Preparation method and application of CAR-T-19 cell

Abstract

The invention relates to a preparation method of CAR-T-19 cells, the method comprises the preparation of a lentiviral vector, the preparation of CD3 + T cells and the transduction of the lentiviral vector, the preparation of the lentiviral vector comprises the following steps: (1) resuspending stromal cells to a stromal cell system with the cell density of (3.5-4.5) * 10 < 6 > / mL by using an OPM-293CD05 culture medium or DEME culture medium, and culturing for 10-36 hours; and (2) replacing the culture medium with an OPM-293 CD05 culture medium or DEME culture medium containing transfection reagent with final concentration of 3.4 mu g / mL-6 mu g / mL and plasmid with final concentration of 1.5 mu g / mL-1. 7mu g / mL, culturing for 20-45 hours, replacing the culture medium with an OPM-293 CD05 culture medium or DEME culture medium containing endonuclease with final concentration of 10-30 (U / mL), culturing for 1-10 hours, and centrifuging to obtain culture supernatant. According to the method, cell preparation conditions are scientifically screened, the preparation time is remarkably shortened, and the prepared CAR-T-19 cells have the advantages of being high in targeted lethality, high in safety and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a preparation method and application of CAR-T-19 cells. Background Art

[0002] Malignant tumors are one of the diseases with the highest incidence (about 23%) and mortality (about 30%) globally, and their incidence shows an increasing trend year by year, seriously threatening human health and life. In 2020, there were 19.29 million newly diagnosed cancer cases globally, 4.57 million newly diagnosed cancer patients in China, and about 715,000 cancer patients died. It is estimated that by 2030, there will be nearly 26 million newly diagnosed cancer cases and nearly 17 million deaths annually. There is an urgent clinical need for safe and effective anti-tumor treatment technologies. The main treatment methods for tumors or cancers include chemotherapy, radiotherapy, surgical resection, etc. Chemotherapy must consider factors such as the patient's condition, age, treatment history, etc., and select appropriate chemotherapy drugs to inhibit tumor growth and extend the life cycle of tumor patients. However, tumor cells are prone to resist the cytotoxic effects mediated by chemotherapy drugs and differentiate into tumor drug-resistant cells to develop tolerance. Approximately 90% of the reasons for the failure of chemotherapy in patients are related to multidrug resistance (MDR) of tumors. Moreover, radiotherapy and chemotherapy have potential risks and side effects such as low selectivity, large side effects, lethal at high doses, vomiting, nausea, etc. Surgical resection of the tumor lesion site can, to a certain extent, relieve the pain suffered by cancer patients due to cancer, but surgical resection is ineffective for cancers located deep in the body and disseminated tumors.

[0003] In recent years, immunotherapy represented by chimeric antigen receptor-modified T cell therapy (CAR-T) has become a research hotspot in the field of tumor treatment. CD19 is widely expressed on the surface of B cells. Tumor cells (such as acute B-lymphoblastic leukemia and B-cell lymphoma, etc.) caused by mutations in B cells and their precursor cells express CD19, making CD19 one of the tumor treatment targets.

[0004] Chimeric antigen receptor T cells targeting CD19 (CAR-T-19 cells) can directly recognize CD19 molecules and kill tumor cells carrying the target, thereby treating tumors, and have achieved remarkable curative effects in the treatment of various recurrent / refractory B-lymphocyte malignancies such as diffuse large B-cell lymphoma (DLBCL) and B-lymphoblastic leukemia (B-ALL). However, the existing CAR-T cells have the following defects. First, they have insufficient recognition and killing ability for tumor cells with low expression, and CAR-T cells are prone to exhaustion and difficult to maintain in the body for a long time, which limits the long-term benefit of CAR-T treatment for patients. The persistence and treatment effect of its treatment need to be improved, and the tumor recurrence rate is high. Second, lentiviral vectors have relatively high residual host cell proteins (HCP) or DNA (HCD) residues, etc., which may affect the safety and effectiveness of CAR-T cell therapy. Third, the preparation cycle of CAR-T cells is relatively long (such as 2-4 weeks), etc., which limits the timely treatment benefit of clinical patients. Therefore, it is necessary to develop safer and more effective CAR-T cell products. Summary of the Invention

[0005] The object of the present invention is to provide a method for preparing CAR-T-19 cells, including the preparation of a lentiviral vector, the preparation of CD3 + T cells, and the transduction of the lentiviral vector. Among them, the preparation of the lentiviral vector and the preparation of CD3 + T cells have no order of precedence. The preparation of the lentiviral vector includes the following steps:

[0006] (1) Resuspend stromal cells with either OPM-293CD05 medium or DEME medium to a stromal cell system with a cell density of (3.5-4.5)×10 6 cells / mL, and then culture it at 37.0°C ± 1°C and CO2 5-8.0% for 10h-36h;

[0007] (2) Replace the medium in the stromal cell system with either OPM-293CD05 medium or DEME medium containing a transfection reagent with a final concentration of 3.4 μg / mL-6 μg / mL and a plasmid with a final concentration of 1.5 μg / mL-1.7 μg / mL, and then culture it at 37.0 ± 1°C and CO2 5-8.0% for 20h-45h. Then, replace the medium with either OPM-293CD05 medium or DEME medium containing an endonuclease with a final concentration of 10-30 (U / mL), and then culture it at 37.0 ± 1°C and CO2 5-8.0% for 1h-10h, centrifuge, and harvest the culture supernatant to obtain the lentiviral vector.

[0008] In a preferred technical solution of the present invention, the stromal cells are selected from any one of 293F and 293T.

[0009] In a preferred technical solution of the present invention, the transfection reagent is selected from any one of polyethyleneimine (PEI), PEI-MAX or a combination thereof.

[0010] In a preferred technical solution of the present invention, the plasmid is selected from any one of pPVLV2_aCD19, pPVLV3_CAR-19, pMDLg / pRRE, pRSV-Rev, pMD.G or a combination thereof.

[0011] In a preferred technical solution of the present invention, the plasmid is a mixed plasmid.

[0012] In a preferred technical solution of the present invention, the mixed plasmid is selected from any one of pPVLV2_aCD19:pMDLg / pRRE:pRSV-Rev:pMD.G or pPVLV3_CAR-19:pMDLg / pRRE:pRSV-Rev:pMD.G, and is prepared according to a mass ratio of 2.3:2.2:1:1.6 to 4:2:2:1, preferably with a mass ratio of 4:2:2:1.

[0013] In a preferred technical solution of the present invention, the P24 protein in the lentiviral vector ≥ 0.9×10 6 pg / mL, preferably ≥ 1.0×10 6 pg / mL.

[0014] In a preferred technical solution of the present invention, the HCP in the lentiviral vector ≤ 4.0×

[0015] 10 4 ng / mL, preferably ≤ 3.8×10 4 ng / mL.

[0016] In a preferred technical solution of the present invention, the HCD in the lentiviral vector ≤ 80 ng / mL, preferably ≤ 50 ng / mL.

[0017] In a preferred technical solution of the present invention, the preparation of the CD3 + T cells includes the preparation of peripheral blood mononuclear cells (PBMCs), the culture of peripheral blood mononuclear cells and the sorting and enrichment.

