WO2021109977A1 - 一种能高效制备且应用安全的嵌合抗原受体t细胞及其制备方法与应用 - Google Patents
一种能高效制备且应用安全的嵌合抗原受体t细胞及其制备方法与应用 Download PDFInfo
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Definitions
- the invention relates to the field of biotechnology, in particular to a chimeric antigen receptor T cell that can be efficiently prepared and applied safely, and a preparation method and application thereof.
- CAR-T therapy is chimeric antigen receptor T-cell immunotherapy, the full English name is Chimeric Antigen Receptor T-Cell Immunotherapy. This is a new type of precision targeted therapy for tumor treatment. In recent years, it has achieved good results in clinical tumor treatment through optimization and improvement. It is a very promising, accurate, fast, efficient, and possible cure for cancer. New tumor immunotherapy methods.
- the current conventional solution is to adopt suitable transfection reagents, maintain the best cell condition or choose the best transfection method; but these changes are still low in transfection efficiency.
- the preparation cost is still high.
- An object of the present invention is to provide a lentiviral recombinant vector.
- the lentiviral recombinant vector provided by the present invention contains a CAR structure coding sequence and a promoter MND that drives the expression of the CAR structure coding sequence;
- the nucleotide sequence of the promoter MND is 2838-3236 of sequence 1;
- the CAR structure in turn includes an extracellular binding domain, one or more hinge domains or spacer domains, a transmembrane domain, one or more intracellular costimulatory signal transduction domains, and a primary signal transduction domain.
- the extracellular domain is selected from at least one of the following target antigen antibodies or antigen-binding fragments, binding ligands or co-receptor extracellular domains: ⁇ folate receptor, 5T4, ⁇ v ⁇ 6 Integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FR ⁇ , GD2, GD3, Glypican-3 (GPC3), HLA-A1 +MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ES
- the hinge domain or spacer domain is selected from at least one of the transmembrane domains of CD8 ⁇ , CD4, CD45, PD1 and CD152; but it is not limited thereto;
- the transmembrane domain is selected from at least one of the transmembrane domains of CD8, CD4, CD45, PD1, and CD152; but not limited thereto;
- the intracellular costimulatory signal transduction domain is selected from CD28, CD54 (ICAM), CD134 (OX40), CD137 (41BB), CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1) and CD278 At least one of (ICOS); but not limited to this;
- the primary signal transduction domain is selected from at least one of the primary signal transduction domain of CD3 ⁇ and the primary signal transduction domain of FcR ⁇ ; but it is not limited thereto.
- the extracellular binding domain (also known as the antigen-specific binding domain) includes the light chain variable region of the CD19 antibody and the heavy chain variable region of the CD19 antibody;
- the hinge domain is a CD8 hinge domain
- the transmembrane domain is a CD8 transmembrane domain
- the intracellular costimulatory signal transduction domain is 4-1BB;
- the primary signal transduction domain is CD3 ⁇ .
- amino acid sequence of the light chain variable region of the CD19 antibody is 23-129 of sequence 3 in the sequence listing;
- amino acid sequence of the heavy chain variable region of the CD19 antibody is the 148-267th position in the sequence 3 in the sequence table;
- the amino acid sequence of the CD8 hinge domain is positions 268-307 of sequence 3 in the sequence listing;
- the amino acid sequence of the CD8 transmembrane domain is positions 308-335 of sequence 3 in the sequence table;
- amino acid sequence of the intracellular costimulatory signal transduction domain 4-1BB is the 336-377th position in the sequence 3 in the sequence table;
- amino acid sequence of the primary signal transduction domain CD3 ⁇ is positions 378-489 of sequence 3 in the sequence listing.
- the nucleotide sequence of the CAR structure coding sequence is 3251-4717 of SEQ ID NO: 1.
- the lentiviral recombination vector is the homologous recombination of DNA molecules at positions 2838-5863 of sequence 1 into a lentiviral expression vector to obtain a vector expressing the CAR structure; in the implementation of the present invention
- the specific lentiviral expression vector used is the pDBR vector.
- the lentiviral recombinant vector in the embodiment of the present invention is the lentiviral recombinant plasmid pLV-M19BBz (M19BBz), and its nucleotide sequence is sequence 1.
- the recombinant virus packaged by the above-mentioned lentiviral recombinant vector is also within the protection scope of the present invention.
- the above-mentioned recombinant virus is a virus capable of expressing the CAR structure shown at positions 3251-4717 of sequence 1, and capable of infecting immune effector cells.
