WO2019242339A1 - 一种包含第三信号受体的嵌合抗原受体及其应用 - Google Patents
一种包含第三信号受体的嵌合抗原受体及其应用 Download PDFInfo
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Definitions
- the invention relates to the technical field of cellular immunotherapy, in particular to a chimeric antigen receptor containing a third signal receptor and application thereof.
- chimeric antigen receptor T cells were first proposed by Gross, Waks, and Eshhar in 1989. They expressed antibodies that recognize TNP on T cells, achieving the activation and effects of antigen-specific, non-MHC-restricted T cells. Enhancement and put forward the concept of the application of CAR-T technology in tumor treatment. According to this principle, embedding tumor-specific antibodies into T cells will give T cells new tumor-killing capabilities. Later, CAR-T technology was introduced into anti-tumor clinical trials. However, early CAR-T cells contained only the first signal because of their intracellular signalling domain, and the selected tumor type was solid tumor. The final clinical results were not Too ideal.
- the structure of CAR consists of an extracellular antigen recognition domain, an extracellular hinge region, a transmembrane region, and an intracellular signaling domain.
- the extracellular antigen recognition domain is usually composed of a single chain antibody, which specifically recognizes the surface molecules of tumor cell membranes, and may also be a ligand or receptor for certain tumor-specific antigens.
- the extracellular hinge region is a space structure that separates the antigen recognition domain from the transmembrane region. The purpose is to provide a suitable spatial location so that the extracellular antigen recognition domain can maintain the correct structure before and after recognition of the antigen and conduct intracellularly. signal.
- the transmembrane region is a domain that ensures the localization of CAR molecules on the membrane surface.
- the intracellular signaling domain is a key part of mediating CAR signaling, usually one or several first signals (recognition of TCR and MHC-I-peptide complexes), and second signals (co-stimulatory receptors and co-stimulatory receptors). Body recognition).
- the first generation CAR contains only the first signal
- the second generation CAR has a first signal and a second signal
- the third generation CAR has a first signal and two second signal domains.
- CAR-T for B cell surface targeting molecules CD19 and CD20 prepared from patient's own blood cells has been relatively mature in the treatment of B cell leukemia, but although the response rate is high, there are a large number of recurrences. Tumor treatment efficiency is relatively low, which is related to the immunosuppressive microenvironment in solid tumors.
- tumor microenvironment In solid tumors, there are a variety of immune cells, tumor cells and stromal cells, which together constitute the tumor microenvironment.
- the tumor microenvironment is usually immunosuppressive. It can inhibit endogenous anti-tumor T cell responses or adoptive T cells (such as CAR-T) at multiple levels. For example, T cells are depleted and tumor-killing cells are lost. Function, eventually T cells are cleared. How to enhance the activation capacity of CAR-T in solid tumors so that it can fight against immunosuppression in the tumor microenvironment is an important idea and direction for the expansion of CAR-T to solid tumors.
- CAR-T domains use novel regulatory molecules such as IL-12, 4 -1BBL, etc. In addition to affecting CAR-T, these molecules will also have non-specific activation of other non-CAR-T cells, potentially causing immune side effects.
- the purpose of the present invention is to overcome the defects in the prior art, provide a chimeric antigen receptor of a third signal receptor and its application, and provide a CAR constructed from a recombinant expression vector such as the chimeric antigen receptor.
- -T cells in which the activation of T cells is subjected to a first signal (recognition of TCR and MHC-I-peptide complex), a second signal (recognition of co-stimulatory receptors and co-stimulatory ligands), and 3
- the regulation of signals cytokine receptor and cytokine recognition
- these three synergistically achieve a large number of T cells to expand, exert effector functions, and clear infections or tumors.
- the present invention adopts the following technical solutions:
- a first object of the present invention is to provide a chimeric antigen receptor including a third signal receptor, and the structure of the chimeric antigen receptor is scFv (X)-(Y) CD3zeta-MN;
- X includes a tumor-targeting antibody or a ligand and a receptor capable of specifically binding to the tumor
- Y is an intracellular region of a co-stimulatory receptor selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226
- M is the intracellular region of the gamma chain family cytokine receptor, the cytokine receptor is selected from IL2Ra, IL2Rb, IL4Ra, IL7Ra, IL9Ra, IL15Ra, IL21Ra
- N is the intracellular region of IL2Rg.