[0018] In a preferred technical solution of the present invention, the preparation of peripheral blood mononuclear cells (PBMC) comprises the following steps: adding a separation medium and a diluent to anticoagulated whole blood, stirring and mixing evenly, adding the mixture to a separation liquid, centrifuging (1000 - 3000 rpm * 10 - 40 min), collecting the cell layer between the interfaces, adding a washing solution, centrifuging and washing, and collecting the cells, wherein the separation medium is selected from any one or a combination of hydroxyethyl starch 40 sodium chloride injection (HES), Percoll, and Ficoll-Paque PLUS; the volume ratio of whole blood to the diluent is 1:1 - 2; the diluent is selected from any one or a combination of sodium chloride injection, Hank's buffer solution, Lactated Ringer's solution, and Dulbecco's phosphate buffer solution; the separation liquid is selected from any one or a combination of hydroxyethyl starch 40 sodium chloride injection (HES), Ficoll, Lymphoprep, Lymphocyte Separation Media, and Cell Separation Media; the osmotic pressure of the separation liquid is 300 mOsmol / Kg - 360 mOsmol / Kg; and the washing solution is selected from any one or a combination of 0.1% human albumin sodium chloride injection, Dulbecco's phosphate buffer solution, and sodium chloride injection.

[0019] In a preferred technical solution of the present invention, the centrifugation conditions in the preparation of peripheral blood mononuclear cells are (1500 - 2500 rpm) * (15 - 30 min), preferably (2000 - 2500 rpm) * (20 - 25 min).

[0020] In a preferred technical solution of the present invention, the washing in the preparation of peripheral blood mononuclear cells is centrifugal washing.

[0021] In a preferred technical solution of the present invention, the centrifugal washing conditions in the preparation of peripheral blood mononuclear cells are (500 - 2000 rpm) * (5 - 20 min), and the washing is performed 1 - 5 times.

[0022] In a preferred technical solution of the present invention, the centrifugal washing conditions in the preparation of peripheral blood mononuclear cells are (1000 - 1800 rpm) * (10 - 15 min), and the washing is performed 2 - 3 times.

[0023] 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.

[0024] In a preferred technical solution of the present invention, the culture, sorting, and enrichment of the mononuclear cells comprise the following steps: adjusting the density of PBMC to (0.5 - 4) × 10 6cells / mL, incubate it at 37°C ± 1°C and 5.0% ± 1% CO2 for 0.5 - 1 h, then centrifuge. Resuspend the collected cells in serum-free medium containing 100 - 600 IU / mL of IL-2 to a density of (2.0 - 4.0)×10 7 cells / mL, and then add CD3 / CD28 magnetic beads to the cell system at a ratio of CD3 / CD28 magnetic beads to T cells of 2 - 5:1, and incubate for 0.5 h - 1.0 h to obtain the product. Among them, the serum-free medium is selected from any one or a combination of X-VIVO15, KBM 581, and GT-T551H3.

[0026] In the preferred technical solution of the present invention, the centrifugation conditions for the culture, sorting, and enrichment of mononuclear cells are (400 - 2500 rpm)×(5 - 30 min), preferably (400 - 2500 rpm)×(10 - 25 min).

[0027] In the preferred technical solution of the present invention, the serum-free medium used for the culture, sorting, and enrichment of mononuclear cells contains 150 - 500 IU / mL of IL-2.

[0028] In the preferred technical solution of the present invention, in the culture, sorting, and enrichment of mononuclear cells, the ratio of CD3 / CD28 magnetic beads to T cells is 3 - 4:1.

[0029] In the preferred technical solution of the present invention, the transduction of the lentiviral vector includes the following steps:

[0030] S-1: Resuspend CD3 + T cells in serum-free medium to a density of (1 - 6)×10 6 cells / mL, incubate it at 37°C ± 1°C and 5.0% ± 1% CO2 for 24 h - 48 h, then replace it with serum-free medium containing a final concentration of 2 - 8 μg / mL of transduction-promoting reagent and a final concentration of 0.2 - 1 MOI of lentiviral vector, and then incubate it at 37°C ± 1°C and 5.0% ± 1% CO2 for 24 h - 48 h to obtain CD3 + T cells transduced with the lentiviral vector. Among them, the transduction-promoting reagent is selected from any one or a combination of Synperonic F 108 and Polybrene;

[0031] S-2: Resuspend CD3 + T cells transduced with the lentiviral vector in serum-free medium to a cell density of (0.2 - 1)×10 6 cells / mL, centrifuge at (500 - 2000 rpm)×(5 - 20 min), collect the cells and resuspend them in serum-free medium to a cell density of (0.2 - 1)×10 6cells / mL, and culture it under the conditions of 37°C and 5% CO2 for 24h - 72h;

[0032] S-3: Resuspend it with serum-free medium to a cell density of (0.2 - 1)×10 6 cells / mL, culture it under the conditions of 37 ± 1°C and 5 ± 0.5% CO2 for 24h - 26h, remove the CD3 / CD28 magnetic beads, centrifuge and wash, then resuspend to obtain it.

[0033] Among them, the serum-free medium described in S-1 to S-3 is selected from any one or a combination of X-VIVO15, KBM 581, and GT-T551 H3.

[0034] In a preferred technical solution of the present invention, the CD3 + T cell density in S-1 is (1.5 - 4)×10 6 cells / mL.

[0035] In a preferred technical solution of the present invention, the final concentration of the transduction promoter in S-1 is 4 - 8 μg / mL.

[0036] In a preferred technical solution of the present invention, the final concentration of the lentiviral vector in S-1 is 0.2 - 0.5 MOI.

[0037] In a preferred technical solution of the present invention, the centrifugal washing solution in S-3 contains physiological saline with 0.1% human albumin.

[0038] In a preferred technical solution of the present invention, the preparation duration of the CAR-T-19 cells ≤ 14 days, preferably 5 days - 12 days, more preferably 5 days - 7 days.

[0039] In a preferred technical solution of the present invention, the cell viability of the CAR-T-19 cells ≥ 80%, preferably ≥ 90%.

[0040] In a preferred technical solution of the present invention, the transduction efficiency of the aCD19CAR+ gene in the CAR-T-19 cells ≥ 15%, preferably ≥ 25%, more preferably ≥ 50%.

[0041] In a preferred technical solution of the present invention, the cell viability of the CAR-T-19 cells after being preserved for 18h - 24h ≥ 75%, preferably ≥ 80%.

[0042] In a preferred technical solution of the present invention, the inserted gene copy number VCN of the CAR-T-19 cells ≤ 5 copies / cell, preferably ≤ 1 copies / cell.

[0043] Another object of the present invention is to provide a CAR-T-19 injection solution, and the injection solution contains (1×10 5 -2×108 ) CAR-T-19 cells per mL and made with normal saline containing 0.5 - 5% human albumin.

[0044] In a preferred technical solution of the present invention, the density of CAR-T-19 cells contained in the injection solution is (1×10 6 -1×10 8 ) cells per mL, preferably (1×10 6 -1×10 7 ) cells per mL.

[0045] In a preferred technical solution of the present invention, the human albumin contained in the normal saline is 0.5 - 5%, preferably 1 - 3%, more preferably 1.5 - 2.5%.

[0046] In a preferred technical solution of the present invention, the cell viability of CAR-T-19 cells ≥ 80%, preferably ≥ 90%.

[0047] In a preferred technical solution of the present invention, the transduction efficiency of the aCD19CAR+ gene of CAR-T-19 cells ≥ 15%, preferably ≥ 25%, more preferably ≥ 50%.

[0048] In a preferred technical solution of the present invention, the cell viability of CAR-T-19 cells after being preserved for 18h - 24h ≥ 75%, preferably ≥ 80%.

[0049] In a preferred technical solution of the present invention, the inserted gene copy number VCN of CAR-T-19 cells ≤ 5 copies / cell, preferably ≤ 1 copies / cell.

[0050] In a preferred technical solution of the present invention, the said injection solution is used for either autologous reinfusion or reinfusion to other individuals, preferably the reinfusion dosage is (0.5 - 5)×10 6 CAR-T-19 cells / Kg.