- the virus is lentivirus, herpes virus, macrophage virus, Epstein-Barr virus, hepatitis B virus, hepatitis C virus or HIV.
- Recombinant cells containing the aforementioned lentiviral recombination vector are also within the protection scope of the present invention.
- the aforementioned recombinant cell is an immune effector cell expressing the aforementioned lentiviral recombinant vector
- the immune effector cells are cytotoxic T lymphocytes, NKT cells, NK cells, helper T cells or gamma delta T cells.
- promoter MND to drive the expression of CAR structure in CAR-T cells is also within the scope of this protection.
- the nucleotide sequence of the promoter MND is 2838-3236 of SEQ ID NO: 1.
- Another object of the present invention is to provide a method for preparing CAR-T cells.
- the method provided by the present invention includes the following steps: transfecting the recombinant virus obtained by packaging the above-mentioned lentiviral recombinant vector into a recombinant cell obtained by transfecting a T cell.
- the present invention also provides the following product for treating tumors or having the function of treating tumors and having a low cytokine storm, which includes the above-mentioned lentiviral recombinant vector or the above-mentioned recombinant cell.
- the invention also provides the following applications:
- the present invention provides the application of the above-mentioned lentiviral recombinant vector, the above-mentioned recombinant virus or the above-mentioned recombinant cell in the preparation of products for treating tumors;
- the present invention provides the application of the above-mentioned lentiviral recombinant vector, the above-mentioned recombinant virus or the above-mentioned recombinant cell in the preparation of products with tumor treatment and low cytokine storm functions.
- the present invention also provides a method for reducing the production of cytokine storm in CAR-T cell therapy, which is to use the promoter MND to drive the expression of CAR structure in T cells;
- the nucleotide sequence of the promoter MND is 2838-3236 of SEQ ID NO: 1.
- the above includes the following steps: 1) preparing the lentiviral recombinant vector in the first object;
- the present invention also provides a method for treating tumors, which includes the following steps:
- the present invention provides genetically engineered immune effector cells comprising a vector designed to express a chimeric antigen receptor that directs cytotoxicity to tumor cells.
- CARs are molecules that combine an antibody based on the specificity of a target antigen (such as a tumor antigen) with a T cell receptor activating intracellular domain to produce A chimeric protein with specific anti-tumor cell immune activity.
- chimeric as used herein describes being composed of parts of different proteins or composed of DNA from different sources.
- the vectors considered herein include promoters such as MND, EF1 ⁇ , CMV, PGK, etc. and polynucleotides encoding CAR (including colony stimulating factor signal peptide, FMC63 single-chain antibody sequence, CD8 hinge region and transmembrane region sequence, 4- 1BB costimulatory signal domain and CD3 ⁇ signal domain as well as T2A and tEGFR).
- the CAR considered herein includes an extracellular domain (also called a binding domain or an antigen-specific binding domain) that binds to a specific target antigen, a transmembrane domain, and an intracellular signal transduction domain.
- the binding of the antigen-binding domain of the CAR to its target antigen on the surface of the target cell results in the aggregation of the CAR and delivers an activation stimulus to the CAR-containing cells.
- the main characteristic of CAR is its ability to redirect immune effector cells specifically, thereby triggering proliferation, cytokine production, phagocytosis, or the ability to mediate the expression of targets in a major histocompatibility complex (MHC)-independent manner
- MHC major histocompatibility complex
- the production of molecules for cell death of antigens uses the cell-specific targeting ability of monoclonal antibodies, soluble ligands or cell-specific co-receptors.
- Figure 1 shows the CAR vector construction of different promoters EF1 ⁇ and MND, (a) CAR vector map using EF1 ⁇ promoter; (b) CAR vector map using MND promoter, (c) CAR vector map of different promoters EF1 ⁇ and MND Schematic diagram of the structure of the CAR carrier.
- FIG. 1 shows the measurement of transfection efficiency.
- Figure 3 is a flow chart of the determination of transfection efficiency.
- Figure 4 shows the difference in the expression of CAR molecules in T cells after transduction of E19BBz and M19BBz.
- Figure 5 shows the antigen-specific tumor clearance by T cells expressing CAR with different promoters and the amount of cytokine produced by CAR-T cells in the corresponding experiments.
- Figure 6 shows the regression effect of CAR-modified T cells under different promoters on tumor cells in tumor model mice after adoptive transfer.
