idarubicin, 2 days); regimen 2, Harringtonine 4-6mg/m 2 for 7 days and cytarabine 100-150mg/m 2 for 7 days; scheme 3, cytarabine 100–150mg/m 2 for 7 days, combined with anthracyclines ( Idarubicin 8–10 mg/m 2 for 2 days) and etoposide 100–150 mg/m 2 for 3 days.
  • the three programs are used alternately for a total of 12-18 months.
  • triple intrathecal chemotherapy (methotrexate, cytarabine and hydrocortisone) was given 4-8 times in total to prevent central nervous system leukemia.
  • EAL was performed in low- and intermediate-risk AML patients with persistently positive MRD status ( ⁇ 0.5%), and in patients with positive MRD status or a 1-copy increase in MRD burden.
  • patients in the combination therapy group received a combination of EAL therapy and chemotherapy.
  • EAL was obtained by collecting 10-80 mL of peripheral blood (determined by absolute peripheral blood lymphocyte count) from each patient.
  • Example 2 place the PBMC suspension in a flask coated with an immobilized anti-CD3 antibody, and perform activation, subculture, and expansion culture according to the preparation method in Example 1; then collect lymphocytes and filter them through a membrane with a pore size of 100 ⁇ m, and resuspend In 100mL of normal saline containing 1% human serum albumin, it is used for intravenous infusion and reinfusion to individual patients.
  • the final product is divided into 1-3 infusion bags according to the total number of cells after culture, and each bag contains 1 ⁇ 10 9 to 2 ⁇ 10 10 CD3+CD8+ T cells.
  • EAL was stored in a 15–25°C incubator and infused into patients within 12 hours, verifying cell viability was always ⁇ 90%. Each transfusion contained ⁇ 3 x 10 9 cells.
  • the study protocol was approved by the Institutional Review Board. Informed consent was obtained from the parents or legal guardians of all patients.
  • CBF AML core binding factor AML
  • RT-PCR reverse transcription polymerase chain reaction
  • Bone marrow samples were collected at diagnosis, prior to each chemotherapy cycle, then every 3-month intervals up to 3 years and every 6-month intervals up to 5 years.
  • the combined treatment group included 26 men and 24 women with a median age of 10.5 years (range, 1-15 years). Among them, there were 42 cases (84.00%) in the low-risk group and 8 cases in the middle-risk group.
  • the mean white blood cell count was 27.9 ⁇ 10 9 (range, 2.1–100 ⁇ 10 9 ), 2 patients had central nervous system leukemia, and 1 patient had a large jaw mass.
  • the combined treatment group received an average of 4 sessions of EAL (range, 1-12 sessions).
  • EAL treatment was chosen to start 6-12 months (average 8 months) after the start of chemotherapy.
  • EAL transfusions A total of 220 EAL transfusions (average of 4 transfusions/patient) were received in the 50 children in the combination therapy group. Analysis of cell phenotype and cell counts from these 220 EAL transfusions showed a mean cell count of 7.5 ⁇ 0.5 ⁇ 10 9 /L and a mean cell viability of 98.9%.
  • the phenotype distribution of T cells was as follows: CD3+, 98.95%; CD3+CD8+, 87.26%.