- the technical measures taken by the present invention also include:
- the X is selected from the group consisting of an anti-CD19 antibody, an anti-CD20 antibody, an EGFR antibody, an HER2 antibody, an EGFRVIII antibody, an anti-PSMA antibody, an anti-BCMA antibody, an anti-CD22 antibody, and an anti-CD30 antibody. It can be understood that X may also be another protein capable of specifically binding to a tumor.
- the X is an anti-CD20 antibody
- the Y is a 4-1BB intracellular region
- the M is selected from the group consisting of an IL2Rb intracellular region, an IL4Ra intracellular region, an IL7Ra intracellular region, an IL9Ra intracellular region, and an IL21Ra cell.
- One of the inner zones are included in the X and the Y.
- the scFv (X)-(Y) CD3zeta is scFv-antihCD20-20BBZ, the sequence of which is shown in SEQ ID No. 1; the sequence of the IL7Ra intracellular region is shown in SEQ ID No. 2; the IL2Rb The sequence of the intracellular region is shown in SEQ ID No. 3; the sequence of the intracellular region of IL4Ra is shown in SEQ ID No. 4; the sequence of the intracellular region of IL9Ra is shown in SEQ ID No. 5; The sequence of the region is shown in SEQ ID No. 6; the sequence of the IL2Rg intracellular region is shown in SEQ ID No. 7.
- the extracellular hinge region of the chimeric antigen receptor is selected from a region of CD8a or IgG; the transmembrane region of the chimeric antigen receptor is selected from one of CD8a, CD28, CD137, or CD3.
- a second object of the present invention is to provide a recombinant expression vector of any of the aforementioned chimeric antigen receptors.
- a third object of the present invention is to provide a CAR-T cell constructed from a recombinant expression vector of any of the aforementioned chimeric antigen receptors.
- a fourth object of the present invention is to provide a method for preparing the above CAR-T cells, which includes the following steps:
- Step 1 Construction of lentiviral vector and virus production
- ScFv (X)-(Y) CD3zeta, M and N form a fusion protein
- lentiviral vectors are added at both ends, and co-transfection with the lentiviral packaging plasmid to obtain scFv (X)-(Y) CD3zeta-MN virus;
- Step 2 Preparation of scFv (X)-(Y) CD3zeta-MN CAR-T cells;
- the isolated and purified human PBMCs were cultured and infected with the scFv (X)-(Y) CD3zeta-MN virus obtained in step 1.
- Cell expansion was performed under appropriate conditions to prepare scFv (X)-(Y) CD3zeta-MN.
- CAR-T cells
- X is a tumor-targeting antibody or other protein
- Y is an intracellular region of a co-stimulatory receptor selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40 , DR3, GITR, CD30, TIM1, SLAM, CD2, CD226
- M is the intracellular region of the gamma chain family cytokine receptor selected from IL2Ra, IL2Rb, IL4Ra, IL7Ra, IL9Ra, IL15Ra, IL21Ra
- N is the intracellular region of IL2Rg.
- the technical measures adopted by the present invention further include:
- the specific steps of constructing the lentiviral vector and virus production include: scFv (X)-(Y) CD3zeta, M and N are formed by an overlap PCR to form a fusion protein, and restriction sites are added at both ends to clone the lentiviral vector. ; The endotoxin-free clone was cloned correctly and transfected with the lentiviral packaging plasmid. The supernatant was collected at a predetermined time, and the virus was concentrated by centrifugation and centrifugation to obtain scFv (X)-(Y) CD3zeta-MN virus.
- scFv (X)-(Y) CD3zeta M and N are formed by an overlap PCR to form a fusion protein, and EcoRI and BamHI restriction sites are added at both ends.
- Clone the pCDH-MSCVEF vector clone the correctly cloned endotoxin-free clone and transfect 293X with the lentiviral packaging plasmid, collect the supernatant after 48 and 72 hours, and centrifuge at 25,000 RPM for 0.4 hours after 0.45 uM filtration to concentrate the virus to obtain scFv ( X)-(Y) CD3zeta-MN virus.
