Preparation technology of regulatable CD19 targeted CAR-T (Chimeric Antigen Receptor-T) cell

Abstract

The invention relates to the field of preparation of CD19 targeted CAR-T cells, in particular to an adjustable CD19 targeted CAR-T cell preparation technology which comprises the following operation steps: S1, T cell collection and separation: S1.1, peripheral blood collection: blood is collected from peripheral veins of a patient, and the general collection amount is determined according to the physical condition of the patient and the amount required by subsequent cell culture and is usually between 50ml and 500ml; s1.2, performing density gradient centrifugal separation on the T cells; s2, CAR gene construction and carrier preparation: S2.1, CAR structure design (taking an inducible promoter system as an example), CD19 targeting CAR-T cells can accurately recognize and combine CD19 antigens on the surfaces of tumor cells, and as CD19 is highly expressed on the surfaces of most blood system malignant tumor cells such as B cell lymphoma and leukemia and is limited in expression in normal tissue cells, the CD19 targeting CAR-T cells can be used for accurately recognizing and combining the CD19 antigens on the surfaces of the tumor cells. Therefore, the CAR-T cells can specifically attack tumor cells like a'biological missile ', and damage to normal tissues is reduced.

Description

Technical Field

[0001] The present invention relates to the field of CD19 targeted CAR-T cell preparation, and in particular to a technology for preparing controllable CD19 targeted CAR-T cells. Background Art

[0002] Traditional cancer treatments mainly include surgery, chemotherapy and radiotherapy. Surgery has a certain effect on early solid tumors, but it is often helpless for malignant tumors that have already metastasized or are in the blood system. Although chemotherapy and radiotherapy can kill tumor cells, they can also cause serious damage to normal tissue cells, resulting in side effects such as nausea, vomiting, hair loss, and bone marrow suppression. Moreover, many tumor cells will gradually develop resistance to chemotherapy drugs, which greatly reduces the therapeutic effect. In this context, a more accurate, efficient and less side-effect treatment method is needed to deal with cancer. CAR-T cell therapy technology came into being. Although CAR-T cell therapy has achieved great success, it also has some problems, the most prominent of which are serious adverse reactions such as cytokine release syndrome (CRS) and neurotoxicity, which may endanger the patient's life. In order to improve the safety and controllability of treatment, we propose a regulatable CD19-targeted CAR-T cell preparation technology to solve the above-mentioned problems. Summary of the invention

[0003] The purpose of the present invention is to solve the shortcomings of the background technology and propose a controllable CD19 targeted CAR-T cell preparation technology.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is: a technology for preparing controllable CD19-targeted CAR-T cells, comprising the following steps: S1. T cell collection and separation: S1.1. Peripheral blood collection: Blood is collected from the patient's peripheral veins. The amount of blood collected is generally determined based on the patient's physical condition and the amount required for subsequent cell culture, usually between 50ml-500ml; S1.2, separation of T cells by density gradient centrifugation; S2. CAR gene construction and vector preparation: S2.1. CAR structure design (taking the inducible promoter system as an example); S2.2. Vector construction: S2.2 (1) If a lentiviral vector is used, the constructed CAR gene is inserted into the lentiviral vector backbone. This process requires the use of molecular biological techniques, such as restriction endonuclease cutting of the vector and CAR gene fragments, and then connecting the CAR gene to the vector using DNA ligase. At the same time, the vector will also contain some marker genes for subsequent screening of successfully transduced T cells; S2.2 (2) For CRISPR / Cas9 gene editing technology, it is necessary to construct a vector system containing the CAR gene, Cas9 protein and guide RNA (gRNA). The gRNA can guide the Cas9 protein to locate to a specific location in the T cell genome for gene editing, such as accurately inserting the CAR gene into the endogenous T cell receptor (TCR) site of the T cell; S3. CAR gene introduction into T cells: S3.1, lentiviral transduction; S3.2, CRISPR / Cas9 gene editing: The constructed CRISPR / Cas9 vector system is introduced into T cells by electroporation or liposome transfection. During electroporation, a short high-voltage pulse is applied to form small holes in the cell membrane, allowing the vector to enter the cell. Liposome transfection uses the fusion of liposomes and cell membranes to deliver the vector into the cell. After introduction, the Cas9 protein edits the T cell genome under the guidance of gRNA and inserts the CAR gene into the specified location. The edited cells then need to be screened to remove cells that have not been successfully edited. S4. Cell culture and expansion: S4.1. Culture condition setting: The transduced or edited T cells are cultured in an incubator at 37°C and a carbon dioxide concentration of about 5%. A culture medium containing multiple cytokines is used, such as interleukin-2 (IL-2), interleukin-7 (IL-7), and interleukin-15 (IL-15). The concentration of IL-2 is generally 10-100 U / mL, and the concentrations of IL-7 and IL-15 are adjusted according to the experimental and treatment needs, usually around 1-10 ng / mL. These cytokines can stimulate the proliferation of T cells and the maturation of CAR-T cells. S4.2, cell proliferation monitoring; S5. Quality control and functional verification: S5.1. Quality Control: S5.1 (1) Purity detection: CAR-T cell surface markers and CAR expression levels were detected by flow cytometry; S5.1 (2) Viability test: Trypan blue staining is used. After mixing the cells with trypan blue solution, they are observed under a microscope. Living cells will not be stained by trypan blue, while dead cells will be stained blue. The proportion of living cells is required to be greater than 90% to ensure the therapeutic effect of CAR-T cells. S5.2, Functional verification; S6. Cell preparation and storage: S6.1. Preparation: After completing quality control and functional validation, resuspend the CAR-T cells in an appropriate formulation buffer; S6.2. Storage method: For short-term storage (several hours to several days), CAR-T cells can be placed in a 4°C refrigerator. If long-term storage is required, liquid nitrogen is generally used for freezing. Before freezing, a cryoprotectant needs to be added, and its final concentration is generally around 5%-10%. After the cells are slowly cooled to -80°C, they are transferred to liquid nitrogen for storage for subsequent treatment.