[0051] In a preferred technical solution of the present invention, the reinfusion dosage of the said injection solution is (0.5 - 5)×10 6 CAR-T-19 cells / Kg.

[0052] Another object of the present invention is to provide a preparation method of a CAR-T-19 injection solution. Resuspend the CAR-T-19 cells prepared by the present invention in normal saline containing 0.5 - 5% human albumin until the density is (1×10 5 -2×10 8 ) cells per mL, then it is obtained.

[0053] In a preferred technical solution of the present invention, the density of CAR-T-19 cells contained in the injection solution is (1×10 6-1×10 8 ) cells / mL, preferably (1×10 6 -1×10 7 ) cells / mL.

[0054] In a preferred technical solution of the present invention, the human albumin contained in the physiological saline is 0.5-5%, preferably 1-3%, and more preferably 1.5-2.5%.

[0055] In a preferred technical solution of the present invention, the cell viability of CAR-T-19 cells ≥80%, preferably ≥90%.

[0056] In a preferred technical solution of the present invention, the transduction efficiency of the aCD19CAR+ gene of CAR-T-19 cells ≥15%, preferably ≥25%, and more preferably ≥50%.

[0057] In a preferred technical solution of the present invention, the cell viability of CAR-T-19 cells stored for 18h-24h ≥75%, preferably ≥80%.

[0058] In a preferred technical solution of the present invention, the copy number of the inserted gene VCN of CAR-T-19 cells ≤5 copies / cell, preferably ≤1 copies / cell.

[0059] In a preferred technical solution of the present invention, the injection is used for either autologous reinfusion or reinfusion to other individuals.

[0060] In a preferred technical solution of the present invention, the reinfusion dosage of the injection is (0.5-5)×10 6 CAR-T-19 cells / Kg.

[0061] Another object of the present invention is to provide the application of the CAR-T-19 injection of the present invention in the preparation of any product for anti-tumor or anti-tumor immunotherapy.

[0062] In a preferred technical solution of the present invention, the tumor is selected from any one of recurrent B-lymphoblastic leukemia (B-ALL), refractory B-lymphoblastic leukemia (B-ALL), acute B-lymphoblastic leukemia patients (B-ALL), diffuse large B-cell lymphoma (DLBCL) or its complications.

[0063] In a preferred technical solution of the present invention, the leukemia is selected from any one of acute lymphoblastic leukemia, acute myeloid leukemia, B-cell chronic lymphocytic leukemia, chronic myeloid leukemia or its complications.

[0064] In a preferred technical solution of the present invention, the acute myeloid leukemia is selected from any one or a combination of acute granulocytic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute basophilic leukemia, acute eosinophilic leukemia, acute megakaryocytic leukemia, acute erythroleukemia, and acute panmyelosis with myelofibrosis.

[0065] In a preferred technical solution of the present invention, the infusion dosage of CAR-T-19 cells or their injection solution is (0.5 - 5)×10 6 CAR-T-19 cells / Kg.

[0066] In a preferred technical solution of the present invention, the CAR-T-19 cells or their injection solution are used for adult patients or pediatric patients.

[0067] Another object of the present invention is to provide a method for immunotherapy using the CAR-T-19 injection solution of the present invention, comprising the following steps:

[0068] (1) Prepare CAR-T-19 cells and their injection solution;

[0069] (2) Autologous infusion or infusion to other individuals of CAR-T-19 cells or their injection solution at a dose of (0.5 - 5)×10 6 CAR-T-19 cells / Kg.

[0070] In a preferred technical solution of the present invention, the CAR-T-19 cells or their injection solution are used for adult patients or pediatric patients.

[0071] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between a liquid and a solid, the percentage is volume / weight percentage; when the present invention relates to the percentage between a solid and a liquid, the percentage is weight / volume percentage; and the rest are weight / weight percentages.

[0072] For the purpose of clearly expressing the present invention, the present invention defines the following terms:

[0073] 90-day objective response rate (Objective Response Rate, ORR): refers to the response rate obtained based on objective measurements within 90 days after the patient receives treatment, including changes in tumor size, changes in blood biochemical indicators, etc.

[0074] 28-day response rate: refers to the sum of the complete remission rate (CR) and partial remission rate (PR) of the patient's condition within 28 days after receiving treatment.

[0075] The present invention uses a cell counter or a cell proliferation kit to detect aCD19CAR+.

[0076] The present invention uses any one of the methods such as Southern hybridization, real-time quantitative PCR (qPCR), digital PCR (dPCR), whole-genome sequencing (WGS), etc. to detect the inserted gene copy number VCN (copies / cell).

[0077] The present invention uses the biological activity of CAR-T-19 cells (RTCA, taking the effector-to-target ratio of 3:1 as an example) to characterize its therapeutic activity.

[0078] Add 100 μl / well of target cells with a density of 2×10 5 cells / ml (using K562 cells and CD19-K562 cells, that is, K562 cells loaded with CD19) for plating, and set the effector-to-target ratio parameter (such as the effector-to-target ratio of 3:1) for baseline measurement. After 16 - 24 h of detecting the plated target cells, add 50 μl / well of effector cells with a density of 1.6×10 7 cell / ml (i.e., CAR-T-19 cells) according to the set effector-to-target ratio, and then track data such as cell morphology, proliferation, and differentiation in real time, dynamically, and quantitatively, that is, the change in impedance generated by the contact between live cells and the microelectrodes in the detection plate. Real-time monitor the generated cell index curve to show the adhesion, spreading, growth, death, etc. states of cells at different time periods. By analyzing the change in the cell index, the killing efficiency of effector cells against target cells can be calculated, and then KT 50 (that is, the time taken for effector cells to kill 50% of target cells) can be calculated through the killing efficiency curve, and the KT 50 value is used as a determination of the biological activity of effector cells. The smaller the KT 50 value, the higher the killing efficiency of effector cells and the higher the biological activity. Conversely, the biological activity of effector cells is lower.

[0079] Compared with the prior art, the present invention has the following beneficial effects:

[0080] The present invention scientifically screens the preparation conditions of CAR-T-19 cells, including the preparation of lentiviral vectors, the preparation of CD3 + T cells, and the optimization of the transduction preparation parameters of lentiviral vectors. First, it significantly improves the purity of lentiviral vectors, significantly reduces the residues of host cell proteins (HCP) and DNA (HCD) in lentiviral vectors, and significantly improves the safety and effectiveness of the prepared CAR-T-19 cells; second, it significantly improves the lentiviral transduction efficiency, significantly shortens the preparation time of CAR-T-19 cells, and is conducive to patients obtaining effective treatment in a timely manner; third, the prepared CAR-T-19 cells have the advantages of high biological activity, strong target killing ability, good stability, and high safety. Detailed implementation mode

[0081] The following further explains and describes the details of the present invention in conjunction with specific embodiments, but does not limit the protection scope of the present invention hereby.

[0082] The mixed plasmid in the specific embodiment is any one of pPVLV2_aCD19:pMDLg / pRRE:pRSV-Rev:pMD.G or pPVLV3_CAR-19:pMDLg / pRRE:pRSV-Rev:pMD.G prepared according to a mass ratio of 4:2:2:1. pMDLg / pRRE is purchased from Pharosvaccine Inc., pRSV-Rev is purchased from Gyeonggi, pMD.G is purchased from Pharosvaccine Inc., Republic of Korea. pPVLV2_aCD19 is prepared with reference to Example 1 of CN110863014A, and SEQ ID NO:2 is the pPVLV2 vector containing the CAR-19 coding sequence; pPVLV3_CAR-19 is prepared using pPVLV3 (purchased from Pharosvaccine Inc., Republic of Korea) and the sequences and methods disclosed in CN110863014A, wherein the amino acid sequence of CAR-19 is SEQ ID NO:16 and the nucleotide sequence is SEQ ID NO:8.