  • the control group consisted of 25 men and 25 women with a median age of 10.6 years (range, 1-15 years). Among them, 42 belonged to the low-risk group (84.00%), and 8 belonged to the intermediate-risk group. At onset, 1 patient had central nervous system leukemia and 1 patient had extensive thoracolumbar infiltration.
  • EAL treatment was initiated when pre-EAL MRD was ⁇ 0.5% and the mean MRD burden was 0.055%.
  • the MRD status of 49 out of 50 patients (98.00%) turned negative, but the MRD status of 1 patient remained positive after combined treatment.
  • MRD status remained negative for an average of 7.9 months during consolidation chemotherapy.
  • the average 5-year EFS rate was 88.9% in the combination therapy group and 69.7% in the control group.
  • the combination therapy group had occasional fever or chills, and no serious inflammatory cytokine cascade leading to organ damage was observed during the treatment, and no cell therapy-related death was observed.

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Abstract

本发明涉及一种活化扩增淋巴细胞原液的制备方法，包括下述步骤：(1)将自体外周全血离心提取获得的外周血单个核细胞(PBMC)与淋巴细胞活化剂置于无血清培养基中共培养，完成淋巴细胞的培养活化，其中，所述外周血单个核细胞在无血清培养基中培养活化的起始密度为(0.2-1.6)×10 6个/ml,所述无血清培养基选自KBM 581、GT-T551 H3的任一种或其组合；(2)将步骤(1)制得的培养活化淋巴细胞按照细胞密度为(0.5-5)×10 6个/ml置于无血清培养基中，进行传代培养，制得淋巴细胞活化培养物，其中，传代培养温度为37.0℃±1.0℃，活化培养环境中含7.5％±1.0％CO 2；(3)在步骤(2)传代培养获得的淋巴细胞活化培养物中加入其体积5-10倍的无血清培养基，进行扩增培养，扩增培养代数为1-5代；(4)离心，洗涤，收集活化扩增的淋巴细胞，即得。本发明制得的活化扩增淋巴细胞具有细胞扩增效率高、细胞活率高、细胞活性稳定、具有较长的有效期、安全有效、副作用小等优点，有效解决了个体化免疫细胞治疗肿瘤技术的关键问题。

Description

一种质量稳定可控的扩增活化淋巴细胞的方法及其用于抗肿瘤用途 技术领域
本发明涉及生物医药领域，具体涉及一种质量稳定可控地扩增活化淋巴细胞的方法及其抗肿瘤用途。
背景技术
恶性肿瘤是威胁人类健康和生命的主要疾病之一，其发病率呈现逐年增高趋势。《2015全球癌症统计》数据显示，全球2015年新增癌症病例数约1410万，死亡人数达820万。中国2015年新发癌症病例429万人，死亡病例达281万人。手术、放疗和化疗成为肿瘤治疗的三大常规手段，但均有特异性不足的特点，导致在清除肿瘤细胞的同时，对正常组织和器官造成不同程度的损伤。20世纪80年代，Rosenberg SA等观察到患非免疫系统肿瘤的动物给予重组IL-2治疗后，动物的淋巴细胞可以导致肿瘤的消退和转移。此后，免疫细胞治疗在临床上的应用迅速展开并成为肿瘤综合治疗的第四种模式。Rosenberg SA等于1988年总结了IL-2与LAK细胞联合治疗214例肿瘤患者，16例患者肿瘤转移灶完全消退，26例患者肿瘤缩小50％以上，该疗法对转移性肾细胞癌、黑色素瘤、结肠癌和非霍奇金淋巴瘤患者呈现一定治疗效果。LAK细胞杀伤力不强，临床应用需要大量输注，且其扩增能力有限，需要在输注细胞的同时大剂量应用IL-2(单次给药1×10 5IU/Kg或[1-6]×10 6IU/m 2)，而大剂量IL-2治疗 过程中可出现明显的患者难以耐受的毒性反应，包括严重的溃疡、肾脏衰竭等。该疗法的应用主要集中于肾癌、黑色素瘤等少数肿瘤，并逐渐不被采用。
美国斯坦福大学医学院骨髓移植研究中心于1991年报道了用IFN-γ、IL-2、抗CD3单克隆抗体和IL-1刺激外周血单个核细胞，诱导产生的细胞对淋巴瘤细胞有强大的杀伤作用而对正常的造血干细胞影响甚微，并将该方法所获得的细胞命名为细胞因子诱导的杀伤细胞(Cytokine-Induced Killer，CIK细胞)，其发挥抗肿瘤作用的效应细胞主要是CD3+CD56+的NKT细胞,兼具T淋巴细胞的抗瘤活性和NK细胞非MHC限制性的特点。研究表明，CIK细胞的扩增效率、肿瘤细胞杀伤能力、体内的活性比LAK细胞明显增高。CIK细胞治疗转移性肾癌、结肠癌、非小细胞肺癌和淋巴瘤已有临床研究报道。
迄今，非特异性免疫细胞治疗与手术、放疗、化疗、靶向治疗、免疫激活剂治疗等肿瘤治疗手段联合使用，临床观察到了大量的非特异性免疫细胞治疗使病人治疗获益的病例，包括预防肿瘤复发、延长患者生存期、与化疗的协同作用、显著改善患者生活质量等，使得非特异性免疫细胞在肿瘤治疗中获得了长久的生命力，备受广泛重视并得到广泛应用。
Yamazaki T等人于2000年在《The Lancet》报道了采用体外培养体系扩增活化自体淋巴细胞预防肝癌术后肿瘤复发的随机对照临床试验研究结果。该研究共计入组了150例接受根治性手术切除的肝癌患者，治疗组(76例)接受细胞免疫治疗，对照组(74例)未接 受任何辅助治疗。治疗组患者在最初6个月内共接受了370次细胞回输，每位患者回输的平均淋巴细胞数为7.1×10 10个，其中，CD3+细胞占78％。45位患者中共出现了62次不良反应(包括发热、头痛、恶心、头晕、瘙痒、心动过速)，所有不良反应均为自限性。未有患者出现肺或肾部症状、任何迹象的感染、肝功能衰退或自身免疫失调。经过平均4.4年(0.2-6.7年)的随访后发现，与对照组相比，治疗组显著降低肝癌复发的风险达41％(95％可信区间为12％-60％，p＝0.01)。治疗组出现首次复发的时间明显晚于对照组。3年内的无复发率，治疗组评估为48％(95％可信区间为37％-59％)，对照组评估为33％(95％可信区间为22％-43％)。5年内的无复发率，治疗组评估为38％[95％可信区间为22％-54％]，对照组评估为22％(95％可信区间为11％-34％)。治疗组患者的无复发存活率(p＝0.01)和疾病特异的存活率(p＝0.04)显著延长。研究结果表明，这种非特异性免疫治疗极大延长了肝癌患者术后无复发生存时间。日本自2000年起将此技术用于临床肿瘤治疗，至2010年每年医治患者约10000例次。韩国于2007年批准该方法进入肝癌和神经胶质母细胞瘤治疗的Ⅲ期临床试验，2012年批准非特异性免疫细胞产品Immuncell-LC上市，显著降低了肝癌术后的复发率，单次回输细胞数量界定为1×10 9-2×10 10个。