[0005] Preferably, the specific steps of separating T cells by density gradient centrifugation mentioned in step S1.2 are: S1.2 (1) The collected peripheral blood is mixed with lymphocyte separation fluid (such as Ficoll-Paque) in a certain proportion and then subjected to density gradient centrifugation. The centrifugation speed is generally 400-1000g and the centrifugation time is about 20-30 minutes. S1.2 (2) During the centrifugation process, the components in the blood will be stratified according to their density, and T cells will be distributed in a specific density layer, so T cells can be separated by aspirating the corresponding cell layer; S1.2 (3) After centrifugation, wash the cells with a buffer (such as PBS) to remove impurities in the separation solution and obtain relatively pure T cells.

[0006] Preferably, the CAR structure design mentioned in step S2.1 specifically includes the following steps: S2.1 (1) Select appropriate single-chain antibody (scFv) to recognize CD19 antigen; S2.1 (2), connects the transmembrane domain to ensure that CAR can be stably anchored on the surface of T cells; S2.1 (3) Select appropriate intracellular signal transduction domains to activate the killing function of T cells; S2.1 (4), insert an inducible promoter upstream of the CAR gene.

[0007] Preferably, the lentiviral transduction mentioned in step S3.1 specifically comprises the following steps: S3.1. (1) Co-culture the lentiviral vector containing the CAR gene with the isolated T cells under appropriate culture conditions. During the transduction process, the virus will integrate the CAR gene into the T cell genome. Generally, the virus titer during transduction is between 10^6-10^8TU / mL (transduction units / mL), the cell density is about 1×10^6-5×10^6 cells / mL, and the transduction time is 12-24 hours; S3.1. (2) After transduction, the non-transduced virus needs to be removed. This can be done by washing the cells. At the same time, culture medium containing antiretroviral drugs is added to prevent the virus from infecting the cells again.

[0008] Preferably, the cell proliferation monitoring mentioned in step S4.2 specifically includes the following steps: S4.2 (1) Regularly observe the growth status of cells and monitor changes in cell number using a cell counter or hemocytometer; S4.2 (2) According to the growth rate of cells, subculture should be carried out in a timely manner to maintain the good growth state of cells; S4.2 (3) During the amplification process, ensure that the CAR expression level of the cells is stable and regularly monitor the expression of CAR by flow cytometry.

[0009] Preferably, the functional verification mentioned in step S5.2 specifically includes the following steps: S5.2 (1), in vitro validation; S5.2(2), In vivo validation (optional).