[0083] OPM-293CD05 medium (OPM, product number: OPM81075-001). HEK293T cells: purchased from the Cell Bank of the Chinese Academy of Sciences. K562 cells (human chronic myeloid leukemia cells): purchased from the Cell Bank of the Chinese Academy of Sciences. IL-2: purchased from Beijing Shuanglu Pharmaceutical Co., Ltd. DEME medium: purchased from Thermo Fisher Scientific. X-VIVO15 serum-free medium: purchased from LONZA. Dynabeads CD3 / CD28: purchased from Thermo Fisher Scientific. Transfection reagent PEI: purchased from Polysciences Incorporate. Endonuclease Benzonase: purchased from Nanjing Novozymes Biotech Co., Ltd. F 108: purchased from SIGMA-ALDRICH. Ficoll: purchased from cytiva.

[0084] Example 1 Preparation of the CAR-T-19 cells and their injection solution of the present invention

[0085] The preparation of the CAR-T-19 cells and their injection solution includes the following steps:

[0086] 1. CD3+ Preparation of T cells

[0087] (1) Peripheral blood was collected from a 16-year-old female patient with relapsed B-lymphoblastic leukemia (BALL) and anticoagulated. 38 ml of the collected peripheral blood was aspirated and placed in a 250 mL centrifuge tube. An equal volume of normal saline was added to the blood sample. After mixing, it was slowly added to two 50 mL centrifuge tubes pre-filled with 15 mL of Ficoll, ensuring a clear interface. Centrifugation was performed (2000 rpm * 20 min). The cell layer at the interface was aspirated and placed in a 50 mL centrifuge tube, mixed 1:1 with normal saline, centrifuged (1200 rpm * 10 min), and the cell pellet was collected and resuspended in 50 mL of normal saline, then centrifuged (1200 rpm * 10 min) to collect the cells;

[0088] (2) 5 mL of X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL of IL-2 was added to the collected cells, and the cell density was resuspended to 0.8×10 6 cells / mL. Then, it was inoculated into a 225 cm 2 cell culture flask and cultured at 37 ± 1°C and 5 ± 0.5% CO2 for 0.5 h - 1 h. Centrifugation was performed (400 g * 10 min), and the cells were collected and resuspended in X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL of IL-2 to a cell density of 3.0×10 7 cells / mL. Then, magnetic beads (Dynabeads CD3 / CD28) were added according to the ratio of magnetic beads to T cells of 3:1, cultured for 0.5 h, and the supernatant was collected to obtain CD3 + T cells;

[0089] 2. Preparation of lentiviral vector

[0090] (1) HEK293T cells were resuspended in DEME medium (pH 7.2 - 7.4) to a cell density of 3.5×10 6 cells / mL and cultured at 37.0°C and 5.0% CO2 for 24 h;

[0091] (2) Replace the medium in the HEK293T cell culture system with OPM-293CD05 medium (pH 7.2 - 7.4) containing PEI at a final concentration of 3.4 μg / mL and mixed plasmids at a final concentration of 1.7 μg / mL. Incubate it at 37.0 ± 1°C and 5.0% CO2 for 44 h, then replace it with OPM-293CD05 medium (pH 7.2 - 7.4) containing Benzonase nuclease at a final concentration of 20 U / mL. Incubate it at 37.0 ± 1°C and 5.0% CO2 for 4 h, centrifuge (500 g * 5 min), harvest the culture supernatant to obtain the lentiviral vector. Among them, the mixed plasmids are composed of pPVLV2_aCD19:pMDLg / pRRE:pRSV-Rev:pMD.G at a mass ratio of 4:2:2:1;

[0092] Detected by the method of the present invention, the P24 protein in the obtained lentiviral vector is 1.08×10 6 pg / mL, the HCP is 3.95×10 4 ng / mL, and the HCD is 61.53 ng / mL;

[0093] 3. Transduction of the lentiviral vector

[0094] (1) Resuspend CD3 + T cells in X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL IL-2 to a density of 4×10 6 cells / mL, and incubate it at 37 ± 1°C and 5 ± 0.5% CO2 for 24 h;

[0095] (2) Replace the medium in the CD3 + T cell culture system with X-VIVO15 serum-free medium containing F108 at a final concentration of 4 μg / mL and the lentiviral vector at a final concentration of 0.46 MOI. Incubate it at 37 ± 1°C and 5 ± 0.5% CO2 for 24 h. Collect the cells, wash them with X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL IL-2, then centrifuge (526 g * 5 min), and resuspend them in X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL IL-2 to a cell density of 0.5×10 6 cells / mL, and incubate it at 37 ± 1°C and 5 ± 0.5% CO2 for 48 h;

[0096] (3) CD3 +The culture medium of the T cell culture system was replaced with X-VIVO15 serum-free medium containing 500 IU / mL IL-2 (pH 7.2 - 7.4), and the cell density was resuspended to 0.5×10 6 cells / mL, and it was cultured at 37 ± 1°C and 5 ± 0.5% CO2 for 24 h;

[0097] (4) After removing the magnetic beads Dynabeads CD3 / CD28 from the cultured cells three times, the cells were washed 3 times by centrifugation (526 g * 5 min) with physiological saline containing 0.1% human albumin, and then resuspended with physiological saline containing 2% human albumin to a CAR-T-19 cell density of 0.5×10 6 cells / mL to prepare the CAR-T-19 injection.

[0098] Detected according to the method of the present invention, the cell viability of CAR-T-19 cells was 91.40%, the gene transduction efficiency of aCD19CAR+ was 37.55%, and the cell viability of CAR-T-19 cells stored for 18 h - 24 h was > 75%.

[0099] From the date of collecting the patient's blood sample to the 5th day, the culture and preparation of CAR-T-19 cells were completed and then autologously transfused into the patient.

[0100] Example 2 Preparation of the CAR-T-19 cells and their injection of the present invention

[0101] The preparation of CAR-T-19 cells and their injection includes the following steps:

[0102] 1. Preparation of CD3 + T cells

[0103] (1) Peripheral blood was collected from a 14-year-old male patient with refractory B-lymphoblastic leukemia (BALL) and anticoagulated. 37 ml of the collected peripheral blood was taken and placed in a 250 mL centrifuge tube. An equal volume of physiological saline was added, and after mixing, it was slowly added to two 50 mL centrifuge tubes each containing 15 mL of Ficoll, ensuring a clear interface, and then centrifuged (2000 rpm * 20 min). The cell layer between the interfaces was taken and placed in a 50 mL centrifuge tube, mixed 1:1 with physiological saline, centrifuged (1200 rpm * 10 min), the cell precipitate was collected and resuspended with 50 mL of physiological saline, and then centrifuged (1200 rpm * 10 min) to collect the cells;

[0104] (2) 5 mL of X-VIVO15 serum-free medium containing 500 IU / mL IL-2 (pH 7.2 - 7.4) was added to the collected cells, and the cell density was resuspended to 0.8×10 6After reaching 2.0×10 cells / mL, inoculate it into a 225 cm 2 cell culture flask, and then place it in an incubator at 37±1°C and 5±0.5% CO2 for 0.5 - 1 h. Centrifuge (400 g * 10 min), collect the cells, and resuspend them in X-VIVO15 serum-free medium containing 500 IU / mL IL-2 (pH 7.2 - 7.4) to a cell density of 2.0×10 7 cells / mL. Then, add magnetic beads (Dynabeads CD3 / CD28) at a ratio of magnetic beads to T cells of 3:1, culture for 0.8 h, collect the supernatant, and obtain CD3 + T cells;

[0105] 2. Preparation of lentiviral vector

[0106] (1) Resuspend HEK293T cells in DEME medium (pH 7.2 - 7.4) to a cell density of 3.5×10 6 cells / mL, and place it in an incubator at 37.0°C and 5.0% CO2 for 24 h;

[0107] (2) Replace the medium of the HEK293T cell culture system with OPM-293CD05 medium (pH 7.2 - 7.4) containing PEI at a final concentration of 3.4 μg / mL and mixed plasmids at a final concentration of 1.7 μg / mL. Place it in an incubator at 37.0±1°C and 5.0% CO2 for 44 h, then replace it with OPM-293CD05 medium (pH 7.2 - 7.4) containing Benzonase at a final concentration of 20 U / mL, and place it in an incubator at 37.0±1°C and 5.0% CO2 for 4 h. Centrifuge (500 g * 5 min), harvest the culture supernatant, and obtain the lentiviral vector. Among them, the mass ratio of pPVLV2_aCD19:pMDLg / pRRE:pRSV-Rev:pMD.G in the mixed plasmids is 4:2:2:1.