无血清细胞培养扩增活化的淋巴细胞(Expanded Activated Lymphocytes)系由患者自体外周血中的单个核细胞制备而成，主要成分为扩增活化的T淋巴细胞(CD3+细胞)，以CD8+杀伤性T淋巴细胞为主要功能细胞，患者单次回输的CD8+T细胞平均数量为 4.70±1.47×10 9个，并在临床实践中显示了很好地肿瘤治疗活性。现有技术公开了无血清培养扩增活化淋巴细胞的方法，但需优化外周血单个核细胞(PBMC)的提取分离、淋巴细胞活化剂的种类及配比、无血清培养基等，以改善该方法制得的淋巴细胞扩增倍数和细胞活率及生物学活性，满足临床细胞质量稳定可控和安全有效的治疗需求。
发明内容
本发明的目的在于提供一种活化扩增淋巴细胞原液的制备方法，包括下述步骤：
(1)将自体外周全血离心提取获得的外周血单个核细胞(PBMC)与淋巴细胞活化剂置于无血清培养基中共培养，完成淋巴细胞的培养活化，其中，所述外周血单个核细胞在无血清培养基中培养活化的起始密度为(0.2-1.6)×10 6个/ml,细胞活化培养温度为37.0℃±1.0℃，活化培养环境中含7.5％±1.0％CO 2，所述淋巴细胞活化剂选自抗人CD2抗体、抗人CD3抗体、抗人CD28抗体、植物血凝素(PHA)的任一种或其组合或将其固化在载体上的含抗体载体，所述无血清培养基选自KBM 581、GT-T551H3的任一种或其组合；
(2)将步骤(1)制得的培养活化淋巴细胞按照细胞密度为(0.5-5)×10 6个/ml置于无血清培养基中，进行传代培养，制得淋巴细胞活化培养物，其中，传代培养温度为37.0℃±1.0℃，活化培养环境中含7.5％±1.0％CO 2，所述无血清培养基选自KBM 581、GT-T551H3的任一种或其组合，传代培养代数为1-5代；
(3)在步骤(2)传代培养获得的淋巴细胞活化培养物中加入其体积5-10倍的无血清培养基，进行扩增培养，其中，扩增培养温度为37.0℃±1.0℃，活化培养环境中含7.5％±1.0％CO 2，所述无血清培养基选自KBM 581、GT-T551 H3的任一种或其组合，扩增培养代数为1-5代；
(4)离心，洗涤，收集活化扩增的淋巴细胞，即得。
本发明的优选技术方案中，所述生物学样品在无血清培养基中培养活化的起始密度为(0.2-1)×10 6个/ml。
本发明的优选技术方案中，所述淋巴细胞活化剂为抗人CD3抗体，优选为2.5μg/ml-5μg/ml，体积为8-15ml，更优选为2.5μg/ml-3.8μg/ml，体积为10-13ml。
本发明的优选技术方案中，所述全血离心提取外周血单个核细胞的方法包括下述步骤：在抗凝处理的全血中加入分离介质和稀释剂，搅拌，混匀，将其加至分离液中，离心1000-3000rpm*10-40min，收集分界面间细胞层，加入洗涤液，离心，洗涤，收集细胞，即得，其中，所述分离介质选自羟乙基淀粉40氯化钠注射液(HES)、Percoll、Ficoll-Paque PLUS的任一种或其组合，全血：稀释剂的体积比为1：1-2，所述稀释剂选自氯化钠注射液、Hank’s缓冲液、Lactated Ringer’s溶液、Dulbecco's磷酸盐缓冲液的任一种或其组合，所述分离液选自羟乙基淀粉40氯化钠注射液(HES)、Ficoll、Lymphoprep、Lymphocyte Separation Media、Cell Separation Media的任一种或其组合，所述分离液的渗透压为300mOsmol/kg-360mOsmol/kg，所 述洗涤液选自0.1％人血白蛋白氯化钠注射液、Dulbecco's磷酸盐缓冲液、氯化钠注射液的任一种或其组合。
本发明的优选技术方案中，离心条件为(1500-2500rpm)*(15-30min)，优选为(2000-2500rpm)*(20-25min)。
本发明的优选技术方案中，洗涤为离心洗涤，洗涤条件为(500-2000rpm)*(5-20min)，离心洗涤1-5次,优选为(1000-1800rpm)*(10-15min)，离心洗涤2-3次。
本发明的优选技术方案中，步骤(2)中传代培养的细胞密度为(1-4)×10 6个/ml，传代培养体系添加无血清培养基后pH 7.00-7.80。
本发明的优选技术方案中，步骤(2)中,传代培养的细胞密度为(2-3)×10 6个/ml，传代培养体系添加无血清培养基后pH 7.02-7.76。
本发明的优选技术方案中，所述分离液的渗透压为310-350mOsmol/kg，优选为316-347mOsmol/kg。
本发明的优选技术方案中，所述传代培养代数为2-3代。
本发明的优选技术方案中，步骤(3)中，当传代培养细胞密度增长至培养活化细胞密度的1-10倍时开始扩增培养，将步骤(2)传代培养获得的淋巴细胞培养物加入其体积共6-8倍的无血清培养基中扩增培养，所述扩增培养体系pH 6.80-7.80。
本发明的优选技术方案中，步骤(3)中，当传代培养细胞密度增长至培养活化细胞密度的1.2-3倍时开始扩增培养，所述扩增培养体系pH 6.88-7.70。
本发明的优选技术方案中，所述扩增培养代数为2-3代。
本发明的优选技术方案中，步骤(4)中的离心条件为(1000-3000rpm)*(1-10min)，优选为(1500-2800rpm)*(2-8min)，更优选为(2000-2500rpm)*(5-6min)。
本发明的优选技术方案中，所述无血清培养基中任选地添加细胞因子IL-2 300-600IU/ml，优选为400-500IU/ml。
本发明的优选技术方案中，所述无血清培养基中pH为6.9-7.9，优选为7.2-7.4。
本发明的优选技术方案中，所述培养设备选自培养箱、摇床、生物反应器的任一种。
本发明的优选技术方案中，活化扩增淋巴细胞扩增倍数≥900倍，优选≥1000倍，更优选≥1100倍。
本发明的优选技术方案中，活化扩增淋巴细胞活率≥95％，优选≥98％。
本发明的优选技术方案中，活化扩增淋巴细胞中的CD8+T细胞数量≥1×10 9个、优选为1×10 9-2×10 10个，更优选为4-9.5×10 9个。
本发明的优选技术方案中，活化扩增后的淋巴细胞生物学活性KT 50≤8.5，优选为KT 50≤4，更优选为KT 50≤0.7141。
本发明的另一目的在于提供一种含有活化扩增淋巴细胞原液的药物组合物，所述组合物由(1-20)×10 7个/ml的活化扩增淋巴细胞、0.5-2％的人血白蛋白和注射用生理盐水组成，活化扩增淋巴细胞活率≥95％，其中，活化扩增淋巴细胞中的CD8+T细胞为(1-20)×10 7 个/ml，所述药物组合物中不含防腐剂和抗生素。