[0010] Preferably, the in vitro validation mentioned in step S5.2 (1) specifically comprises the following steps: ①. Co-culture CAR-T cells with tumor cell lines expressing CD19 antigen (such as Raji cells) in vitro at a certain ratio (such as 1:1, 5:1); ② After a certain period of culture (such as 4-24 hours), collect the culture supernatant, detect the LDH content therein, and calculate the lysis rate of tumor cells.

[0011] Preferably, the in vivo validation mentioned in step S5.2 (1) specifically comprises the following steps: ① Establish a CD19-positive tumor model in an animal model (such as immunodeficient mice) and inject CAR-T cells into the mice by intravenous injection; ②. Observe the growth of mouse tumors, measure tumor volume and weight, and detect changes in cytokines in mice to verify the anti-tumor effect of CAR-T cells in vivo.

[0012] Preferably, the proportion of CAR-positive cells mentioned in the purity detection step S5.1 (1) is generally required to be above 70%-90%.

[0013] Preferably, the cryoprotectant mentioned in step S6.2 may be dimethyl sulfoxide.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. The CD19-targeted CAR-T cells of the present invention can accurately recognize and bind to the CD19 antigen on the surface of tumor cells. Since CD19 is highly expressed on the surface of most blood malignant tumor cells such as B-cell lymphomas and leukemias, but is limitedly expressed in normal tissue cells, this allows CAR-T cells to specifically attack tumor cells like "biological missiles" and reduce damage to normal tissues. For example, when treating B-cell acute lymphoblastic leukemia (B-ALL), CAR-T cells can efficiently recognize CD19 on the surface of leukemia cells without causing obvious toxic effects on other non-CD19 expressing cells.

[0015] 2. This cell preparation technology can be transformed using the patient's own T cells. T cells are collected from the patient's peripheral blood, and then genetically engineered in vitro to express CAR targeting CD19. The CAR-T cells prepared in this way have the characteristics of the patient's own immune cells. After being infused back into the patient's body, they can better adapt to the patient's immune system and reduce the probability of immune rejection. In addition, the preparation parameters of CAR-T cells can be flexibly adjusted according to the patient's specific condition, such as the number of tumor cells, the patient's physical condition and other factors, to achieve individualized precision treatment.

[0016] 3. Through technical means such as inducible promoter systems (such as tetracycline regulatory systems) and split CAR structures, the activity of CAR-T cells can be controlled by external signals or drugs. During the treatment process, if the patient has serious adverse reactions, such as cytokine release syndrome (CRS) or neurotoxicity, the doctor can reduce the activity of CAR-T cells and alleviate adverse reactions by giving corresponding regulatory drugs (such as stopping the administration of doxycycline in the Tet-on system). This controllability greatly improves the safety of treatment and enables doctors to better manage the risks during treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the preparation process of a controllable CD19-targeted CAR-T cell preparation technology of the present invention. DETAILED DESCRIPTION

[0018] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art may think of other obvious variations.