[0108] Detected according to the method of the present invention, the P24 protein in the prepared lentiviral vector is 0.98×10 6 pg / mL, the HCP is 3.83×10 4 ng / mL, and the HCD is 42.15 ng / mL.

[0109] 3. Transduction of lentiviral vector

[0110] (1) Resuspend CD3+ T cells in X-VIVO15 serum-free medium containing 500 IU / mL IL-2 (pH 7.2 - 7.4) to a cell density of 2×10 6 cells / mL, and place it in an incubator at 37±1°C and 5±0.5% CO2 for 24 h;

[0111] (2) Replace the culture medium in the CD3+ T cell culture system with X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing F 108 at a final concentration of 2 μg / mL and lentiviral vector at a final concentration of 0.23 MOI. Incubate it at 37 ± 1 °C and 5 ± 0.5% CO2 for 24 h. Collect the cells, wash them with X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL IL-2, centrifuge (526 g * 5 min), and then resuspend the cells with X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL IL-2 to a cell density of 0.5×10 6 cells / mL. Incubate it at 37 ± 1 °C and 5 ± 0.5% CO2 for 48 h;

[0112] (3) Replace the culture medium in the CD3+ T cell culture system with X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL IL-2, adjust the cell density to 0.5×10 6 cells / mL, and incubate it at 37 ± 1 °C and 5 ± 0.5% CO2 for 24 h;

[0113] (4) After removing the magnetic beads Dynabeads CD3 / CD28 from the cultured cells three times, wash them by centrifugation (526 g * 5 min) with physiological saline containing 0.1% human albumin three times, and then resuspend the CAR-T-19 cells with physiological saline containing 2% human albumin to a cell density of 1×10 6 cells / mL to prepare the CAR-T-19 injection.

[0114] After detection, the cell viability of CAR-T-19 cells is 93.80%, the gene transduction efficiency of aCD19CAR+ is 34.79%, and the cell viability of cells stored for 18 h - 24 h > 75%.

[0115] From the date of collecting the patient's blood sample to the 5th day, complete the culture and preparation of CAR-T-19 cells and reinfuse them into the patient's own body.

[0116] Example 3 The preparation of the CAR-T-19 cells and their injection of the present invention

[0117] The preparation of CAR-T-19 cells and their injection includes the following steps:

[0118] 1. Preparation of CD3 + T cells

[0119] (1) Peripheral blood was collected from a 17-year-old female patient with relapsed B-lymphoblastic leukemia (BALL) and anticoagulated. 37 ml of the collected peripheral blood was aspirated and placed in a 250 mL centrifuge tube. An equal volume of normal saline was added to the blood sample, and after mixing, it was slowly added to two 50 mL centrifuge tubes each containing 15 mL of Ficoll, ensuring a clear interface, and centrifuged (2000 rpm * 20 min). The cell layer at the interface was aspirated and placed in a 50 mL centrifuge tube, mixed 1:1 with normal saline, centrifuged (1200 rpm * 10 min), the cell pellet was collected, resuspended in 50 mL of normal saline, and centrifuged (1200 rpm * 10 min) to collect the cells;

[0120] (2) 5 mL of X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL IL-2 was added to the collected cells to resuspend the cell density to 0.8×10 6 cells / mL, and then it was inoculated into a 225 cm 2 cell culture flask. Then it was cultured at 37 ± 1°C and 5 ± 0.5% CO2 for 0.5 h - 1 h, centrifuged (400 g * 10 min), the cells were collected, resuspended in X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL IL-2 to a cell density of 4.0×10 7 cells / mL, and then magnetic beads (Dynabeads CD3 / CD28) were added according to the ratio of magnetic beads to T cells of 3:1, mixed for 1.0 h, the supernatant was collected, and CD3 + T cells were prepared;

[0121] 2. Preparation of lentiviral vector

[0122] (1) HEK293T cells were placed in DEME medium (pH 7.2 - 7.4) to prepare a cell system with a cell density of 3.5×10 6 cells / mL, and it was cultured at 37.0°C and 5.0% CO2 for 24 h;

[0123] (2) Replace the medium of the HEK293T cell culture system with OPM-293CD05 medium (pH 7.2 - 7.4) containing PEI at a final concentration of 3.4 μg / mL and a mixed plasmid at a final concentration of 1.7 μg / mL. Incubate it at 37.0 ± 1°C and 5.0% CO₂ for 44 h, then replace it with OPM-293CD05 medium (pH 7.2 - 7.4) containing Benzonase at a final concentration of 20 U / mL. Incubate it at 37.0 ± 1°C and 5.0% CO₂ for 4 h, centrifuge (500 g * 5 min), harvest the culture supernatant to obtain the lentiviral vector. Among them, the mass ratio of pPVLV2_aCD19:pMDLg / pRRE:pRSV-Rev:pMD.G in the mixed plasmid is 4:2:2:1;

[0124] Detected by the method of the present invention, the P24 protein in the prepared lentiviral vector is 1.06×10 6 pg / mL, the HCP is 3.59×10 4 ng / mL, and the HCD is 84.55 ng / mL.

[0125] 3. Transduction of the lentiviral vector

[0126] (1) Resuspend CD3⁺ T cells in X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL IL-2 to a density of 1×10 6 cells / mL, and incubate it at 37 ± 1°C and 5 ± 0.5% CO₂ for 24 h;

[0127] (2) Replace the medium of the CD3⁺ T cell culture system with X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing F 108 at a final concentration of 8 μg / mL and the lentiviral vector at a final concentration of 0.2 MOI. Incubate it at 37 ± 1°C and 5 ± 0.5% CO₂ for 24 h. After collecting the cells, wash them with X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL IL-2, then centrifuge (526 g * 5 min), and resuspend the cells in X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL IL-2 to a density of 0.5×10 6 cells / mL, and incubate it at 37 ± 1°C and 5 ± 0.5% CO₂ for 48 h;

[0128] (3) Replace the X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL IL-2 to adjust the cell density to 0.5×10 6cells / mL, and culture them sub - passaged for 24 h under the conditions of 37 ± 1 °C and 5 ± 0.5% CO2;

[0129] (4) After removing the magnetic beads Dynabeads CD3 / CD28 from the cultured cells three times, wash them 3 times by centrifugation (526 g * 5 min) with physiological saline containing 0.1% human albumin, and then resuspend the CAR - T - 19 cells with physiological saline containing 2% human albumin at a density of 2×10 6 cells / mL to prepare the CAR - T - 19 injection.

[0130] Detected according to the method of the present invention, the cell viability of the prepared CAR - T - 19 cells is 93.90%, the gene transduction efficiency of aCD19CAR+ is 38.99%, and the cell viability after preservation for 18 h - 24 h is > 75%.