本发明的优选技术方案中，所述组合物由(2-18)×10 9个/ml的活化扩增淋巴细胞浓度、1-1.5％人血白蛋白浓度和注射用生理盐水组成，活化扩增淋巴细胞活率≥98％，活化扩增淋巴细胞中的CD8+T细胞为1×10 9-2×10 10个。
本发明的优选技术方案中，生物学样品的细胞扩增倍数≥900倍，优选为≥1000倍，更优选为≥1100倍。
本发明的优选技术方案中，活化扩增淋巴细胞生物学活性KT50≤8.5，优选为KT50≤4，更优选为KT50≤0.7141。
本发明的优选技术方案中，患者单次回输活化扩增淋巴细胞总数≤2×10 10个，有效期内细胞活率≥85％。
本发明的优选技术方案中，组合物于15-25℃保存12h内的活化扩增淋巴细胞活率≥85％。
本发明的另一目的在于提供一种含有活化扩增淋巴细胞原液的药物组合物的制备方法，将活化扩增淋巴细胞原液重悬于含有人血白蛋白的注射用生理盐水中。
本发明的另一目的在于提供本发明的活化扩增淋巴细胞用于免疫治疗的方法，包括下述步骤：
(1)从个体获得含有淋巴细胞的生物学样品；
(2)采用本发明的制备方法活化、传代、扩增所述淋巴细胞， 获得活化扩增淋巴细胞；
(3)将所述活化扩增淋巴细胞或其药物组合物回输给所述个体。
本发明的另一目的在于提供本发明的活化扩增淋巴细胞用于免疫治疗的给药方案，包括下述方案：
(1)第一疗程，受试者接受3-6次静脉输注，每周1次；
(2)第二疗程，受试者接受3-6次静脉输注，每两周1次；
(3)第三疗程，受试者接受3-6次静脉输注，每三周1次；
(4)第四疗程，受试者接受7-10次静脉输注，每四周1次。
本发明优选的技术方案中，包括下述方案：
(1)第一疗程，受试者接受4-5次静脉输注，每周1次；
(2)第二疗程，受试者接受4-5次静脉输注，每两周1次；
(3)第三疗程，受试者接受4-5次静脉输注，每三周1次；
(4)第四疗程，受试者接受8-9次静脉输注，每四周1次。
本发明的优选技术方案中，每次静脉输注的活化扩增淋巴细胞数≥2x10 8个淋巴细胞，优选为≥(2-10)x10 9个淋巴细胞。
本发明的另一目的在于提供本发明的活化扩增淋巴细胞用于制备抗肿瘤免疫治疗的药物中的应用。
本发明优选的技术方案中，所述肿瘤选自肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、脑肿瘤、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子 宫体瘤、骨肉瘤、骨癌、胰腺癌、皮肤癌、前列腺癌、子宫癌、肛区癌、睾丸癌、输卵管癌、子宫内膜癌、阴道癌、阴户癌、霍奇金病、非霍奇金淋巴瘤、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病、儿童实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮状癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症的任一种或其组合。
本发明优选的技术方案中，所述肺癌选自小细胞肺癌、非小细胞肺癌的任一种或其并发症。
本发明优选的技术方案中，所述白血病选自急性淋巴性白血病、急性骨髓性白血病、B细胞慢性淋巴性白血病、慢性骨髓性白血病的任一种或其并发症。
本发明优选的技术方案中，所述急性骨髓性白血病选自急性粒细胞性白血病、急性早幼粒细胞白血病、急性粒单核细胞白血病、急性嗜碱性粒细胞白血病、急性嗜酸性粒细胞白血病、急性巨核细胞白血病、急性红白血病、急性全骨髓增生伴骨髓纤维化的任一种或其组合。
本发明优选的技术方案中，所述免疫治疗选自抗肿瘤免疫治疗或肿瘤根治术后免疫治疗的任一种或其组合。
本发明优选的技术方案中，所述免疫治疗选自原发性肝细胞癌根治术后免疫治疗。
本发明优选的技术方案中，所述药物用于成年患者或儿童患者。
本发明的另一目的在于提供本发明的活化扩增淋巴细胞联合手术、放疗、化疗的任一种在抗肿瘤或其免疫治疗中的应用。
本发明的另一目的在于提供活化扩增淋巴细胞用于制备增强抗病毒能力的药物中的应用。
本发明的另一目的在于提供活化扩增淋巴细胞用于制备增强治疗自身免疫性疾病的药物中的应用。
本发明的优选技术方案中，所述自身免疫性疾病选自系统性红斑狼疮、类风湿性关节炎、硬皮病、甲状腺机能亢进、青少年糖尿病、原发性血小板紫癜、自身免疫性溶血性贫血、溃疡性结肠炎、皮肤病的任一种或其并发症。
除非另有说明，本发明采用扩增活化的淋巴细胞生物学活性(RTCA，以效靶比40:1为例)来表征活化扩增淋巴细胞的治疗活性。将消化好的靶细胞(如HepG2等)按照每孔加入100μl的密度为2×10 5cells/ml靶细胞提前铺板，并设置好效靶比参数(如效靶比为40:1)进行基线测量。在靶细胞铺板检测16-24h后，按照设定的效靶比加入特定密度(每孔加入50μl密度为1.6×10 7cell/ml)的效应细胞(即扩增活化的淋巴细胞)后，实时、动态、定量跟踪细胞形态和增殖分化等数据，即活细胞与检测板中的微电极接触产生电阻抗的改变。当在含有贴壁细胞的孔中加入效应细胞后，贴壁生长在微电极表面的细胞引起贴壁电极界面阻抗的改变，这种改变与细胞指数(Cell Index)的改变呈正相关性。仪器专用软件将电阻信号转化为特定的 细胞指数，软件实时监测产生的细胞指数曲线，以展现细胞在不同时间段内的黏附、伸展、生长、死亡等状态，通过分析细胞指数的改变，即可计算出效应细胞对靶细胞(如HepG2细胞)的杀伤效率，通过杀伤效率曲线，计算出KT 50(即效应细胞杀伤50％靶细胞所用时间)，并以KT 50值作为判定效应细胞(即活化扩增淋巴细胞)生物学活性的指标。KT 50值越小，说明杀伤效率越高，生物学活性越高。反之，则生物学活性越低。
除非另有说明，本发明涉及液体与液体之间的百分比时，所述的百分比为体积/体积百分比；本发明涉及液体与固体之间的百分比时，所述百分比为体积/重量百分比；本发明涉及固体与液体之间的百分比时，所述百分比为重量/体积百分比；其余为重量/重量百分比。
与现有技术相比，本发明具有下述有益技术效果：
1、本发明制得的活化扩增淋巴细胞具有细胞扩增效率高、细胞活率高、细胞活性稳定、具有较长的有效期、安全有效、副作用小等优点，有效解决了个体化免疫细胞治疗肿瘤技术的关键问题，一是使用具有稳定细胞培养效率的无血清培养体系，持续培养12天而始终保持细胞的杀伤活性和扩增能力，且避免了使用血清可能导致的病源微生物污染及其它对细胞生长的不利因素；二是细胞扩增效率高，不需要使用单核细胞采集机，高效扩增1000倍以上，避免了传统免疫细胞治疗需要使用单采机采取大量起始外周血单个核细胞对患者整体免疫细胞系统的破坏作用；三是细胞活性高、稳定性好，细胞可保存12h而不影响其治疗效果；四是解决了个体化产品的标准化质量控 制体系、规模化生产的技术问题，获得相对质量均一的细胞产品的技术工艺，显著提高治疗靶向性和治疗精准性及患者的依从性。
2、本发明的活化扩增淋巴细胞用于肿瘤患者的治疗后，可以预防肿瘤术后复发，延长肿瘤术后复发期，显著延长肿瘤患者生存期并显著改善患者生存质量，且治疗过程中，减少化疗药物剂量、放疗剂量以及放化疗治疗过程中出现的恶心、呕吐、脱发、失眠等不良反应，无血象降低、肝肾功能损害等严重不良反应，减少肿瘤导致疼痛，显著提高了患者的生活质量。