[0019] Embodiment 1: like Figure 1 A controllable CD19-targeted CAR-T cell preparation technology is shown, comprising the following steps: S1. T cell collection and separation: S1.1. Peripheral blood collection: Blood is collected from the patient's peripheral veins. The amount of blood collected is generally determined based on the patient's physical condition and the amount required for subsequent cell culture. It is usually between tens of milliliters and hundreds of milliliters. This is because peripheral blood is an important source of T cells, and the collection is relatively convenient and less traumatic to the patient. S1.2. Separation of T cells by density gradient centrifugation: After the collected peripheral blood is mixed with lymphocyte separation fluid (such as Ficoll-Paque) in a certain proportion, density gradient centrifugation is performed. The centrifugation speed is generally 400g and the centrifugation time is about 20 minutes. During the centrifugation process, the components in the blood will be stratified according to different densities, and T cells will be distributed in a specific density layer. Therefore, T cells can be separated by aspirating the corresponding cell layer, and then the cells can be washed with a buffer solution (such as PBS) to remove impurities in the separation fluid and obtain relatively pure T cells; S2. CAR gene construction and vector preparation: S2.1. CAR structure design (taking the inducible promoter system as an example); S2.1 (1) Select a suitable single-chain antibody (scFv) to recognize the CD19 antigen. This scFv is usually obtained by genetically engineering an antibody that can specifically bind to CD19, so that it can perform antigen recognition function in a single-chain form; S2.1 (2), connecting to a transmembrane domain, such as the transmembrane portion of CD28 or CD8 protein, to ensure that CAR can be stably anchored on the surface of T cells; S2.1 (3) Select appropriate intracellular signal transduction domains, such as the CD3ζ chain, which can transmit antigen recognition signals to the interior of T cells and activate the killing function of T cells; S2.1 (4), insert an inducible promoter upstream of the CAR gene, such as the promoter sequence in the tetracycline regulation system (Tet-on / Tet-off); S2.2. Vector construction: S2.2, (1) If a lentiviral vector is used, the constructed CAR gene is inserted into the lentiviral vector backbone. This process requires the use of molecular biological techniques, such as restriction endonuclease cutting the vector and CAR gene fragments, and then connecting the CAR gene to the vector using DNA ligase. At the same time, the vector will also contain some marker genes for subsequent screening of successfully transduced T cells; S2.2 (2) For CRISPR / Cas9 gene editing technology, it is necessary to construct a vector system containing the CAR gene, Cas9 protein and guide RNA (gRNA). The gRNA can guide the Cas9 protein to locate to a specific location in the T cell genome for gene editing, such as accurately inserting the CAR gene into the endogenous T cell receptor (TCR) site of the T cell; S3. CAR gene introduction into T cells: S3.1 Lentiviral transduction: S3.1. (1) Co-culture the lentiviral vector containing the CAR gene with the isolated T cells under appropriate culture conditions. During the transduction process, the virus will integrate the CAR gene into the T cell genome. The transduction efficiency can be improved by optimizing the virus titer, cell number and transduction time factors. Generally, the virus titer during transduction is between 10^6TU / mL (transduction units / mL), the cell density is about 1×10^6 cells / mL, and the transduction time is 12 hours; S3.1. (2) After transduction, it is necessary to remove the untransduced virus, which can be done by washing the cells. At the same time, add culture medium containing antiretroviral drugs to prevent the virus from infecting the cells again; S3.2, CRISPR / Cas9 gene editing: The constructed CRISPR / Cas9 vector system is introduced into T cells by electroporation or liposome transfection. During electroporation, a short high-voltage pulse is applied to form small holes in the cell membrane, allowing the vector to enter the cell. Liposome transfection uses the fusion of liposomes and cell membranes to deliver the vector into the cell. After introduction, the Cas9 protein edits the T cell genome under the guidance of gRNA and inserts the CAR gene into the specified location. The edited cells then need to be screened to remove cells that have not been successfully edited. S4. Cell culture and expansion: S4.1. Culture condition setting: The transduced or edited T cells are cultured in an incubator at 37°C and a carbon dioxide concentration of about 5%. A culture medium containing multiple cytokines is used, such as interleukin-2 (IL-2), interleukin-7 (IL-7), and interleukin-15 (IL-15). The concentration of IL-2 is generally 10 / mL, and the concentrations of IL-7 and IL-15 are adjusted according to the experimental and treatment needs, usually around 1ng / mL. These cytokines can stimulate the proliferation of T cells and the maturation of CAR-T cells. S4.2. Cell proliferation monitoring: S4.2 (1) Regularly observe the growth status of cells and monitor changes in cell number using a cell counter or hemocytometer; S4.2 (2) According to the growth rate of cells, subculture should be carried out in a timely manner to maintain the good growth state of cells; S4.2 (3) During the expansion process, it is necessary to ensure that the CAR expression level of the cells is stable. The expression of CAR can be regularly detected by flow cytometry. Generally, the cells need to be expanded to the number required for treatment. For most CAR-T cell therapies, the number of cells needs to reach millions or even billions. S5. Quality control and functional verification: S5.1. Quality Control: S5.1 (1) Purity detection: Use flow cytometry to detect CAR-T cell surface markers, for example, detect the expression ratio of CD3, CD4, and CD8 markers to ensure that CAR-T cells are the main component and the proportion of impurity cells (such as B cells and NK cells) is low. At the same time, detect the expression level of CAR; S5.1 (2) Viability test: Trypan blue staining is used. After mixing the cells with trypan blue solution, they are observed under a microscope. Living cells will not be stained by trypan blue, while dead cells will be stained blue. The proportion of living cells is required to be greater than 90% to ensure the therapeutic effect of CAR-T cells. S5.2. Functional verification: S5.2 (1) In vitro validation: ①. CAR-T cells are co-cultured with tumor cell lines expressing CD19 antigen (such as Raji cells) in vitro at a certain ratio (1:1), and the killing efficiency of CAR-T cells on tumor cells is detected by lactate dehydrogenase (LDH) release experiment; ② After a certain period of culture (4 hours), collect the culture supernatant, detect the LDH content, and calculate the lysis rate of tumor cells. At the same time, apoptosis of tumor cells can also be observed through cell apoptosis detection (such as Annexin V-FITC / PI staining) to verify the killing function of CAR-T cells; S5.2(2), In vivo validation (optional): ① Establish a CD19-positive tumor model in an animal model (such as immunodeficient mice) and inject CAR-T cells into the mice by intravenous injection; ②. Observe the growth of mouse tumors, measure tumor volume and weight, and detect changes in cytokines in mice to verify the anti-tumor effect of CAR-T cells in vivo; S6. Cell preparation and storage: S6.1. Preparation: After completing quality control and functional verification, the CAR-T cells are resuspended in a suitable preparation buffer, which usually contains physiological salts, buffers (such as HEPES) and human serum albumin to maintain cell stability; S6.2. Storage method: For short-term storage (8h), CAR-T cells can be placed in a 4°C refrigerator. If long-term storage is required, liquid nitrogen is generally used for freezing. Before freezing, a cryoprotectant (such as dimethyl sulfoxide, DMSO) needs to be added. The final concentration is generally around 5%. After the cells are slowly cooled to -80°C, they are transferred to liquid nitrogen for storage for subsequent treatment.