- Figure 7 shows the transfection efficiency of the two CAR-T cells in clinical trials, Relative MFI, the expansion rate of T cell culture in vitro and the expansion rate of CAR-T in vivo.
- Figure 8 is a summary of the patient's response after treatment in the clinical trial, (a) the patient's body temperature change within 14 days after reinfusion; (b) the highest fever temperature of the patient after treatment; (c) the CRS series statistics of the patient after treatment .
- Figure 9 shows the long-term clinical results of CAR-T cells produced by two promoters after treatment.
- the lentiviral recombinant plasmid pLV-M19BBz (M19BBz) containing the MND promoter (as shown in Figure 1b and 1c below) has a nucleotide sequence of sequence 1 (full gene synthesis).
- This recombinant plasmid can also be used for sequence 1 to 2838- Homologous recombination of 5863 DNA molecules into the pDBR vector (Calderón-Gómez, E., et al., Reprogrammed quiescent B cells provide an effective cellular therapy against encephalomyelitis.European encephalomyelitis.European of Immunology, 2011.41(6) 1696-1708.).
- positions 2838-3236 of sequence 1 are MND promoters
- positions 3251-3316 are the nucleic acid encoding colony stimulating factor signal peptide (amino acid sequence is positions 1-22 of sequence 3)
- positions 3317-3637 are extra-membrane antigen binding Region CD19 antibody light chain variable region VL19 encoding nucleic acid (amino acid sequence is sequence 3 23-129)
- 3638-3691 is Linker
- 3692-4051 is extra-membrane antigen binding region CD19 antibody heavy chain can be Variable region VH19 encoding nucleic acid (VH19 amino acid sequence is sequence 3, positions 148-267; Sequence 1, positions 3317-4051 are FMC63 single-chain antibody coding nucleic acid), and positions 4052-4171 are CD8 hinge region coding nucleic acid (amino acid sequence 3 268-307), 4172-4255 are the CD8 transmembrane region coding nucleic acid (amino acid sequence 3, 308-335), and 4256-
- the lentiviral recombinant plasmid pLV-E19BBz (E19BBz) containing the EF1 ⁇ promoter (as shown in Figure 1a and 1c above) has the nucleotide sequence of sequence 2.
- the recombinant plasmid is the MND promoter from the 2838-3236 of sequence 1 Replace with the promoter EF1 ⁇ (sequence 2 at positions 2838-4131 are the promoter EF1 ⁇ ), and the remaining nucleotide sequence remains unchanged, to obtain a fragment.
- the above-mentioned recombinant vector is prepared according to the following method: separately clone the synthetic promoter sequence and the polynucleotide sequence of CAR, design primers to connect the two parts by overlapping PCR, and after the above sequence is successfully spliced, cloned into the pDBR vector (GenBank: FR822201.1) to construct pLV-E19BBz.
- pDBR vector GenBank: FR822201.1
- the promoter EF1 ⁇ of pLV-E19BBz (E19BBz) was replaced with MND and named pLV-M19BBz (M19BBz).
- M19BBz pLV-M19BBz
- the lentiviral recombinant plasmids pLV-E19BBz (E19BBz) and pLV-M19BBz (M19BBz) were packaged respectively to obtain the corresponding lentiviral particles. Specific steps are as follows:
- each petri dish 500 ⁇ l buffer (purchased from Polyplus Transfection, product catalog number B161116), 6 ⁇ g of the lentiviral recombinant plasmid prepared in Example 1 pLV-E19BBz (E19BBz) or pLV-M19BBz (M19BBz) , 3 ⁇ g psPAX2 (purchased from Wuhan Miaoling Biotechnology Co., Ltd., product catalog number P026) and 1.5 ⁇ g pMD2.G (purchased from Guangzhou Jisai Biotechnology Co., Ltd., product catalog number 161220L08), mix well, and then add to the system Add packaging reagents (purchased from Polyplus Transfection, product catalog number 114-15), 25 ⁇ l/10 cm petri dish, mix again, and let stand at room temperature for 10 minutes to obtain a mixed solution.
- 500 ⁇ l buffer purchased from Polyplus Transfection, product catalog number B161116)
- the culture supernatant is collected as the virus stock solution, and the collected virus stock solution is filtered into a 50 ml centrifuge tube with a 0.45 ⁇ m filter, and centrifuged at a high speed of 18500 g at 4° C. for 2 hours. The supernatant is discarded, and DMEM complete medium (the volume ratio of the added medium to the virus stock solution is 1:500) is added to the pellet to resuspend the virus particles, which is the virus concentrate.