3、本发明的淋巴细胞扩增方法具有操作简便、质量稳定可控，适宜工业化生产等优点。
附图说明
图1实施例1第一次传代培养时EAL细胞生长状况(250×镜下观察)图2实施例1第一次扩增培养时EAL细胞生长状况(250×镜下观察)图3实施例1第二次扩增培养时EAL细胞生长状况(250×镜下观察)
具体实施方式
以下结合实施例对本发明做进一步的说明，但并不因此将本发明限制在所描述的实施例范围中。
实施例1淋巴细胞活化扩增原液制备
(1)分离外周血单个核细胞(PBMC)并接种活化培养(第0天)
患者1：肝癌患者，年龄：55岁，性别：男。
所述无血清细胞培养基为：GT-T551 H3无血清细胞培养基(培养基中IL-2的浓度为500IU/ml,pH 7.2-7.4)
将抽取的患者全血37ml放入一新250ml离心管中，加入与血样等体积的0.9％氯化钠注射液，混匀后，将其缓慢加到已分装好15ml Ficoll的50ml离心管中(2根离心管)，保证分界面清晰，2000rpm离心20min，离心机设置加速7减速0。吸取界面间细胞层到一新50ml离心管，用0.9％氯化钠注射液1:1混匀，1200rpm离心10min。离心完成后，倾去上清，振开细胞沉淀后，用50ml0.9％氯化钠注射液重悬，1200rpm离心10min。离心完成后，倾去上清，振开细胞沉淀后，加5ml无血清细胞培养基，混匀后，取10μl细胞悬液进行细胞计数。用无血清细胞培养基重悬细胞浓度为0.8×10 6个/ml后，接种到包被有抗人CD3抗体的225cm 2细胞培养瓶中，取样，进行无菌检查并进行标记，并将其放入细胞培养箱进行培养。
淋巴细胞活化扩增原液制备过程中，培养箱培养条件：37℃，7.5％CO 2。
(2)第一次传代培养(第3天)
当培养基颜色变浅变黄时(以采血当日为第0天计，接种培养后第3天)，从培养箱取出细胞培养瓶；显微镜下观察细胞形态：生长状况良好(见图1)。添加50ml无血清细胞培养基倒入细胞培养瓶中，将其放回细胞培养箱进行培养。
(3)第二次传代培养(第4天)
当培养基颜色变浅变黄时，从培养箱取出细胞培养瓶；显微镜下观察细胞形态：生长状况良好。添加140ml无血清细胞培养基，将其放回细胞培养箱进行培养。
(4)第一次扩增培养(第5天)
从培养箱取出细胞培养瓶，显微镜下观察细胞形态：生长状况良好(见图2)，取样进行细胞计数，当活细胞密度不低于1.0×10 6个/ml时，将细胞培养液和760ml无血清细胞培养基，倒入细胞培养袋中，将其放回细胞培养箱进行培养。
(5)第二次扩增培养(第8天)
当培养液颜色偏黄时，取一新细胞培养袋至生物安全柜，通过注射器套筒倒入1000ml无血清细胞培养基(袋A)，封死管口。从培养箱取出含有培养中细胞的细胞培养袋(袋B)，通过无菌接合机将两袋连通，首先使袋A中液体全部流入袋B中，混匀后再放回1000ml至袋A，两袋中液体量均为1000ml。封死管口，抽取样品进行无菌检查。显微镜下观察细胞形态(见图3)，将两袋细胞放回培养箱中进行培养。
(6)纯化、离心浓缩收集细胞制成细胞原液(第12天)
两个细胞培养袋后第4天，从培养箱中取出一细胞培养袋至生物安全柜，将细胞悬液均分到4个250ml离心管中，2000rpm离心5分钟。倾去上清振散沉淀。再从培养箱中取出同一批次另一细胞培养袋至生物安全柜，将细胞悬液均分到原来的4个250ml离心管中，2000rpm离心5分钟。倾去上清振散沉淀，用250ml含0.1％人血白 蛋白的0.9％氯化钠注射液将一个管中细胞分三次冲洗合并到另一个管中，四管合并至两管，2000rpm离心5分钟。倾去上清，振散沉淀，再用200ml 0.1％人血白蛋白氯化钠注射液将两个管中细胞分三次冲洗合并到一个管中，混匀后，取样进行细胞计数，2000rpm离心5分钟，倾去上清后，即为活化扩增淋巴细胞原液，获得2.30×10 10个活化扩增的淋巴细胞。
(7)组合物的制备
活化扩增淋巴细胞按照浓度为8×10 7个/ml重悬细胞原液于含有1％人血白蛋白的注射用生理盐水中，即得。
实施例2淋巴细胞活化扩增原液制备
(1)分离PBMC并接种活化培养(第0天)
患者2：乳腺癌患者，年龄：50，性别：女。
所述无血清细胞培养基为：KBM 581无血清细胞培养基(培养基中IL-2的浓度为500IU/ml,pH 7.2-7.4)
将63ml全血放入一新250ml离心管中，加入与血样等体积的氯化钠注射液，混匀后缓慢加到已分装好15ml Ficoll的50ml离心管中(4根离心管)，保证分界面清晰，2000rpm离心20min，离心机设置加速7减速0。吸取界面间细胞层到一新50ml离心管，用0.9％氯化钠注射液1:1混匀，1200rpm离心10min。离心完成后倾去上清，振开细胞沉淀后用50ml氯化钠注射液重悬，1200rpm离心10min。离心完成后倾去上清，振开细胞沉淀后加5ml无血清细胞培养基，混 匀后取10μl细胞悬液进行细胞计数。用无血清细胞培养基重悬细胞浓度为0.2×10 6个/ml后，接种到包被有抗人CD3抗体的225cm 2细胞培养瓶中。取样，进行无菌检查并进行标记，并将其放入细胞培养箱进行培养。
淋巴细胞活化扩增原液制备过程中，培养箱培养条件：37℃，7.5％CO 2。
(2)第一次传代培养(第4天)
当培养基颜色变浅变黄时(以采血当日为第0天计，接种培养后第4天)，从培养箱取出细胞培养瓶；显微镜下观察细胞形态：生长状况良好。进行细胞计数，添加50ml无血清细胞培养基倒入细胞培养瓶中，将其放回细胞培养箱进行培养。
(3)第二次传代培养(第5天)
当培养基颜色变浅变黄时，从培养箱取出细胞培养瓶；显微镜下观察细胞形态：生长状况良好。进行细胞计数，添加140ml无血清细胞培养基倒入细胞培养瓶中，将其放回细胞培养箱进行培养。
(4)第一次扩增培养(第6天)
从培养箱取出细胞培养瓶，显微镜下观察细胞形态：生长状况良好，取样进行细胞计数，当活细胞密度不低于1×10 6个/ml时，将细胞培养液和750ml无血清细胞培养基倒入细胞培养袋中，将其放回细胞培养箱进行培养。
(5)第二次扩增培养(第8天)
当培养液颜色偏黄时，取一新细胞培养袋至生物安全柜，通过注射器套筒倒入1000ml无血清细胞培养基(袋A)，封死管口。从培养箱取出含有培养中细胞的细胞培养袋(袋B)，通过无菌接合机将两袋连通，首先使袋A中液体全部流入袋B中，混匀后再放回1000ml至袋A，两袋中液体量均为1000ml。封死管口，抽取样品进行无菌检查。显微镜下观察细胞形态，将两袋细胞放回培养箱中进行培养。
(6)纯化、离心浓缩收集细胞制成细胞原液(第12天)
两个细胞培养袋后第4天，从培养箱中取出一细胞培养袋至生物安全柜，将细胞悬液均分到4个250ml离心管中，2000rpm离心5分钟。倾去上清振散沉淀。再从培养箱中取出同一批次另一细胞培养袋至生物安全柜，将细胞悬液均分到原来的4个250ml离心管中，2000rpm离心5分钟。倾去上清振散沉淀，用250ml 0.9％氯化钠注射液将一个管中细胞分三次冲洗合并到另一个管中，四管合并至两管，2000rpm离心5分钟。倾去上清振散沉淀，然后用200ml氯化钠注射液将两个管中细胞分三次冲洗合并到一个管中，混匀后取样进行细胞计数，然后2000rpm离心5分钟，倾去上清后即为活化扩增淋巴细胞原液，获得2.84×10 10个活化扩增的淋巴细胞。