[0020] Embodiment 2: like Figure 1 A controllable CD19-targeted CAR-T cell preparation technology is shown, comprising the following steps: S1. T cell collection and separation: S1.1. Peripheral blood collection: Blood is collected from the patient's peripheral veins. The amount of blood collected is generally determined based on the patient's physical condition and the amount required for subsequent cell culture. It is usually between tens of milliliters and hundreds of milliliters. This is because peripheral blood is an important source of T cells, and the collection is relatively convenient and less traumatic to the patient. S1.2. Separation of T cells by density gradient centrifugation: After the collected peripheral blood is mixed with lymphocyte separation fluid (such as Ficoll-Paque) in a certain proportion, density gradient centrifugation is performed. The centrifugation speed is generally 1000g and the centrifugation time is about 30 minutes. During the centrifugation process, the components in the blood will be stratified according to different densities, and T cells will be distributed in a specific density layer. Therefore, T cells can be separated by aspirating the corresponding cell layer. After that, the cells can be washed with a buffer solution (such as PBS) to remove impurities in the separation fluid and obtain relatively pure T cells. S2. CAR gene construction and vector preparation: S2.1. CAR structure design (taking the inducible promoter system as an example); S2.1 (1) Select a suitable single-chain antibody (scFv) to recognize the CD19 antigen. This scFv is usually obtained by genetically engineering an antibody that can specifically bind to CD19, so that it can perform antigen recognition function in a single-chain form; S2.1 (2), connecting to a transmembrane domain, such as the transmembrane portion of CD28 or CD8 protein, to ensure that CAR can be stably anchored on the surface of T cells; S2.1 (3) Select appropriate intracellular signal transduction domains, such as the CD3ζ chain, which can transmit antigen recognition signals to the interior of T cells and activate the killing function of T cells; S2.1 (4), insert an inducible promoter upstream of the CAR gene, such as the promoter sequence in the tetracycline regulation system (Tet-on / Tet-off); S2.2. Vector construction: S2.2, (1) If a lentiviral vector is used, the constructed CAR gene is inserted into the lentiviral vector backbone. This process requires the use of molecular biological techniques, such as restriction endonuclease cutting the vector and CAR gene fragments, and then connecting the CAR gene to the vector using DNA ligase. At the same time, the vector will also contain some marker genes for subsequent screening of successfully transduced T cells; S2.2 (2) For CRISPR / Cas9 gene editing technology, it is necessary to construct a vector system containing the CAR gene, Cas9 protein and guide RNA (gRNA). The gRNA can guide the Cas9 protein to locate to a specific location in the T cell genome for gene editing, such as accurately inserting the CAR gene into the endogenous T cell receptor (TCR) site of the T cell; S3. CAR gene introduction into T cells: S3.1 Lentiviral transduction: S3.1. (1) Co-culture the lentiviral vector containing the CAR gene with the isolated T cells under appropriate culture conditions. During the transduction process, the virus will integrate the CAR gene into the T cell genome. The transduction efficiency can be improved by optimizing the virus titer, cell number and transduction time factors. Generally, the virus titer during transduction is between 10^8TU / mL (transduction units / mL), the cell density is about 5×10^6 cells / mL, and the transduction time is 24 hours; S3.1. (2) After transduction, it is necessary to remove the untransduced virus, which can be done by washing the cells. At the same time, add culture medium containing antiretroviral drugs to prevent the virus from infecting the cells again; S3.2, CRISPR / Cas9 gene editing: The constructed CRISPR / Cas9 vector system is introduced into T cells by electroporation or liposome transfection. During electroporation, a short high-voltage pulse is