- the virus titer was detected.
- the titers of E19BBz virus and M19BBz virus were 2.6 ⁇ 10 8 /ml and 4.3 ⁇ 10 8 /ml, respectively.
- the E19BBz virus stock solution and the M19BBz virus stock solution obtained by quantification (4) using the p24 ELISA kit (TAKARA, 632200) were detected.
- the result is shown in Figure 2b. It can be seen that the amount of p24 in the M19BBz virus stock solution is greater than the E19BBz virus stock solution.
- lymphocyte separation solution (Dongfang Huahui; 25710) into a new centrifuge tube, and then slowly add 2 times the volume (ie 30ml) of the blood sample diluted in step (1) to the upper layer of lymphocyte separation solution.
- the centrifugation parameters are 2000rpm, 20min, rise 7 fall 4, 25°C;
- the monocyte layer is transferred to a new centrifuge tube and washed with saline;
- step (3) Purify CD3+ T cells with the kit for monocytes obtained in step (3) (Miltenyi Biotec: 130-050-101) to obtain CD3+ T cells.
- E19BBz virus or M19BBz virus (MOI of 0.25, 0.5, 1, 2) prepared in step 1 (5) above to the culture flask with CD3+ T cells at room temperature. , 4, 8), then put the culture flask in a centrifuge, and centrifuge at 2000rpm, 2h, up 4 down 4, 35°C centrifugation parameters to complete the lentiviral transduction, and get CAR-T of M19BBz with different MOI Cells and CAR-T cells of E19BBz with different MOIs;
- step (8) of the above one were tested by flow cytometry, and the transduction rate was tested by CD4-FITC (Biolegend; 357405) and 7AAD (Biolegend; 420403).
- FIG. 3 is a representative graph with MOI value of 0.25. It can be seen that the T cell transduction rate of M19BBz is higher than that of E19BBz.
- the cells on the fifth day (MOI value of 0.5) cultured in step (8) of the above one were tested by flow cytometry, and the biotinylated Erbitux (anti-EGFR monoclonal antibody, Merck: Erbitux) and SA-PE (Biolegend: 405204) or SA-APC (Biolegend: 405207) detects the expression of tEGFR, as well as the detection of specific antibodies with antigen CD19Fc-FITC (ACRO: P15391-1) and FMC63scFv (Bioswan: 019-01-647M) CAR molecule expression. Untransduced CD3+ T cells were used as controls.
- MFI Fluorescence Intensity
- tEGFR Detect the expression of tEGFR (erb).
- M19BBz positive cell MFI/negative cell MFI
- E19BBz E19BBz
- FMC63scFv antibody scFv
- scFv FMC63scFv antibody
- the target cells were first stained with 1mg/ml calcein (Calcein-AM, Invitrogen, C3099), and then in 200 ⁇ l cell culture medium (RPMI 1640, Gibco, 22400-089+10% FBS ExCell Bio, FND500) target cells were added to 2 ⁇ 10 4, each CAR-T cells were added to each 2 ⁇ 10 5, after 37 °C for four hours, the supernatant was detected calcein released (detected using a microplate reader Multiskan Sky, excitation detection condition The wavelength is 490nm and the emission wavelength is 515nm.).
- Spontaneous release group the target cells directly detect the supernatant after the same time, the value of this group is the spontaneous release of fluorescence;
- Specific killing efficiency (%) (fluorescence value of experimental group-fluorescence value of spontaneous release group)/(fluorescence value of complete release group-fluorescence value of spontaneous release group) ⁇ 100%
- NOD-SCID mice were used to establish a Raji-luciferase tumor model.
- This model was constructed by infusion of Raji human B-cell lymphoma cells containing luc (luciferase) labeled Raji-luciferase (by Yikang (Beijing) Pharmaceutical Provided by Science and Technology Co., Ltd.), the tumor cells in the logarithmic growth phase were injected into the blood vessels of the mouse through the tail vein of the mouse, and the details are as follows:
- the Raji-luciferase tumor model cells were prepared into a cell suspension with a concentration of 1 ⁇ 10 7 /ml with PBS, and inoculated into mice through the tail vein, 100 ⁇ l/mouse; each mouse was inoculated with 1 ⁇ 10 6 cells.
- the mice will be injected with luciferin for three days. After anesthesia, the fluorescence intensity will be detected on the equipment (IVIS Lumina, Series III, PE). After the detection, mice with the same fluorescence intensity will be screened and then randomly divided and administered (six mice in each group).