(7)组合物的制备
活化扩增淋巴细胞按照浓度为6×10 7个/ml重悬细胞原液于含有1.5％人血白蛋白的注射用生理盐水中，即得。
实施例3淋巴细胞活化扩增原液制备
(1)分离PBMC并接种活化培养(第0天)
患者3：非小细胞肺癌患者，年龄：53，性别：男。
所述无血清细胞培养基为：KBM 581(CORNING)无血清细胞培养基(培养基中IL-2的浓度为400IU/ml,pH7.2-7.4)
将抽取患者的58ml全血放入一新250ml离心管中，加入与血样等体积的0.9％氯化钠注射液，混匀后缓慢加到已分装好15ml Ficoll的50ml离心管中(4根离心管)，保证分界面清晰，2000rpm离心20min，离心机设置加速7减速0。吸取界面间细胞层到一新50ml离心管，用0.9％氯化钠注射液1:1混匀，1200rpm离心10min。离心完成后倾去上清，振开细胞沉淀后用50ml氯化钠注射液重悬，1200rpm离心10min。离心完成后倾去上清，振开细胞沉淀后加5ml无血清细胞培养基，混匀后取10μl细胞悬液进行细胞计数。用无血清细胞培养基重悬细胞浓度为0.3×10 6个/ml后，接种到包被有抗人CD3抗体的225cm 2细胞培养瓶中。取样，进行无菌检查并进行标记，并将其放入细胞培养箱进行培养。
淋巴细胞活化扩增原液制备过程中，培养箱培养条件：37℃，5％CO 2。
(2)第一次传代培养(第3天)
当培养基颜色变浅变黄时(以采血当日为第0天计，接种培养后第3天)，从培养箱取出细胞培养瓶；显微镜下观察细胞形态：生长状况良好。进行细胞计数，添加50ml无血清细胞培养基倒入细胞培养瓶中，将其放回细胞培养箱进行培养。
(3)第二次传代培养(第4天)
当培养基颜色变浅变黄时，从培养箱取出细胞培养瓶；显微镜下观察细胞形态：生长状况良好。进行细胞计数，添加140ml无血清细胞培养基倒入细胞培养瓶中，将其放回细胞培养箱进行培养。
(4)第一次扩增培养(第6天)
从培养箱取出细胞培养瓶，显微镜下观察细胞形态：生长状况良好，取样进行细胞计数，当活细胞密度不低于1×10 6个/ml时，将细胞培养液和750ml无血清细胞培养基倒入细胞培养袋中，将其放回细胞培养箱进行培养。
(5)第二次扩增培养(第8天)
当培养液颜色偏黄时，取一新细胞培养袋至生物安全柜，通过注射器套筒倒入1000ml无血清细胞培养基(袋A)，封死管口。从培养箱取出含有培养中细胞的细胞培养袋(袋B)，通过无菌接合机将两袋连通，首先使袋A中液体全部流入袋B中，混匀后再放回1000ml至袋A，两袋中液体量均为1000ml。封死管口，抽取样品进行无菌检查。显微镜下观察细胞形态，将两袋细胞放回培养箱中进行培养。
(6)纯化、离心浓缩收集细胞制成细胞原液(第12天)
两个细胞培养袋后第4天，从培养箱中取出一细胞培养袋至生物安全柜，将细胞悬液均分到4个250ml离心管中，2000rpm离心5分钟。倾去上清振散沉淀。再从培养箱中取出同一批次另一细胞培养袋至生物安全柜，将细胞悬液均分到原来的4个250ml离心管中，2000rpm离心5分钟。倾去上清振散沉淀，用250ml 0.9％氯化钠注射 液将一个管中细胞分三次冲洗合并到另一个管中，四管合并至两管，2000rpm离心5分钟。倾去上清，振散沉淀，再用200ml氯化钠注射液将两个管中细胞分三次冲洗合并到一个管中，混匀后取样进行细胞计数，2000rpm离心5分钟，倾去上清后即为扩增活化淋巴细胞原液，获得2.23×10 10个活化扩增的淋巴细胞。
(7)组合物的制备
活化扩增淋巴细胞按照浓度为8×10 7个/ml重悬细胞原液于含有1％人血白蛋白的注射用生理盐水中，即得。
实施例4无血清培养基的筛选
采用与实施例1相同的活化扩增工艺，体外比较研究三种无血清培养基KBM 581(CORNING)、GT-T551H3(TAKARA)、TexMACS GMP Medium(Miltentyi biotec)对EAL细胞制备的影响。
采集3个正常人外周血分离PBMC，进行细胞计数和表型检测，每份PBMC三等分后分别用3种无血清培养基，接种于已包被活化抗体细胞培养瓶进行培养，每4-6天进行一次细胞计数并补充相应细胞培养基，12天后结束培养并进行细胞计数、活率、表型检测、杀伤活性检测。
使用无血清培养基TexMACS GMP Medium培养细胞，细胞在培养6～8天后细胞扩增缓慢，无法满足淋巴细胞体外扩增质量要求。
表1
Figure PCTCN2022142940-appb-000001
Figure PCTCN2022142940-appb-000002
表2
Figure PCTCN2022142940-appb-000003
表3
Figure PCTCN2022142940-appb-000004
Figure PCTCN2022142940-appb-000005
实施例5患者的活化淋巴细胞扩增效果
(1)采集了共计50例患者的外周血，不同性别、年龄和肿瘤类型均有分布。患者基本信息见表4-表6。
表4
性别 男 女 总计
数量 25 25 50
表5
年龄 21-40 41-60 61-80 合计
人数 5 24 21 50
表6
肿瘤类型 人数
血液肿瘤 9
消化道肿瘤 9
肝胆肿瘤 11
呼吸系统肿瘤 9
妇科肿瘤 5
乳腺癌肿瘤 4
泌尿系统肿瘤 3
合计 50
淋巴细胞扩增活化原液的制备参考实施例1。
使用细胞计数板对细胞进行计数，以测定扩增比例。通过台盼蓝染色以及细胞计数获得扩增的细胞活率。通过流式细胞术检测扩增细胞的表型检测使用FITC标记的CD3抗体、APC标记的CD8抗体测定。
表7
Figure PCTCN2022142940-appb-000006
Figure PCTCN2022142940-appb-000007
(2)用上述扩增细胞进行了787次临床杀伤试验，检测细胞的生物学活性KT50，即杀伤达到50％的时间，时间越短代表生物学效力越好。KT50小于1h的次数占到2.29％、KT50在1-2h的次数占到34.81％、KT50在2-3h的次数占到31.77％、KT50在3-4h的次数占到18.3％、KT50在4-5h的次数占到8.39％、KT50在5-6h的次数占到2.92％、KT50在6-8h的次数占到1.52％。
试验例1本发明活化扩增淋巴细胞用于免疫治疗的研究
EAL治疗组的入选标准如下：患者经组织学或细胞学诊断为胃癌、肺癌等，预期寿命>12周，美国东部肿瘤协作组(ECOG)活动状态(performance status,PS)评分0-2；ECOG评分>2分患者被排除在外。所有患者均同意其治疗方案并签署知情同意书。
EAL治疗组：50例，患者年龄、性别、患病信息见实施例5，自体输入扩增活化淋巴细胞制剂。治疗方案：第一疗程，受试者接受4次静脉输注，每一周1次；第二疗程，受试者接受4次静脉输注，每两周1次；第三疗程，接受4次静脉输注，每三周1次；第四疗程，受试者接受8次静脉输注，每四周1次。