applied to form small holes in the cell membrane, allowing the vector to enter the cell. Liposome transfection uses the fusion of liposomes and cell membranes to deliver the vector into the cell. After introduction, the Cas9 protein edits the T cell genome under the guidance of gRNA and inserts the CAR gene into the specified location. The edited cells then need to be screened to remove cells that have not been successfully edited. S4. Cell culture and expansion: S4.1. Culture condition setting: The transduced or edited T cells are cultured in an incubator at 37°C and a carbon dioxide concentration of about 5%. A culture medium containing multiple cytokines is used, such as interleukin-2 (IL-2), interleukin-7 (IL-7), and interleukin-15 (IL-15). The concentration of IL-2 is generally 100 U / mL, and the concentrations of IL-7 and IL-15 are adjusted according to the experimental and treatment needs, usually around 10 ng / mL. These cytokines can stimulate the proliferation of T cells and the maturation of CAR-T cells. S4.2. Cell proliferation monitoring: S4.2 (1) Regularly observe the growth status of cells and monitor changes in cell number using a cell counter or hemocytometer; S4.2 (2) According to the growth rate of cells, subculture should be carried out in a timely manner to maintain the good growth state of cells; S4.2 (3) During the expansion process, it is necessary to ensure that the CAR expression level of the cells is stable. The expression of CAR can be regularly detected by flow cytometry. Generally, the cells need to be expanded to the number required for treatment. For most CAR-T cell therapies, the number of cells needs to reach millions or even billions. S5. Quality control and functional verification: S5.1. Quality Control: S5.1 (1) Purity detection: Use flow cytometry to detect CAR-T cell surface markers, for example, detect the expression ratio of CD3, CD4, and CD8 markers to ensure that CAR-T cells are the main component and the proportion of impurity cells (such as B cells and NK cells) is low. At the same time, detect the expression level of CAR; S5.1 (2) Viability test: Trypan blue staining is used. After mixing the cells with trypan blue solution, they are observed under a microscope. Living cells will not be stained by trypan blue, while dead cells will be stained blue. The proportion of living cells is required to be greater than 90% to ensure the therapeutic effect of CAR-T cells. S5.2, Functional Verification: S5.2 (1) In vitro validation: ①. CAR-T cells are co-cultured with tumor cell lines expressing CD19 antigen (such as Raji cells) in vitro at a certain ratio (5:1), and the killing efficiency of CAR-T cells on tumor cells is detected by lactate dehydrogenase (LDH) release experiment; ② After a certain period of culture (24 hours), collect the culture supernatant, detect the LDH content, and calculate the lysis rate of tumor cells. At the same time, apoptosis of tumor cells can also be observed through cell apoptosis detection (such as Annexin V-FITC / PI staining) to verify the killing function of CAR-T cells; S5.2(2), In vivo validation (optional): ① Establish a CD19-positive tumor model in an animal model (such as immunodeficient mice) and inject CAR-T cells into the mice by intravenous injection; ②. Observe the growth of mouse tumors, measure tumor volume and weight, and detect changes in cytokines in mice to verify the anti-tumor effect of CAR-T cells in vivo; S6. Cell preparation and storage: S6.1. Preparation: After completing quality control and functional verification, the CAR-T cells are resuspended in a suitable preparation buffer, which usually contains physiological salts, buffers (such as HEPES) and human serum albumin to maintain cell stability; S6.2. Storage method: For short-term storage (3 days), CAR-T cells can be placed in a 4°C refrigerator. If long-term storage is required, liquid nitrogen is generally used for freezing. Before freezing, a cryoprotectant (such as dimethyl sulfoxide, DMSO) needs to be added. The final concentration is generally around 10%. After the cells are slowly cooled to -80°C, they are transferred to liquid nitrogen for storage for subsequent treatment.