[0131] From the date of collecting the patient's blood sample to the 5th day, complete the culture and preparation of CAR - T - 19 cells and reinfuse them into the patient's own body.

[0132] Example 4 Preparation of the CAR - T - 19 cells and their injection of the present invention

[0133] The preparation of CAR - T - 19 cells and their injection includes the following steps:

[0134] 1. CD3 + Preparation of T cells

[0135] (1) Collect peripheral blood from a 16 - year - old female patient with relapsed B - lymphoblastic leukemia (BALL) and anticoagulate it. Aspirate 42 ml of the collected peripheral blood, place it in a 250 mL centrifuge tube, add physiological saline with the same volume as the blood sample, mix well, and then slowly add it to two 50 mL centrifuge tubes filled with 15 mL Ficoll respectively, ensuring a clear interface, and centrifuge (2000 rpm * 20 min). Place the cell layer between the interfaces aspirated into a 50 mL centrifuge tube, mix it 1:1 with physiological saline, centrifuge (1200 rpm * 10 min), collect the cell precipitate, resuspend it with 50 mL physiological saline, and centrifuge (1200 rpm * 10 min) to collect the cells;

[0136] (2) Add 5 mL of X - VIVO15 serum - free medium (pH 7.2 - 7.4) containing 500 IU / mL IL - 2 to the collected cells to resuspend the cell density to 0.8×10 6 cells / mL, and then inoculate it into a 225 cm 2In a cell culture flask, place it at 37 ± 1 °C and 5 ± 0.5% CO2 for 0.5 h - 1 h, centrifuge (400 g * 10 min), collect the cells and resuspend them in X-VIVO15 serum-free medium containing 500 IU / mL IL-2 (pH 7.2 - 7.4) to a cell density of 3.0×10 7 cells / mL. Then, add magnetic beads (Dynabeads CD3 / CD28) according to a magnetic bead:T cell number ratio of 3:1, mix for 0.5 h, collect the supernatant, and prepare CD3 + T cells;

[0137] 2. Preparation of lentiviral vector

[0138] (1) Resuspend HEK293T cells in DEME medium (pH 7.2 - 7.4) to a cell density of 3.5×10 6 cells / mL, and culture them at 37.0 °C and 5.0% CO2 for 24 h;

[0139] (2) Replace the medium in the HEK293T cell culture system with OPM-293CD05 medium (pH 7.2 - 7.4) containing a final concentration of 3.4 μg / mL of PEI and a final concentration of 1.7 μg / mL of mixed plasmids. Place it at 37.0 ± 1 °C and 5.0% CO2 for 44 h, then replace it with OPM-293CD05 medium (pH 7.2 - 7.4) containing a final concentration of 20 U / mL of Benzonase nuclease. Place it at 37.0 ± 1 °C and 5.0% CO2 for 44 h, then add Benzonase nuclease at a final concentration of 20 U / mL and culture for 4 h. Centrifuge (500 g * 5 min) to harvest the culture supernatant and obtain the lentiviral vector. Among them, the mass ratio of pPVLV2_aCD19:pMDLg / pRRE:pRSV-Rev:pMD.G in the plasmid is 4:2:2:1.

[0140] Detected according to the method of the present invention, the P24 protein in the prepared lentiviral vector > 1×10 6 pg / mL, HCP < 5×10 4 ng / mL, HCD < 50 ng / mL.

[0141] 3. Transduction of lentiviral vector

[0142] (1) Resuspend CD3 + T cells to a density of 1.5×10 6cells / mL, and culture it under the conditions of 37±1°C and 5±0.5% CO2 for 24 h;

[0143] (2) Replace the medium of the CD3 + T cell culture system with X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing Polybrene at a final concentration of 2 μg / mL and lentiviral vector at a final concentration of 0.46 MOI. Culture it under the conditions of 37±1°C and 5±0.5% CO2 for 24 h. After collecting the cells, wash them with X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL IL-2, centrifuge (526 g * 5 min), and then resuspend the cells with X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL IL-2 to a cell density of 0.5×10 6 cells / mL, and culture it under the conditions of 37±1°C and 5±0.5% CO2 for 48 h;

[0144] (3) Replace the medium of the CD3 + T cell culture system with X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL IL-2, and resuspend the cells to a cell density of 0.5×10 6 cells / mL, and culture it under the conditions of 37±1°C and 5±0.5% CO2 for 24 h;

[0145] (4) After removing the magnetic beads Dynabeads CD3 / CD28 from the cultured cells three times, wash them 3 times by centrifugation (526 g * 5 min) with physiological saline containing 0.1% human albumin, and then resuspend the CAR-T-19 cells with physiological saline containing 2% human albumin to a cell density of 0.5×10 6 cells / mL to prepare the CAR-T-19 injection.

[0146] Detected according to the method of the present invention, the cell viability of CAR-T-19 cells is 90.8%, the gene transduction efficiency of aCD19CAR+ is 52.92%, and the cell viability of CAR-T-19 stored for 18 h - 24 h > 75%.

[0147] From the date of collecting the patient's blood sample to the 5th day, complete the culture and preparation of CAR-T-19 cells and reinfuse them into the patient's autologous body.

[0148] Example 5 Preparation of the CAR-T-19 cells and their injection of the present invention

[0149] The preparation of CAR-T-19 cells and their injection includes the following steps:

[0150] 1. CD3+ Preparation of T cells

[0151] (1) Peripheral blood was collected from a 22-year-old female patient with refractory B-lymphocyte leukemia (BALL) and anticoagulated. 38 ml of the collected peripheral blood was aspirated and placed in a 250 mL centrifuge tube. An equal volume of normal saline was added to the blood sample. After mixing, it was slowly added to two 50 mL centrifuge tubes each containing 15 mL of Ficoll, ensuring a clear interface, and centrifuged (2000 rpm * 20 min). The cell layer between the interfaces was aspirated and placed in a 50 mL centrifuge tube, mixed 1:1 with normal saline, centrifuged (1200 rpm * 10 min), the cell pellet was collected, resuspended with 50 mL of normal saline, and centrifuged (1200 rpm * 10 min) to collect the cells;

[0152] (2) 5 mL of X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL of IL-2 was added to the collected cells and resuspended to a cell density of 0.8×10 6 cells / mL. It was inoculated into a 225 cm 2 cell culture flask and then cultured at 37 ± 1°C and 5 ± 0.5% CO2 for 0.5 h - 1 h, centrifuged (400 g * 10 min), and the cells were collected and resuspended with X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL of IL-2 to a cell density of 3.0×10 7 cells / mL. Then, magnetic beads (Dynabeads CD3 / CD28) were added according to the ratio of magnetic beads to T cells of 3:1, cultured for 0.5 h, and the supernatant was collected to obtain CD3 + T cells;

[0153] 2. Preparation of lentiviral vector

[0154] (1) HEK293T cells were resuspended in DEME medium (pH 7.2 - 7.4) to a cell density of 3.5×10 6 cells / mL and cultured at 37.0°C and 5.0% CO2 for 24 h;

[0155] (2) Replace the medium of the HEK293T cell culture system with OPM-293CD05 medium (pH 7.2 - 7.4) containing PEI at a final concentration of 3.4 μg / mL and the mixed plasmid at a final concentration of 1.7 μg / mL. Incubate it at 37.0 ± 1 °C and 5.0% CO₂ for 44 h, then replace it with OPM-293CD05 medium (pH 7.2 - 7.4) containing Benzonase at a final concentration of 20 U / mL. Incubate it at 37.0 ± 1 °C and 5.0% CO₂ for 4 h, centrifuge (500 g * 5 min), harvest the culture supernatant to obtain the lentiviral vector. Among them, the mass ratio of pPVLV3_CAR-19:pMDLg / pRRE:pRSV-Rev:pMD.G in the mixed plasmid is 4:2:2:1;

[0156] Detected by the method of the present invention, the P24 protein in the obtained lentiviral vector > 1 × 10 6 pg / mL, HCP < 5 × 10 4 ng / mL, HCD < 50 ng / mL.