- Control group Inject 200 ⁇ l of physiological saline containing 2% (mass volume percentage content, unit is g/ml) human albumin (Hebei Daan Pharmaceutical Co., Ltd., S200443042);
- M19BBz group (CAR-T M19BBz): Inject 200 ⁇ l of a solution containing 5 ⁇ 10 6 M19BBz CAR-T cells (MOI value is 0.25) (convert the total cell number according to the transfection rate, and resuspend the cells in 2% Human albumin in normal saline);
- E19BBz group (CAR-T E19BBz): Inject 200 ⁇ l of a solution containing 5 ⁇ 10 6 E19BBz CAR-T cells (MOI value is 0.25) (convert the total number of cells according to the transfection rate, and resuspend the cells in a 2% Human albumin in normal saline);
- the leukocytes were prepared according to the method of 1 and 3 in Example 1, and prepared multiple batches of M19BBz CAR-T cells (a certain batch of MOI value is 1) and E19BBz CAR-T cells (a certain batch of MOI value 1) (each is a different batch of cells), CAR-T cells were subjected to flow cytometry to detect the transduction rate (by biotinylated Erbitux (anti-EGFR monoclonal antibody, Merck: Love) before reinfusion on the fourteenth day). Biduo) and SA-PE (Biolegend: 405204) or SA-APC (Biolegend: 405207) to detect the expression of tEGFR), and the MFI value. Patients undergo reinfusion and stay in the hospital for observation until they are discharged.
- the number of chimeric antigen receptors on the surface of CAR-T cells can be changed, the safety of the product can be further adjusted, cytokine storms can be reduced, and safer products can be provided.
- the experiment of the present invention proves that, in order to overcome the shortcomings of low transfection efficiency and large amount of cytokine produced in the current CAR-T treatment process, the present invention can significantly increase the transfection rate, and the process development of clinical-grade cell production is easier to carry out.
- Reduce production costs reduce the density of CAR molecules on the cell surface and reduce the intensity of the reaction, thereby reducing the occurrence of CRS and making CAR-T treatment safer.
- the specifics are as follows: by improving the initiation of the CAR structure by the promoter, that is, using the MND promoter to promote the CAR structure, a new chimeric antigen receptor is prepared, and T cells expressing the chimeric antigen receptor are prepared.
- the T cells have an impact
- the CAR molecule has the characteristic of less expression on the cell surface.
- it has the advantage of lessening the cell killing effect. This can greatly reduce the occurrence of cytokine storms and effectively improve the safety.