对照组：20例，同期进入医院治疗的癌症患者(胃癌、小细胞肺癌、肝癌、白血病患者各5例)，并经历了化疗或放疗，常规治疗方案；排除有细胞治疗史且ECOG>2的患者。治疗期间，研究对象的化疗方案包括：CE(依托泊苷+卡铂)、EP(依托泊苷+顺铂)、I P(伊立替康+顺铂)、PP(紫杉醇+顺铂/奈达铂)和DP(多西紫杉醇+顺铂)、CAV(环磷酰胺+法莫霉素+长春新碱)、NP(纳维滨+顺铂)、培美曲塞单用，单用多西紫杉醇，单用替莫唑胺胶囊，单用足叶乙甙胶囊。治疗病史不完整或失去随访的患者不进行统计。分析免疫细胞治疗的有效性和安全性。
(1)疗效评价
EEAL治疗组的中位OS时间明显长于对照组。EAL免疫治疗延长了癌症患者生存期1-7年；改善化疗导致用药患者的恶心、呕吐、脱发、腹泻、食欲不振、发烧、寒颤、头痛、瘙痒、皮疹、心动过速等副作用及不良反应，无血象降低、肝肾功能损害等严重不良反应，显著提高并改善了患者的生活质量。
(2)安全性评价
EAL治疗组：EAL治疗患者中仅出现1级或2级自限性不良事件 (见表8)；没有患者出现肺部或肾脏症状、感染症状、肝功能恶化或自身免疫性疾病；没有治疗相关死亡记录。
对照组：出现血象降低、损害肝肾功等严重不良反应，且有较严重恶心、呕吐、腹泻、脱发等不良反应。
表8
不良事情 免疫治疗患者
发烧 2(4.00％)
寒冷 1(2.00％)
头痛 1(2.00％)
恶心 0
瘙痒 0
皮疹 1(2.00％)
心动过速 0
腹泻 0
试验例2本发明活化扩增淋巴细胞的联合用药研究
1、患者入组
在100名患有低水平微小残留病(MRD)的低危或中危儿童急性髓系白血病(AML)患者中开展了临床治疗研究。
低水平MRD定义为每次骨髓穿刺时白血病母细胞的比例<0.5％。排除了高危AML、急性早幼粒细胞白血病、疾病复发、诱导化疗后MRD持续阴性的低危疾病以及治疗6个月后MRD>0.5％的患者。根据父母的意愿和经济状况，将参与本研究的100名患者分为2组。联合治疗组(n＝50)接受化疗和EAL治疗，化疗组(n＝50)接受单独化疗。见表9。每位患者的父母或法定监护人都提供了化疗和EAL治疗的书面 知情同意书。
2、治疗方案
(1)化疗方案
诱导化疗包括7天100-150mg/m 2的阿糖胞苷，联合蒽环类药物(2天伊达比星8-10mg/m 2)和3天100-150mg/m 2的依托泊苷。2个周期的诱导后开始巩固化疗。巩固由三个方案组成：方案1，高剂量阿糖胞苷(Ara-c 3g/m 2，3天)和蒽环霉素(8-10mg/m 2伊达比星，2天)；方案2，7天4-6mg/m 2的Harringtonine和7天100-150mg/m 2的阿糖胞苷；方案3，7天100–150mg/m 2的阿糖胞苷，联合蒽环类药物(伊达比星8–10mg/m 2，2天)和3天100–150mg/m 2的依托泊苷。三种方案交替使用，共12-18个月。小剂量Ara-c巩固化疗的每个周期中，给予三联鞘内化疗(甲氨蝶呤、阿糖胞苷和氢化可的松)共4-8次，以预防中枢神经系统白血病。
(2)EAL疗法
对MRD状态持续阳性(<0.5％)的低危和中危AML患者，以及MRD状态变为阳性或MRD负荷增加1个拷贝的患者进行EAL治疗。在本研究中，联合治疗组的患者接受了EAL治疗和化疗的联合治疗。EAL是通过从每位患者收集10-80mL外周血(根据绝对外周血淋巴细胞计数确定)。然后，将PBMC悬浮液置于涂有固定化抗CD3抗体的烧瓶中，按照实施例1的制备方法进行活化、传代、扩增培养；然后收集淋巴细胞并通过孔径为100μm的膜过滤，并重悬于100mL含有1％人血清白蛋白的生理盐水中，用于静脉输注回输给患者个体。最终产品根据 培养后细胞总数分为1-3个输液袋，每袋含1×10 9至2×10 10个CD3+CD8+T细胞。
EAL储存在15–25℃恒温器中，12小时内输注给患者，验证细胞活力始终≥90％。每次输血含有≥3×10 9个细胞。研究方案经伦理审查委员会批准。已获得所有患者父母或法定监护人的知情同意。
3、监测和跟进
监测措施：核心结合因子AML(CBF AML)的MRD通过实时定量逆转录聚合酶链反应(RT-PCR)测定进行监测。对于非CBF AML患者，根据白血病相关免疫表型(LAIP)通过流式细胞术检测MRD。
监测和随访时间点：在诊断时、每个化疗周期之前、然后每3个月间隔至3年和每6个月间隔至5年收集骨髓样本。
4、结果
参与研究的100名儿童在性别、年龄、遗传特征、风险分层、化疗8个月后的MRD状态或化疗强度方面，未观察到显著组间差异。
联合治疗组包括26名男性和24名女性，中位年龄为10.5岁(范围，1-15岁)。其中，低危组42例(84.00％)，中危组8例。
发病时，平均白细胞计数为27.9×10 9(范围，2.1-100×10 9)，2名患者患有中枢神经系统白血病，1名患者出现大下颌肿块。联合治疗组平均接受EAL治疗4次(范围，1-12次)。在本研究中，选择在化疗开始后6-12个月(平均8个月)开始EAL治疗。
联合治疗组的50名儿童共接受220次EAL输血(平均4次输血/患者)。对这220次EAL输血的细胞表型和细胞计数的分析表明，平 均细胞计数为7.5±0.5×10 9/L，平均细胞活力为98.9％。T细胞表型分布如下：CD3+，98.95％；CD3+CD8+，87.26％。
对照组包括25名男性和25名女性，中位年龄为10.6岁(范围，1-15岁)。其中，42人属于低危组(84.00％)，8人属于中危组。发病时，1例患者患有中枢神经系统白血病，1例患者有广泛的胸腰椎浸润。
表9
  联合治疗组 化疗组
N 50 50
年龄(年)    
≥10岁(n) 28 30
＜10岁(n) 26 20
性别    
男(n) 26 25
女(n) 24 25
风险分层    
低风险(n) 42 42
中风险(n) 8 8
化疗周期(n) 9.10±0.38 10.18±0.58
CBF-AML化疗8个月后MRD(％) 0.05±0.02 0.05±0.11
非CBFAML化疗8个月后MRD(％) 0.08±0.20 0.06±0.31
(2)巩固治疗期间两组MRD阴性率
当EAL治疗前MRD<0.5％且平均MRD负荷为0.055％时开始EAL治疗。EAL治疗和化疗后，50名患者中有49名(98.00％)的MRD状态转为阴性，但联合治疗后1名患者的MRD状态仍为阳性。联合治疗组中，巩固化疗期间MRD状态保持阴性的平均时间为7.9个月。
对照组中，30名患者(60.00％)的MRD状态转为阴性。在巩固化疗期间，MRD状态保持阴性的时间平均为4.8个月。
联合治疗组98.00％的患者在巩固化疗期间MRD状态转为阴性，显著高于对照组患者。且联合治疗组的MRD状态保持阴性的时间为7.9个月，显着长于化疗组。
(3)两组生存率比较
联合治疗组的中位EFS为81个月，对照组为50个月。
联合治疗组的平均5年EFS率为88.9％，对照组为69.7％。
联合治疗组中，4名患者(8.00％)出现疾病复发。其中，3名患者出现骨髓复发，1名患者在中枢神经系统复发后出现骨髓复发。
对照组中，12名患者(24.0％)疾病复发，均为骨髓复发。复发的中位时间为15.3个月(范围，6-34)。联合治疗组的中位复发时间为35.8个月，明显长于化疗组。
(4)不良反应
联合治疗组偶有发烧或发冷，在治疗过程中，未观察到严重的炎性细胞因子级联导致器官损伤，也没有细胞治疗相关的死亡。