[0157] 3. Transduction of the lentiviral vector

[0158] (1) Resuspend CD3 + T cells in X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL IL-2 to a density of 4 × 10 6 cells / mL, and incubate it at 37 ± 1 °C and 5 ± 0.5% CO₂ for 24 h;

[0159] (2) Replace the medium of the CD3 + T cell culture system with X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing F108 at a final concentration of 4 μg / mL and the lentiviral vector at a final concentration of 0.46 MOI. Then add it and incubate it at 37 ± 1 °C and 5 ± 0.5% CO₂ for 24 h, collect the cells, centrifuge (526 g * 5 min), wash them with X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL IL-2, and then resuspend the cells in X-VIVO15 serum-free medium (pH 7.2 - 7.4) containing 500 IU / mL IL-2 to a density of 0.5 × 10 6 cells / mL, and incubate it at 37 ± 1 °C and 5 ± 0.5% CO₂ for 48 h;

[0160] (3) Replace the culture medium of the culture system with X-VIVO15 serum-free medium containing 500 IU / mL IL-2 (pH 7.2 - 7.4), resuspend the cell density to 0.5×10 6 cells / mL, and culture it at 37 ± 1°C and 5 ± 0.5% CO2 for 24 h;

[0161] (4) After removing the magnetic beads Dynabeads CD3 / CD28 from the cultured cells three times, centrifuge (526 g * 5 min) and wash 3 times with physiological saline containing 0.1% human albumin, then resuspend the CAR-T-19 cell density to 0.5×10 6 cells / mL with physiological saline containing 2% human albumin to prepare the CAR-T-19 injection.

[0162] Detected according to the method of the present invention, the cell viability of the prepared CAR-T-19 cells is greater than 90%, the gene transduction efficiency of aCD19CAR+ is greater than 30%, and the cell viability after preservation for 18 h - 24 h is greater than 75%.

[0163] From the date of collecting the patient's blood sample to the 5th day, the culture and preparation of CAR-T-19 cells are completed and then infused back into the patient's own body.

[0164] Test Example 1 Efficacy study of the CAR-T-19 injection of the present invention for the treatment of patients with acute B-lymphoblastic leukemia

[0165] According to the following inclusion criteria and exclusion criteria, 40 patients with acute B-lymphoblastic leukemia (BALL) aged 2 - 24 years are enrolled, including 17 male patients and 23 female patients, with an average age of 8.85 years.

[0166] Inclusion criteria:

[0167] 1. Voluntarily participate in the clinical study; the patient himself or his legal guardian fully understands and is informed of this study and signs the informed consent form (ICF); willing to follow and complete all test procedures.

[0168] 2. Age 25 years old (inclusive) or younger, gender is not limited;

[0169] 3. Bone marrow examination clearly diagnoses acute B-lymphoblastic leukemia and meets one of the following conditions;

[0170] (1) Relapsed B-ALL: ① Relapsed within 12 months after the first remission; ② Relapsed after first-line / multiple-line salvage chemotherapy; ③ Relapsed after autologous or allogeneic hematopoietic stem cell transplantation.

[0171] (2) Refractory B-ALL: ① Those who have not achieved a complete remission after two courses of chemotherapy with a standardized induction regimen, or ② Those who have not achieved a complete remission after first-line / multiple salvage chemotherapy;

[0172] (3) Subjects with Ph+ ALL can be included in the following situations: ① Recurrence or refractoriness after at least two TKIs (tyrosine kinase inhibitors) treatment; For example, Ph+ ALL subjects with t315i mutation are resistant to first-generation TKI drugs and second-generation TKI drugs. In the absence of effective TKI drug treatment, it is not required that the subject must receive at least two TKI drug treatments; ② Unable to tolerate TKI treatment; ③ There are contraindications to TKI treatment.

[0173] 4. Tumor cells in bone marrow (BM) or peripheral blood (PB) express CD19 measured within 3 months before screening;

[0174] 5. Good organ function, meeting the following criteria:

[0175] ① ALT ≤ 5 times the upper limit of normal value (ULN);

[0176] ② Total bilirubin ≤ 2 times ULN (≤ 3 times ULN for Gilbert's syndrome);

[0177] ③ When not receiving oxygen, there is no dyspnea greater than grade 1, and oxygen saturation > 95%;

[0178] ④ Left ventricular ejection fraction (LVEF) ≥ 50%;

[0179] ⑤ Serum creatinine ≤ 1.5 times ULN.

[0180] 6. Karnofsky score (≥ 16 years old) ≥ 70 or Lansky (< 16 years old) score ≥ 50;

[0181] 7. Have sufficient venous access (for apheresis or venous blood sampling), and there are no other contraindications to blood cell separation;

[0182] 8. Women of childbearing age have negative blood / urine pregnancy tests within 3 days before apheresis and before cell infusion. Any male and female patients with fertility must agree to use effective contraceptive methods throughout the study and for at least 2 years after the study treatment is administered. In the judgment of the researcher, a patient is considered fertile if: he / she is biologically capable of having children and has normal sexual life;

[0183] 9. For patients who have previously received allogeneic hematopoietic stem cell transplantation, the peripheral blood of their previous donors is used for the preparation of CAR-T-19 cells. The conditions that the donors need to meet are as follows:

[0184] ① Have previously donated bone marrow / hematopoietic stem cells as a transplant donor for this subject;

[0185] ②At least 8 years old at the time of screening;

[0186] ③Voluntarily participate in the clinical study; the subject (and legal guardian, if applicable) fully understands and is informed of this study and signs the informed consent form (ICF); willing to follow and complete all trial procedures;

[0187] ④Have sufficient venous access (for apheresis or venous blood sampling) and no other contraindications to blood cell separation.

[0188] Exclusion criteria:

[0189] 1. Isolated extramedullary relapse;

[0190] 2. Suffers from genetic diseases, except Down syndrome;

[0191] 3. Suffers from Burkitt lymphoma / leukemia;

[0192] 4. Has active central nervous system disease;

[0193] 5. Has active central nervous system leukemia at the time of screening (defined as CNS-3 and CNS-2 with neurological symptoms as defined in the NCCN guidelines and judged by the researcher to be active central leukemia);

[0194] 6. Has a history of other malignancies or has other malignancies at the same time (except for adequately treated cervical carcinoma in situ, basal cell or squamous cell skin cancer, locally treated prostate cancer after radical surgery, thyroid cancer, ductal carcinoma in situ after radical surgery);

[0195] 7. Has or is suspected of having uncontrolled fungal, bacterial, viral or other infections;

[0196] 8. Received HSCT within 3 months before screening, or had grade II to IV active graft-versus-host disease (GVHD) or moderate to severe chronic GVHD within 4 weeks before screening;

[0197] 9. Received the following anti-tumor treatments before apheresis: ①Received standardized chemotherapy, targeted therapy and other drug treatments within 14 days or at least 5 half-lives (whichever is shorter); ②Received radiotherapy within 14 days; ③Received intrathecal therapy within 7 days; ④Received donor lymphocyte infusion (DLI) within 4 weeks; ⑤Received blinatumomab within 14 days.

[0198] Trial results:

[0199] (1) Prepare CAR-T-19 cells from 40 patients according to the methods of Examples 1-5 and detect them according to the method of the present invention. The cell viability of CAR-T-19 cells is 80%-99%, the gene transduction efficiency of aCD19CAR+ is greater than 30%, the cell viability after storage for 18 h is greater than 75%, the inserted gene copy number VCN < 5 copies / cell, and the VCN of 16 patients < 1 copy / cell.