- the specific steps of preparing the scFv (X)-(Y) CD3zeta-MN CAR-T cells include: after separating and purifying human PBMC, inoculating it into a culture plate with appropriate stimulation conditions, and culturing for a predetermined time, The scFv (X)-(Y) CD3zeta-MN virus produced in step 1 was infected, and the cells were expanded according to appropriate stimulation conditions. After 2 rounds of stimulation and expansion, the cells obtained were scFv (X)-(Y) CD3zeta -MN CAR-T cells.
- the stimulating conditions of cultured, isolated and purified human PBMC are anti-hCD3 and anti-hCD28, and the stimulating conditions of expanded cells are stimulated with artificial antigen presenting cells or anti-hCD3 / 28 every 6 days.
- the X is selected from an anti-CD19 antibody, an anti-CD20 antibody, an EGFR antibody, a HER2 antibody, and an EGFRVIII antibody.
- the X is an anti-CD20 antibody
- the Y is 4-1BB
- the M is selected from one of IL2Rb, IL4Ra, IL7Ra, IL9Ra, and IL21Ra.
- the scFv (X)-(Y) CD3zeta is scFv-antihCD20-20BBZ, the sequence of which is shown in SEQ ID No. 1; the sequence of the IL7Ra intracellular region is shown in SEQ ID No. 2; the IL2Rb The sequence of the intracellular region is shown in SEQ ID No. 3; the sequence of the intracellular region of IL4Ra is shown in SEQ ID No. 4; the sequence of the intracellular region of IL9Ra is shown in SEQ ID No. 5; The sequence of the region is shown in SEQ ID No. 6; the sequence of the IL2Rg intracellular region is shown in SEQ ID No. 7.
- the lentiviral packaging plasmid in step 1 includes VSV-g, pMD, Gag / Pol, RSV-REV, and a Beckman ultracentrifuge and a SW28 rotor are used for centrifugation.
- a fifth object of the present invention is to provide a preparation containing the above CAR-T cells or a CAR-T cell prepared by the above-mentioned preparation method; further, the preparation further includes a pharmaceutically acceptable diluent or excipient. Agent.
- a sixth object of the present invention is to provide an application of the above-mentioned chimeric antigen receptor, the above-mentioned CAR-T cell, or the CAR-T cell prepared by the above-mentioned preparation method in preparing a medicine for treating or preventing tumor.
- the tumor is a solid tumor
- examples of the solid tumor include, but are not limited to, lymphoma, kidney tumor, neuroblastoma, germ cell tumor, osteosarcoma, chondrosarcoma, soft tissue sarcoma, liver tumor, thymoma, Pulmonary blastoma, pancreatoblastoma, hemangioma, etc.
- the present invention has the following beneficial effects:
- the CAR-T cells according to the present invention significantly improve the tumor killing ability and expansion ability, and significantly improve the killing ability of solid tumors / metastatic tumors.
- the CAR-T cells according to the invention include a third signal receptor (IL2Ra , IL2Rb, IL4Ra, IL7Ra, IL9Ra, IL15Ra, IL21Ra, etc.), which are not conventionally used ligands or secretory factors, such as the third signal receptor IL7Ra, which is mainly expressed in memory CD4 and CD8 T cells. Long-term survival and the formation of memory T cells have an important role. Integrating the third signal receptor signal into CAR-T has the potential to enhance the effect, and only affects CAR-T cells, reducing the immune side effects risks of.
- the present invention improves the activation capacity and survival capacity of CAR-T cells in tumors compared with the currently used CAR-T technology, and the activation capacity and expansion capacity are significantly improved. Enhancement, thereby increasing the efficacy of CAR-T cells to have more excellent anti-tumor efficacy.