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明权利要求保护的范围。

Claims (20)

  1. 一种活化扩增淋巴细胞原液的制备方法，包括下述步骤：
    (1)将自体外周全血离心提取获得的外周血单个核细胞(PBMC)与淋巴细胞活化剂置于无血清培养基中共培养，完成淋巴细胞的培养活化，其中，所述外周血单个核细胞在无血清培养基中培养活化的起始密度为(0.2-1.6)×10 6个/ml,细胞活化培养温度为37.0℃±1.0℃，活化培养环境中含7.5％±1.0％CO 2，所述淋巴细胞活化剂选自抗人CD2抗体、抗人CD3抗体、抗人CD28抗体、植物血凝素(PHA)的任一种或其组合或将其固化在载体上的含抗体载体，所述无血清培养基选自KBM 581、GT-T551 H3的任一种或其组合；
    (2)将步骤(1)制得的培养活化淋巴细胞按照细胞密度为(0.5-5)×106个/ml置于无血清培养基中，进行传代培养，制得淋巴细胞活化培养物，其中，传代培养温度为37.0℃±1.0℃，活化培养环境中含7.5％±1.0％CO 2，所述无血清培养基选自KBM 581、GT-T551 H3的任一种或其组合，传代培养代数为1-5代；
    (3)在步骤(2)传代培养获得的淋巴细胞活化培养物中加入其体积5-10倍的无血清培养基，进行扩增培养，其中，扩增培养温度为37.0℃±1.0℃，活化培养环境中含7.5％±1.0％CO 2，所述无血清培养基选自KBM 581、GT-T551 H3的任一种或其组合，扩增培养代数为1-5代；
    (4)离心，洗涤，收集活化扩增的淋巴细胞，即得。
  2. 如权利要求1所述的方法，其特征在于，所述全血离心提取外 周血单个核细胞的方法包括下述步骤：在抗凝处理的全血中加入分离介质和稀释剂，搅拌，混匀，将其加至分离液中，离心1000-3000rpm*10-40min，收集分界面间细胞层，加入洗涤液，离心，洗涤，收集细胞，即得，其中，所述分离介质选自羟乙基淀粉40氯化钠注射液(HES)、Percoll、Ficoll-Paque PLUS的任一种或其组合，全血：稀释剂的体积比为1：1-2，所述稀释剂选自氯化钠注射液、Hank’s缓冲液、Lactated Ringer’s溶液、Dulbecco's磷酸盐缓冲液的任一种或其组合，所述分离液选自羟乙基淀粉40氯化钠注射液(HES)、Ficoll、Lymphoprep、Lymphocyte Separation Media、Cell Separation Media的任一种或其组合，所述分离液的渗透压为300mOsmol/kg-360mOsmol/kg，所述洗涤液选自0.1％人血白蛋白氯化钠注射液、Dulbecco's磷酸盐缓冲液、氯化钠注射液的任一种或其组合。
  3. 如权利要求1-2任一项所述的方法，其特征在于，离心条件为(1500-2500rpm)*(15-30min)，优选为(2000-2500rpm)*(20-25min)，洗涤为离心洗涤，洗涤条件为(500-2000rpm)*(5-20min)，离心洗涤1-5次,优选为(1000-1800rpm)*(10-15min)，离心洗涤2-3次。
  4. 如权利要求1-3任一项所述的方法，其特征在于，所述无血清培养基中任选地添加细胞因子IL-2 300-600IU/ml，优选为400-500IU/ml。
  5. 如权利要求1-4任一项所述的方法，其特征在于，活化扩增淋 巴细胞扩增倍数≥900倍，优选≥1000倍，更优选≥1100倍。
  6. 如权利要求1-5任一项所述的方法，其特征在于，活化扩增淋巴细胞活率≥95％，优选≥98％。
  7. 如权利要求1-6任一项所述的方法，其特征在于，活化扩增淋巴细胞中的CD8+T细胞数量≥1×10 9个、优选为1×10 9-2×10 10个，更优选为4-9.5×10 9个。
  8. 如权利要求1-7任一项所述的方法，其特征在于，活化扩增后的淋巴细胞生物学活性KT50≤8.5，优选为KT50≤4，更优选为KT50≤0.7141。
  9. 一种含有如权利要求1-8任一项所述制备方法制备得到的活化扩增淋巴细胞原液的药物组合物，所述组合物由(1-20)×10 7个/ml的活化扩增淋巴细胞、0.5-2％的人血白蛋白和注射用生理盐水组成，活化扩增淋巴细胞活率≥95％，其中，活化扩增淋巴细胞中的CD8+T细胞为(1-20)×10 7个/ml，所述药物组合物中不含防腐剂和抗生素。
  10. 如权利要求9所述的药物组合物，其特征在于，生物学样品的细胞扩增倍数≥900倍，优选为≥1000倍，更优选为≥1100倍。
  11. 如权利要求9-10任一项所述的药物组合物，其特征在于，活化扩增淋巴细胞生物学活性KT50≤8.5，优选为KT50≤4，更优选为KT50≤0.7141。
  12. 如权利要求9-11任一项所述的药物组合物，其特征在于，患者单次回输活化扩增淋巴细胞总数≤2×10 10个，有效期内细胞活率≥85％。
  13. 如权利要求9-12任一项所述的药物组合物，其特征在于，组合物于15-25℃保存12h内的活化扩增淋巴细胞活率≥85％。
  14. 如权利要求9-13任一项所述的含有活化扩增淋巴细胞原液的药物组合物的制备方法，将活化扩增淋巴细胞原液重悬于含有人血白蛋白的注射用生理盐水中。
  15. 含有如权利要求1-8任一项所述制备方法制备得到的活化扩增淋巴细胞用于免疫治疗的方法，包括下述步骤：
    (1)从个体获得含有淋巴细胞的生物学样品；
    (2)采用本发明的制备方法活化、传代、扩增所述淋巴细胞，获得活化扩增淋巴细胞；
    (3)将所述活化扩增淋巴细胞或其药物组合物回输给所述个体。
  16. 含有如权利要求1-8任一项所述制备方法制备得到的活化扩增淋巴细胞用于免疫治疗的给药方案，其特征在于，包括下述方案：
    (1)第一疗程，受试者接受3-6次静脉输注，每周1次；
    (2)第二疗程，受试者接受3-6次静脉输注，每两周1次；
    (3)第三疗程，受试者接受3-6次静脉输注，每三周1次；
    (4)第四疗程，受试者接受7-10次静脉输注，每四周1次。
  17. 如权利要求16所述的给药方案，其特征在于，包括下述方案：
    (1)第一疗程，受试者接受4-5次静脉输注，每周1次；
    (2)第二疗程，受试者接受4-5次静脉输注，每两周1次；
    (3)第三疗程，受试者接受4-5次静脉输注，每三周1次；
    (4)第四疗程，受试者接受8-9次静脉输注，每四周1次。
  18. 如权利要求15-17任一项所述的给药方案，其特征在于，每次静脉输注的活化扩增淋巴细胞数≥2x10 8个淋巴细胞，优选为≥(2-10)x10 9个淋巴细胞。
  19. 含有如权利要求1-8任一项所述制备方法制备得到的活化扩增淋巴细胞用于制备抗肿瘤免疫治疗的药物、增强抗病毒能力的药物、增强治疗自身免疫性疾病的药物中的任一种的应用。
  20. 含有如权利要求1-8任一项所述制备方法制备得到的活化扩增淋巴细胞联合手术、放疗、化疗的任一种在抗肿瘤或其免疫治疗中的应用。
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