[0200] (2) Investigation of the biological activity of CAR-T-19 cells

[0201] Using K562 cells and CD19-K562 cells (K562 cells loaded with CD19) as target cells, co-culture K562 cells and CD19-K562 cells with CAR-T-19 cells at an effector-to-target ratio of 3:1 respectively, and detect the killing efficiency by flow cytometry. The killing efficiency of the patient's CAR-T-19 cells against CD19-positive target cells is higher than that against K562 cells (CD19-negative cells). Among them, the killing efficiency of the CAR-T-19 cells of 8 patients is 50%-65%; the killing efficiency of the CAR-T-19 cells of 23 patients is 20%-49%; the killing efficiency of the CAR-T-19 cells of 4 patients is 5%-19%.

[0202] (4) Prepare CAR-T-19 cell injections from 40 patients according to the methods of Examples 1-5 and perform autologous reinfusion. The curative effect results are as follows:

[0203] The 28-day response rate and its 95% CI (Confidence Interval) are 91.11% (78.78%-97.52%), and the 90-day objective response rate (ORR) and its 95% CI are 97.78% (88.23%).

[0204] The incidence rate of grade 3 and above cytokine release syndrome in the overall cytokine release syndrome (CRS) is 17.8%. The overall incidence rate of CAR-T cell-related encephalopathy syndrome is 4.4%, among which the incidence rate of grade 3 and above CAR-T cell-related encephalopathy syndrome is 4.4%. There is no DLT occurrence, no serious adverse events occur, and no death events are observed.

[0205] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or deformations according to the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing CAR-T-19 cells, comprising preparing a lentiviral vector, CD3 + Preparation of T cells and transduction of lentiviral vectors, where Preparation of lentiviral vector and CD3 + There is no order of precedence for the preparation of T cells. The preparation of the lentiviral vector comprises the following steps: (1) Resuspend the stromal cells in either OPM-293CD05 medium or DEME medium to a cell density of (3.5-4.5)×10 6 The matrix cell system was prepared by culturing the cells at 37.0°C ± 1°C and CO2 5-8.0% for 10-36 hours. (2) The culture medium in the matrix cell system is replaced with either OPM-293CD05 culture medium or DEME culture medium containing a final concentration of transfection reagent of 3.4 μg / mL-6 μg / mL and a final concentration of plasmid of 1.5 μg / mL-1.7 μg / mL, and then cultured at 37.0±1°C and CO2 5-8.0% for 20 h-45 h. The culture medium is then replaced with either OPM-293CD05 culture medium or DEME culture medium containing a final concentration of endonuclease of 10-30 (U / mL), and then cultured at 37.0±1°C and CO2 5-8.0% for 1 h-10 h. The culture supernatant is harvested after centrifugation.

2. The method according to claim 1, wherein the plasmid is selected from any one of pPVLV2_aCD19, pPVLV3_CAR-19, pMDLg / pRRE, pRSV-Rev, pMD.G or a combination thereof.

3. The method according to any one of claims 1 to 2, wherein the HCD in the lentiviral vector is ≤80 ng / mL, preferably ≤50 ng / mL.

4. The method according to any one of claims 1 to 3, wherein the CD3 + The preparation of T cells includes the preparation, culture, and sorting and enrichment of peripheral blood mononuclear cells (PBMCs), among which: The preparation of peripheral blood mononuclear cells (PBMC) comprises the following steps: adding separation medium and diluent to anticoagulated whole blood, stirring, mixing, adding the separation medium and diluent to the anticoagulated whole blood, centrifuging at 1000-3000rpm*10-40min, collecting the cell layer between the interfaces, adding washing solution, centrifuging, washing, collecting cells, and obtaining, wherein the separation medium is selected from any one or a combination of hydroxyethyl starch 40 sodium chloride injection (HES), Percoll, Ficoll-Paque PLUS, the volume ratio of whole blood: diluent is 1:1-2, the diluent is selected from any one or a combination of sodium chloride injection, Hank's buffer, Lactated Ringer's solution, Dulbecco's phosphate buffer, and the separation solution is selected from hydroxyethyl starch 40 sodium chloride injection (HES), Ficol l, Lymphoprep, Lymphocyte Separation Media, Cell l Separation Media or a combination thereof, the osmotic pressure of the separation solution is 300mOsmol / Kg-360mOsmol / Kg, and the washing solution is selected from 0.1% human albumin sodium chloride injection, Dulbecco's phosphate buffer, sodium chloride injection or a combination thereof.

5. The method according to any one of claims 1 to 4, wherein the culturing and sorting and enrichment of mononuclear cells comprises the following steps: adjusting the PBMC density to (0.5-4)×10 6 The cells were cultured at 37°C ± 1°C and 5.0% ± 1% CO2 for 0.5-1h, centrifuged, and the collected cells were resuspended in serum-free medium containing 100-600 IU / mL of IL-2 to a density of (2.0-4.0)×10 7 / mL, then add CD3 / CD28 magnetic beads to the cell system at a ratio of 2-5:1 of CD3 / CD28 magnetic beads: T cells, and incubate for 0.5h-1.0h to obtain, wherein, The serum-free culture medium is selected from any one of X-VIVO15, KBM581, GT-T551 H3 or a combination thereof.

6. The preparation method according to any one of claims 1 to 5, wherein the lentiviral vector transduction comprises the following steps: S-1: CD3 + T cells were resuspended in serum-free medium to a density of (1-6) × 10 6 After culturing at 37°C ± 1°C and 5.0% ± 1% CO2 for 24 h-48 h, replace the serum-free medium containing a final concentration of a transduction-promoting agent of 2-8 μg / mL and a final concentration of a lentiviral vector of 0.2-1 MOI, and then culture the medium at 37°C ± 1°C and 5.0% ± 1% CO2 for 24 h-48 h to obtain CD3 T cells transduced with lentiviral vectors. + T cells, among which The transduction promoting agent is selected from any one of Synperonic F108 and Polybrene or a combination thereof; S-2: Resuspend the lentiviral vector-transduced CD3 + T cells to a cell density of (0.2-1)×10 6 / mL, centrifuge (500-2000rpm)*(5-20min), collect the cells and resuspend them in serum-free medium to a cell density of (0.2-1)×10 6 / mL, and culture them at 37°C, 5% CO2 for 24h-72h; S-3: Resuspend in serum-free medium to a cell density of (0.2-1)×10 6 / mL, place it at 37±1℃, 5±0.5% CO2 and culture it for 24h-26h, remove the CD3 / CD28 magnetic beads, wash it by centrifugation, and resuspend it to obtain. Wherein, the serum-free culture medium described in S-1 to S-3 is selected from any one of X-VIVO15, KBM 581, GT-T551 H3 or a combination thereof.

7. According to the preparation method according to any one of claims 1 to 6, the preparation time of the CAR-T-19 cells is ≤14 days, preferably 5 days to 12 days, and more preferably 5 days to 7 days.

8. A CAR-T-19 injection, the injection containing a density of (1×10 5 -2×10 8 ) cells / mL of CAR-T-19 cells and normal saline containing 0.5-5% human albumin.

9. Use of the CAR-T-19 injection as described in claim 8 for preparing any product for anti-tumor or anti-tumor immunotherapy.

10. The use according to claim 9, wherein the tumor is selected from any one of relapsed B-lymphocytic leukemia (B-ALL), refractory B-lymphocytic leukemia (B-ALL), acute B-lymphocytic leukemia (BALL), diffuse large B-cell lymphoma (DLBCL) or its complications.