- the preparation method obtains Chimeric antigen receptor T cells also have high transduction efficiency, and can be stably and persistently expressed on the surface of T cells, so that when they bind to the antigen on the surface of the target cell, they can play a killing effect on the target cell, and in addition, the expression can be sustained. It can also make its killing effect lasting; make it safer clinically for the treatment of cancer.
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Abstract
Description
Claims (18)
- 慢病毒重组载体,其含有CAR结构和驱动所述CAR结构表达的启动子MND;所述启动子MND的核苷酸序列为序列1第2838-3236位;所述CAR结构依次包括胞外结合结构域、一个或多个铰链结构域或间隔结构域、跨膜结构域、一个或多个胞内共刺激信号转导结构域以及初级信号转导结构域。
- 根据权利要求1所述的慢病毒重组载体,其特征在于:所述胞外结合结构域选自如下靶抗原的抗体或其抗原结合片段、束缚配体或者共受体的胞外结构域中至少一种:α叶酸受体、5T4、αvβ6整合素、BCMA、B7-H3、B7-H6、CAIX、CD19、CD20、CD22、CD30、CD33、CD44、CD44v6、CD44v7/8、CD70、CD79a、CD79b、CD123、CD138、CD171、CEA、CSPG4、EGFR、包括ErbB2的EGFR家族、EGFRvIII、EGP2、EGP40、EPCAM、EphA2、EpCAM、FAP、胎儿型AchR、FRα、GD2、GD3、磷脂酰肌醇聚糖-3、HLA-A1+MAGE1、HLA-A2+MAGE1、HLA-A3+MAGE1、HLA-A1+NY-ESO-1、HLA-A2+NY-ESO-1、HLA-A3+NY-ESO-1、IL-11Rα、IL-13Rα2、λ、Lewis-Y、κ、间皮素、Muc1、Muc16、NCAM、NKG2D配体、NY-ESO-1、PRAME、PSCA、PSMA、ROR1、SSX、生存素、TAG72、TEM和VEGFR2;所述铰链结构域或间隔结构域选自CD8α、CD4、CD45、PD1和CD152的跨膜结构域中的至少一种;所述跨膜结构域选自CD8、CD4、CD45、PD1和CD152的跨膜结构域中的至少一种;所述胞内共刺激信号转导结构域选自CD28、CD54(ICAM)、CD134(OX40)、CD137(41BB)、CD152(CTLA4)、CD273(PD-L2)、CD274(PD-L1)和CD278(ICOS)中至少一种;所述初级信号转导结构域选自CD3ζ的初级信号转导结构域和FcRγ的初级信号转导结构域中至少一种。
- 根据权利要求1或2所述的慢病毒重组载体,其特征在于:所述胞外结合结构域包括CD19抗体的轻链可变区和CD19抗体的重 链可变区;所述铰链结构域为CD8铰链结构域;所述跨膜结构域为CD8跨膜结构域;所述胞内共刺激信号转导结构域为4-1BB;所述初级信号转导结构域为CD3ζ。
- 根据权利要求3所述的慢病毒重组载体,其特征在于:所述CD19抗体的轻链可变区的氨基酸序列为序列表中序列3第23-129位;所述CD19抗体的重链可变区的氨基酸序列为序列表中序列3第148-267位;所述CD8铰链结构域的氨基酸序列为序列表中序列3第268-307位;所述CD8跨膜结构域的氨基酸序列为序列表中序列3第308-335位;所述胞内共刺激信号转导结构域4-1BB的氨基酸序列为序列表中序列3第336-377位;所述初级信号转导结构域CD3ζ的氨基酸序列为序列表中序列3第378-489位。
- 根据权利要求1-4中任一所述的慢病毒重组载体,其特征在于:所述CAR结构的核苷酸序列为序列1第3251-4717位。
- 根据权利要求5所述的慢病毒重组载体,其特征在于:所述慢病毒表达载体为pDBR载体;所述慢病毒重组载体的核苷酸序列为序列1。
- 由权利要求1-6中任一所述的慢病毒重组载体包装得到的重组病毒。
- 含有权利要求1-6中任一所述的慢病毒重组载体的重组细胞。
- 根据权利要求8所述的重组细胞,其特征在于:所述重组细胞为表达权利要求1-6中任一所述的慢病毒重组载体的免疫效应细胞。
- 根据权利要求9所述的重组细胞,其特征在于:所述免疫效应细胞为细胞毒性T淋巴细胞、NKT细胞、NK细胞、辅助性T细胞或gamma delta T细胞。
- 启动子MND在CAR-T细胞中用作驱动CAR结构表达的应用;所述启动子MND的核苷酸序列为序列1第2838-3236位。
- 一种制备CAR-T细胞的方法,包括如下步骤:将由权利要求1-6中任一所述的慢病毒重组载体包装得到的重组病毒转染T细胞得到的重组细胞。
- 一种治疗肿瘤或具有治疗肿瘤且低细胞因子风暴功能的产品,其包括权利要求1-6中任一所述的慢病毒重组载体或权利要求8-10中任一所述重组细胞。
- 权利要求1-6中任一所述的慢病毒重组载体、权利要求7所述重组病毒或权利要求8-10中任一所述重组细胞在制备治疗肿瘤的产品中的应用;
- 权利要求1-6中任一所述的慢病毒重组载体、权利要求7所述重组病毒或权利要求8-10中任一所述重组细胞在制备具有治疗肿瘤且低细胞因子风暴功能的产品中的应用。
- 一种降低CAR-T细胞治疗中细胞因子风暴产生量的方法,为用启动子MND驱动CAR结构在T细胞中表达;所述启动子MND的核苷酸序列为序列1第2838-3236位。
- 根据权利要求16所述的方法,其特征在于:所述方法包括如下步骤:1)制备权利要求1-6中任一所述的慢病毒重组载体;2)将所述慢病毒重组载体导入自体T细胞中,得到CAR-T细胞;3)将所述CAR-T细胞回输体内,实现降低CAR-T细胞治疗中细胞因子风暴产生量。
- 一种治疗肿瘤的方法,包括如下步骤:1)制备权利要求1-6中任一所述的慢病毒重组载体;2)将所述慢病毒重组载体导入自体T细胞中,得到CAR-T细胞;3)将所述CAR-T细胞回输体内,实现治疗肿瘤。
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