- FIG. 1 is a schematic diagram illustrating a molecular structure of a chimeric antigen receptor (CAR) containing a third signal receptor in each embodiment of the present invention
- FIG. 2 is a schematic diagram of a virus titer measured after 293 cells were infected by BBZIL2RbIL2Rg virus according to an embodiment of the present invention
- FIG. 3 is a schematic diagram of a virus titer measured after 293 cells were infected by BBZIL4RaIL2Rg virus according to an embodiment of the present invention
- FIG. 4 is a schematic diagram of a virus titer measured after 293 cells were infected by BBZIL7RaIL2Rg virus according to an embodiment of the present invention
- FIG. 5 is a schematic diagram of a virus titer measured after BBZIL9RaIL2Rg virus infects 293 cells according to an embodiment of the present invention
- FIG. 6 is a schematic diagram of a virus titer measured after 293 cells were infected by BBZIL21RaIL2Rg virus according to an embodiment of the present invention
- FIG. 7 is a schematic diagram of the results of phenotypic analysis of BBZ CAR-T cells and BBZIL2RbIL2Rg CAR-T cells in an embodiment of the present invention
- FIG. 8 is a schematic diagram of the results of phenotypic analysis of BBZ CAR-T cells and BBZIL4RaIL2Rg CAR-T cells in an embodiment of the present invention
- FIG. 9 is a schematic diagram of the results of phenotypic analysis of BBZ CAR-T cells and BBZIL7RaIL2Rg CAR-T cells in an embodiment of the present invention.
- FIG. 10 is a schematic diagram of the results of phenotypic analysis of BBZ CAR-T cells and BBZIL9RaIL2Rg CAR-T cells in an embodiment of the present invention
- FIG. 11 is a schematic diagram of the results of phenotypic analysis of BBZ CAR-T cells and BBZIL21RaIL2Rg CAR-T cells in an embodiment of the present invention
- FIG. 12 is a schematic diagram of the expansion capacity of BBZ CAR-T cells and BBZIL7RaIL2Rg CAR-T cells in an embodiment of the present invention
- FIG. 13 is a schematic diagram of tumor killing ability of BBZ CAR-T cells and BBZIL7RaIL2Rg CAR-T cells in an embodiment of the present invention
- FIG. 14 is a schematic diagram of the anti-tumor ability of BBZ CAR-T cells and BBZIL7RaIL2Rg CAR-T cells in an embodiment of the present invention
- FIG. 15 is a schematic diagram of the in vivo viability of BBZ CAR-T cells and BBZIL7RaIL2Rg CAR-T cells in an embodiment of the present invention.
- the present invention provides a chimeric antigen receptor including a third signal receptor.
- the structure of the chimeric antigen receptor is scFv (X)-(Y) CD3zeta-MN; wherein X is a tumor-targeting antibody or other protein ; Y is the intracellular region of a co-stimulatory receptor selected from the group consisting of ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226; M is the intracellular region of the gamma chain family cytokine receptor selected from IL2Ra, IL2Rb, IL4Ra, IL7Ra, IL9Ra, IL15Ra, IL21Ra; N is the IL2Rg intracellular region.
- the invention also relates to a CAR-T cell constructed from any of the above-mentioned chimeric antigen receptor recombinant expression vectors and a preparation method thereof, a preparation containing the CAR-T cell, and an application of the CAR-T cell.
- the chimeric antigen receptor (CAR) molecules containing a third signal receptor are BBZIL2RbIL2Rg, BBZIL4RaIL2Rg, BBZIL7RaIL2Rg, BBZIL9RaIL2Rg, and BBZIL21RaIL2Rg, and their structures are shown in FIG. 1.
- the preparation of the 20BBZIL2RbIL2Rg CAR-T cells described in this example includes the following steps:
- ScFv-antihCD20-20BBZ (SEQ ID No. 1), IL2Rb intracellular region (SEQ ID No. 3) and IL2Rg intracellular region (SEQ ID No. 7) were formed by overlapping PCR to form a fusion protein, and EcoRI and BamHI were added at both ends.
- the pCDH-MSCVEF vector was cloned at the restriction site. Endotoxin-free clones that were sequenced correctly were cloned with lentiviral packaging plasmids (VSV-g, pMD, Gag / Pol, RSV-REV) to transfect 293X. Supernatants were collected after 48 and 72 hours.
- the Beckman ultracentrifuge and SW28 rotor were centrifuged at 25,000 RPM for 2 hours to concentrate the virus, namely pCDH-MSCVEF-20BBZIL2RbIL2Rg virus (referred to as 20BBZIL2RbIL2Rg virus) for subsequent CAR-T cell production.
- pCDH-MSCVEF-20BBZ virus (20BBZ virus for short) was produced, and the obtained 20BBZIL2RbIL2Rg virus was used to infect 293 cells, and the virus titer was measured, as shown in FIG. 2.