[0021] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. A technology for preparing regulatable CD19-targeted CAR-T cells, characterized in that: The steps include: S1. T cell collection and separation: S1.

1. Peripheral blood collection: Blood is collected from the patient's peripheral veins. The amount of blood collected is generally determined based on the patient's physical condition and the amount required for subsequent cell culture, usually between 50ml-500ml; S1.2, separation of T cells by density gradient centrifugation; S2. CAR gene construction and vector preparation: S2.

1. CAR structure design (taking the inducible promoter system as an example); S2.

2. Vector construction: S2.2 (1) If a lentiviral vector is used, the constructed CAR gene is inserted into the lentiviral vector backbone. This process requires the use of molecular biological techniques, such as restriction endonuclease cutting of the vector and CAR gene fragments, and then connecting the CAR gene to the vector using DNA ligase. At the same time, the vector will also contain some marker genes for subsequent screening of successfully transduced T cells; S2.2 (2) For CRISPR / Cas9 gene editing technology, it is necessary to construct a vector system containing the CAR gene, Cas9 protein and guide RNA (gRNA). The gRNA can guide the Cas9 protein to locate to a specific location in the T cell genome for gene editing, such as accurately inserting the CAR gene into the endogenous T cell receptor (TCR) site of the T cell; S3. CAR gene introduction into T cells: S3.1, lentiviral transduction; S3.2, CRISPR / Cas9 gene editing: The constructed CRISPR / Cas9 vector system is introduced into T cells by electroporation or liposome transfection. During electroporation, a short high-voltage pulse is applied to form small holes in the cell membrane, allowing the vector to enter the cell. Liposome transfection uses the fusion of liposomes and cell membranes to deliver the vector into the cell. After introduction, the Cas9 protein edits the T cell genome under the guidance of gRNA and inserts the CAR gene into the specified location. The edited cells then need to be screened to remove cells that have not been successfully edited. S4. Cell culture and expansion: S4.

1. Culture condition setting: The transduced or edited T cells are cultured in an incubator at 37°C and a carbon dioxide concentration of about 5%. A culture medium containing multiple cytokines is used, such as interleukin-2 (IL-2), interleukin-7 (IL-7), and interleukin-15 (IL-15). The concentration of IL-2 is generally 10-100 U / mL, and the concentrations of IL-7 and IL-15 are adjusted according to the experimental and treatment needs, usually around 1-10 ng / mL. These cytokines can stimulate the proliferation of T cells and the maturation of CAR-T cells. S4.2, cell proliferation monitoring; S5. Quality control and functional verification: S5.

1. Quality Control: S5.1 (1) Purity detection: CAR-T cell surface markers and CAR expression levels were detected by flow cytometry; S5.1 (2) Viability test: Trypan blue staining is used. After mixing the cells with trypan blue solution, they are observed under a microscope. Living cells will not be stained by trypan blue, while dead cells will be stained blue. The proportion of living cells is required to be greater than 90% to ensure the therapeutic effect of CAR-T cells. S5.2, Functional verification; S6. Cell preparation and storage: S6.

1. Preparation: After completing quality control and functional validation, resuspend the CAR-T cells in an appropriate formulation buffer; S6.

2. Storage method: For short-term storage (several hours to several days), CAR-T cells can be placed in a 4°C refrigerator. If long-term storage is required, liquid nitrogen is generally used for freezing. Before freezing, a cryoprotectant needs to be added, and its final concentration is generally around 5%-10%. After the cells are slowly cooled to -80°C, they are transferred to liquid nitrogen for storage for subsequent treatment.