- the preparation of the 20BBZIL4RaIL2Rg CAR-T cells described in this example includes the following steps:
- ScFv-antihCD20-20BBZ (SEQ ID No. 1), IL4Ra intracellular region (SEQ ID No. 4) and IL2Rg intracellular region (SEQ ID No. 7) were formed by an overlap PCR to form a fusion protein, and EcoRI and BamHI were added at both ends.
- the pCDH-MSCVEF vector was cloned at the restriction site. Endotoxin-free clones that were sequenced correctly were cloned with lentiviral packaging plasmids (VSV-g, pMD, Gag / Pol, RSV-REV) to transfect 293X. Supernatants were collected after 48 and 72 hours.
- the Beckman ultracentrifuge and SW28 rotor were centrifuged at 25,000 RPM for 2 hours to concentrate the virus, namely pCDH-MSCVEF-20BBZIL4RaIL2Rg virus (referred to as 20BBZIL4RaIL2Rg virus) for subsequent CAR-T cell production.
- pCDH-MSCVEF-20BBZ virus 20BBZ virus for short
- 20BBZIL4RaIL2Rg virus was used to infect 293 cells, and the virus titer was measured, as shown in FIG. 3.
- the preparation of the 20BBZIL7RaIL2Rg CAR-T cells described in this example includes the following steps:
- ScFv-antihCD20-20BBZ (SEQ ID No. 1), IL7Ra intracellular region (SEQ ID No. 2) and IL2Rg intracellular region (SEQ ID No. 7) were formed by an overlap PCR to form a fusion protein, and EcoRI and BamHI were added at both ends.
- the pCDH-MSCVEF vector was cloned at the restriction site. Endotoxin-free clones that were sequenced correctly were cloned with lentiviral packaging plasmids (VSV-g, pMD, Gag / Pol, RSV-REV) to transfect 293X. Supernatants were collected after 48 and 72 hours.
- the Beckman ultracentrifuge and SW28 rotor were centrifuged at 25,000 RPM for 2 hours to concentrate the virus, namely pCDH-MSCVEF-20BBZIL7RaIL2Rg virus (referred to as 20BBZIL7RaIL2Rg virus) for subsequent CAR-T cell production.
- the control pCDH-MSCVEF-20BBZ virus (20BBZ virus for short) was produced, and the obtained 20BBZIL7RaIL2Rg virus was used to infect 293 cells, and the virus titer was measured, as shown in FIG. 4.
- the preparation of the 20BBZIL9RaIL2Rg CAR-T cells described in this example includes the following steps:
- ScFv-antihCD20-20BBZ (SEQ ID No. 1), IL9Ra intracellular region (SEQ ID No. 5) and IL2Rg intracellular region (SEQ ID No. 7) were formed by overlapping PCR to form a fusion protein, and EcoRI and BamHI were added at both ends.
- the pCDH-MSCVEF vector was cloned at the restriction site. Endotoxin-free clones that were sequenced correctly were cloned with lentiviral packaging plasmids (VSV-g, pMD, Gag / Pol, RSV-REV) to transfect 293X. Supernatants were collected after 48 and 72 hours.
- the Beckman ultracentrifuge and SW28 rotor were centrifuged at 25,000 RPM for 2 hours to concentrate the virus, namely pCDH-MSCVEF-20BBZIL9RaIL2Rg virus (referred to as 20BBZIL9RaIL2Rg virus) for subsequent CAR-T cell production.
- pCDH-MSCVEF-20BBZ virus 20BBZ virus for short
- 20BBZIL9RaIL2Rg virus was used to infect 293 cells, and the virus titer was measured, as shown in FIG. 5.
- the preparation of the 20BBZIL21RaIL2Rg CAR-T cells described in this example includes the following steps:
- ScFv-antihCD20-20BBZ (SEQ ID No. 1), IL21Ra intracellular region (SEQ ID No. 6) and IL2Rg intracellular region (SEQ ID No. 7) were formed by overlapping PCR to form a fusion protein, and EcoRI and BamHI were added at both ends.
- the pCDH-MSCVEF vector was cloned at the restriction site. Endotoxin-free clones that were sequenced correctly were cloned with lentiviral packaging plasmids (VSV-g, pMD, Gag / Pol, RSV-REV) to transfect 293X. Supernatants were collected after 48 and 72 hours.
- the Beckman ultracentrifuge and SW28 rotor were centrifuged at 25,000 RPM for 2 hours to concentrate the virus, namely pCDH-MSCVEF-20BBZIL21RaIL2Rg virus (referred to as 20BBZIL21RaIL2Rg virus) for subsequent CAR-T cell production.
- pCDH-MSCVEF-20BBZ virus 20BBZ virus for short
- 20BBZIL21RaIL2Rg virus was used to infect 293 cells, and the virus titer was measured, as shown in FIG. 6.
- the 20BBZ CAR-T cells and 20BBZIL7RaIL2Rg CAR-T cells prepared in step 2 in Example 3 were continuously cultured for 14 days, and stimulated with artificial antigen presenting cells every 6 days. The cell count was shown in FIG. 12. It can be seen from the figure that 20BBZIL7RaIL2Rg CAR-T cells have stronger proliferation ability than 20BBZCAR-T cells.
- 20BBZ CAR-T cells and 20BBZIL7RaIL2Rg CAR-T cells prepared in step 2 in Example 3 were seeded into a 96-well plate, and Raji tumors were added according to CAR-T: tumor cell ratios 1: 1, 1: 2, and 1: 4. Cells were compared for survival ratio of tumor cells after 24 and 48 hours. The results are shown in FIG. 13. It can be seen from the figure that 20BBZIL7RaIL2Rg CAR-T cells have similar tumor-killing ability compared to 20BBZ CAR-T cells.
- the present invention enables CAR-T cells to activate, survive, and expand CAR
- the enhancement ability is significantly enhanced, and it has more excellent anti-tumor efficacy.
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Abstract
Description
Claims (10)
- 一种包含第三信号受体的嵌合抗原受体,其特征在于,所述嵌合抗原受体的结构为scFv(X)-(Y)CD3zeta-MN;其中,X包括肿瘤靶向抗体或能与肿瘤特异结合的配体、受体;Y为共刺激受体的胞内区,所述共刺激受体选自ICOS、CD28、CD27、HVEM、LIGHT、CD40L、4-1BB、OX40、DR3、GITR、CD30、TIM1、SLAM、CD2、CD226;M为gamma chain家族细胞因子受体的胞内区,所述细胞因子受体选自IL2Ra、IL2Rb、IL4Ra、IL7Ra、IL9Ra、IL15Ra、IL21Ra;N为IL2Rg胞内区。
- 根据权利要求1所述的一种包含第三信号受体的嵌合抗原受体,其特征在于,所述X选自anti-CD19抗体、anti-CD20抗体、EGFR抗体、HER2抗体、EGFRVIII抗体、anti-PSMA抗体、anti-BCMA抗体、anti-CD22抗体、anti-CD30抗体。
- 根据权利要求1所述的一种包含第三信号受体的嵌合抗原受体,其特征在于,所述X为anti-CD20抗体,所述Y为4-1BB胞内区,所述M选自IL2Rb胞内区、IL4Ra胞内区、IL7Ra胞内区、IL9Ra胞内区、IL21Ra胞内区中的一种。
- 根据权利要求3所述的一种包含第三信号受体的嵌合抗原受体,其特征在于,所述scFv(X)-(Y)CD3zeta为scFv-antihCD20-20BBZ,其序列如SEQ ID No.1所示;所述IL7Ra胞内区的序列如SEQ ID No.2所示;所述IL2Rb胞内区的序列如SEQ ID No.3所示;所述IL4Ra胞内区的序列如SEQ ID No.4所示;所述IL9Ra胞内区的序列如SEQ ID No.5所示;所述IL21Ra胞内区的序列如SEQ ID No.6所示;所述IL2Rg胞内区的序列如SEQ ID No.7所示。
- 一种由如权利要求1~4中任一项所述的嵌合抗原受体的重组表达载体构建的CAR-T细胞。
- 一种如权利要求5所述的CAR-T细胞的制备方法,其特征在于,包括如下步骤:步骤一、慢病毒载体的构建及病毒生产;将scFv(X)-(Y)CD3zeta,M和N形成融合蛋白,两端加入慢病毒载体,并和慢病毒包装质粒共同转染,获得scFv(X)-(Y)CD3zeta-MN病毒;步骤二、scFv(X)-(Y)CD3zeta-MN CAR-T细胞的制备;分离纯化后的人PBMC经过培养后,感染步骤一获得的scFv(X)-(Y)CD3zeta-MN病毒,在合适的条件下进行细胞扩增,制备scFv(X)-(Y)CD3zeta-MN CAR-T细胞。