2. A controllable CD19-targeted CAR-T cell preparation technology according to claim 1, characterized in that: The specific steps of separating T cells by density gradient centrifugation mentioned in step S1.2 are: S1.2 (1) The collected peripheral blood is mixed with lymphocyte separation fluid (such as Ficoll-Paque) in a certain proportion and then subjected to density gradient centrifugation. The centrifugation speed is generally 400-1000g and the centrifugation time is about 20-30 minutes. S1.2 (2) During the centrifugation process, the components in the blood will be stratified according to their density, and T cells will be distributed in a specific density layer, so T cells can be separated by aspirating the corresponding cell layer; S1.2 (3) After centrifugation, wash the cells with a buffer (such as PBS) to remove impurities in the separation solution and obtain relatively pure T cells.

3. The method for preparing a controllable CD19-targeted CAR-T cell according to claim 1, characterized in that: The CAR structure design mentioned in step S2.1 specifically includes the following steps: S2.1 (1) Select appropriate single-chain antibody (scFv) to recognize CD19 antigen; S2.1 (2), connects the transmembrane domain to ensure that CAR can be stably anchored on the T cell surface; S2.1 (3) Select appropriate intracellular signal transduction domains to activate the killing function of T cells; S2.1 (4), insert an inducible promoter upstream of the CAR gene.

4. The method for preparing a controllable CD19-targeted CAR-T cell according to claim 1, characterized in that: The lentiviral transduction mentioned in step S3.1 specifically includes the following steps: S3.

1. (1) Co-culture the lentiviral vector containing the CAR gene with the isolated T cells under appropriate culture conditions. During the transduction process, the virus will integrate the CAR gene into the T cell genome. Generally, the virus titer during transduction is between 10^6-10^8TU / mL (transduction units / mL), the cell density is about 1×10^6-5×10^6 cells / mL, and the transduction time is 12-24 hours; S3.

1. (2) After transduction, the non-transduced virus needs to be removed. This can be done by washing the cells. At the same time, culture medium containing antiretroviral drugs is added to prevent the virus from infecting the cells again.

5. The technology for preparing controllable CD19-targeted CAR-T cells according to claim 1, characterized in that: The cell proliferation monitoring mentioned in step S4.2 specifically includes the following steps: S4.2 (1) Regularly observe the growth status of cells and monitor changes in cell number using a cell counter or hemocytometer; S4.2 (2) According to the growth rate of cells, subculture should be carried out in a timely manner to maintain the good growth state of cells; S4.2 (3) During the amplification process, ensure that the CAR expression level of the cells is stable and regularly monitor the expression of CAR by flow cytometry.

6. The method for preparing a controllable CD19-targeted CAR-T cell according to claim 1, characterized in that: The functional verification mentioned in step S5.2 specifically includes the following steps: S5.2 (1), in vitro validation; S5.2(2), In vivo validation (optional).

7. The technology for preparing controllable CD19-targeted CAR-T cells according to claim 6, characterized in that: The in vitro validation mentioned in step S5.2 (1) specifically includes the following steps: ①. Co-culture CAR-T cells with tumor cell lines expressing CD19 antigen (such as Raji cells) in vitro at a certain ratio (such as 1:1, 5:1); ② After a certain period of culture (such as 4-24 hours), collect the culture supernatant, detect the LDH content therein, and calculate the lysis rate of tumor cells.

8. The technology for preparing controllable CD19-targeted CAR-T cells according to claim 6, characterized in that: The in vivo validation mentioned in step S5.2 (1) specifically includes the following steps: ① Establish a CD19-positive tumor model in an animal model (such as immunodeficient mice) and inject CAR-T cells into the mice by intravenous injection; ②. Observe the growth of mouse tumors, measure tumor volume and weight, and detect changes in cytokines in mice to verify the anti-tumor effect of CAR-T cells in vivo.

9. The method for preparing a controllable CD19-targeted CAR-T cell according to claim 1, characterized in that: The proportion of CAR-positive cells mentioned in the purity detection step S5.1 (1) is generally required to be above 70%-90%.

10. The technology for preparing controllable CD19-targeted CAR-T cells according to claim 1, characterized in that: The cryoprotectant mentioned in step S6.2 may be dimethyl sulfoxide.

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