- 根据权利要求6所述的CAR-T细胞的制备方法,其特征在于,所述慢病毒载体的构建及病毒生产的具体步骤包括:将scFv(X)-(Y)CD3zeta,M和N通过overlap PCR形成融合蛋白,两端加入酶切位点克隆慢病毒载体;将测序正确的克隆无内毒素大提,和慢病毒包装质粒共同转染,预定时间后收取上清,过滤、离心浓缩病毒,获得scFv(X)-(Y)CD3zeta-MN病毒。
- 根据权利要求6所述的CAR-T细胞的制备方法,其特征在于,所述scFv(X)-(Y)CD3zeta-MN CAR-T细胞的制备的具体步骤包括:将人PBMC经过分离纯化后,接种到具有合适刺激条件的培养板,培养预定时间后,感染步骤一生产的scFv(X)-(Y)CD3zeta-MN病毒,按照合适的刺激条件进行细胞扩增,经过2轮刺激扩增后,获得的细胞即为scFv(X)-(Y)CD3zeta-MN CAR-T细胞。
- 一种含有如权利要求5所述的CAR-T细胞的制剂。
- 如权利要求1~4中任一项所述的嵌合抗原受体,如权利要求5所述的CAR-T细胞在制备治疗或预防肿瘤药物中的应用。
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
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CN202210196195.6A CN114516922A (zh) | 2018-06-20 | 2019-03-13 | 一种包含第三信号受体的嵌合抗原受体及其应用 |
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JP2020571580A JP7433656B2 (ja) | 2018-06-20 | 2019-03-13 | 第3シグナル受容体を含むキメラ抗原受容体およびその使用 |
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US17/127,283 US11524034B2 (en) | 2018-06-20 | 2020-12-18 | Chimeric antigen receptor comprising third signal receptor and use thereof |
US18/047,750 US20230134345A1 (en) | 2018-06-20 | 2022-10-19 | Chimeric antigen receptor comprising third signal receptor and use thereof |
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WO2021207150A1 (en) * | 2020-04-06 | 2021-10-14 | Celledit Llc | Genetically modified immune cells expressing a chimeric antigen receptor and having reduced proinflammatory cytokine signaling |
EP4083073A4 (en) * | 2020-02-28 | 2023-07-12 | Nanjing Bioheng Biotech Co., Ltd | NOVEL CHIMERIC ANTIGEN RECEPTOR AND USE THEREOF |
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CN110615843B (zh) * | 2018-06-20 | 2023-05-09 | 上海隆耀生物科技有限公司 | 一种包含第三信号受体的嵌合抗原受体及其应用 |
CN111518219B (zh) * | 2020-05-08 | 2021-06-22 | 浙江大学 | 嵌合性抗原受体及表达其的巨噬细胞、调节巨噬细胞极化的方法和应用 |
CN112062864A (zh) * | 2020-09-18 | 2020-12-11 | 樊克兴 | 靶向bcma肿瘤抗原受体修饰t细胞的制备方法和应用 |
WO2023274355A1 (zh) * | 2021-07-02 | 2023-01-05 | 上海交通大学 | 改造的间充质干细胞和免疫效应细胞联合治疗肿瘤 |
AU2022346040A1 (en) * | 2021-09-17 | 2024-03-14 | The Board Of Trustees Of The Leland Stanford Junior University | Chimeric antigen receptors comprising interleukin-9 receptor signaling domain |
WO2023133360A2 (en) * | 2022-01-10 | 2023-07-13 | Phenomic Ai | Anti-collagen triple helix repeat containing 1 (cthrc1) antibodies and methods of using the same |
CN117402231B (zh) * | 2023-12-14 | 2024-04-09 | 南方医科大学南方医院 | 一种自发传递il-21信号的受体及其应用 |
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CN112673025B (zh) | 2022-04-01 |
US11524034B2 (en) | 2022-12-13 |
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CN112673025A (zh) | 2021-04-16 |
EP3812401A4 (en) | 2022-04-20 |
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