Chimeric antigen receptor comprising co-stimulatory receptor and application thereof

Abstract

The present application relates to a kind of chimeric antigen receptors comprising costimulatory receptors, the structure of the chimeric antigen receptor is scFv (X)-(Y) CD3 zeta-2A-(Z);Wherein, X includes tumor targeting antibody or can be combined with tumor specific ligand, receptor;Y is the intracellular region of costimulatory receptor, Z is costimulatory receptor, the costimulatory receptor is selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226.The present application also provides a kind of CAR-T cell constructed by the recombinant expression vector of the above-mentioned chimeric antigen receptor and its preparation method and application.The CAR-T cell described in the present application significantly improves the tumor killing ability and expansion capacity.

Description

[0001] This application is a divisional application of the Chinese patent application with the application date of March 13, 2019, the application number of 201980041270.8, and the invention name of "Chimeric Antigen Receptor Containing Costimulatory Receptor and Application". TECHNICAL FIELD

[0002] The present application relates to the technical field of cellular immunotherapy, and particularly relates to a chimeric antigen receptor containing a costimulatory receptor and application thereof. BACKGROUND

[0003] It has been an important direction of application of immunology in translational medicine to conquer tumor by immunological treatment. With the development of various omics (genomics, proteomics, etc.), the immunogenicity of tumor cells due to mutation has been widely recognized, which lays a theoretical foundation for tumor immunotherapy. At the same time, with the accumulation of tumor immunology research itself, tumor immunotherapy has made great progress in recent years, and a series of new immunotherapy methods have gradually entered the clinic. The current research of tumor immunology lays the central position of T cell killing in tumor immunotherapy, and the chimeric antigen receptor T cell (CAR-T cell) is a tumor killing cell artificially modified by combining the target recognition of antibody and the tumor killing function of T cell.

[0004] The concept of chimeric antigen receptor T cells was first proposed by Gross, Waks and Eshhar in 1989, who expressed an antibody recognizing TNP on T cells, achieving the activation and enhancement of the effect of antigen-specific, non-MHC restricted T cells, and proposing the concept of CAR-T technology in tumor treatment. According to this principle, the antibody with tumor specificity is embedded in T cells, which will give T cells new tumor killing ability. After that, CAR-T technology was introduced into anti-tumor clinical trials, but the early CAR-T cells had only the first signal in the intracellular signal transduction domain, and the selected tumor type was solid tumor, and the final clinical results were not ideal. In 2008, Fred Hutchison Cancer Research Institute and other institutions used CAR-T to treat B-cell lymphoma, although the treatment results were not ideal, but the key of this clinical experiment was to prove that CAR-T treatment targeting CD20-expressing B cells was relatively safe. Subsequently, in 2010, the NCI reported a successful case of B-cell lymphoma treatment using CAR-T against CD19, the patient's lymphoma was controlled, normal B cells were also eliminated, and serum Ig was significantly reduced, providing theoretical and practical support for the effectiveness of CAR-T in treating B-cell-derived lymphoma. In 2011, Dr. Carl June of the University of Pennsylvania led a team to use CAR-T specific to CD19 to treat B-cell-derived chronic lymphocytic leukemia, showing a "cure" effect, and subsequently launched a clinical experiment on relapsed and refractory acute lymphoblastic leukemia, also achieved good effect. Due to this breakthrough progress and the development of other immune regulation methods, Science magazine ranked tumor immunotherapy as the first in the 2013 science and technology breakthrough progress. This success has caused widespread impact around the world, and countries have begun to carry out a large number of scientific research and clinical trials based on CAR-T for tumor treatment.

[0005] The structure of CAR is composed 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 tumor cell membrane surface molecules, and can also be a ligand or receptor of some tumor-specific antigens, etc. The extracellular hinge region is a spatial structure for separating the antigen recognition domain and the transmembrane region, and its purpose is to provide a suitable spatial position so that the extracellular antigen recognition domain can maintain the correct structure before and after recognizing the antigen and transmit the intracellular signal. The transmembrane region is a domain to ensure the localization of CAR molecules on the membrane surface. The intracellular signaling domain is the key part of CAR signal transduction, which is usually a combination of one or more first signals (TCR and MHC-I-peptide complex recognition), second signals (costimulatory receptor and costimulatory ligand recognition). The first generation of CAR contains only the first signal, the second generation of CAR has a first signal and a second signal, and the third generation of CAR has a first signal and two second signal domains. Although CAR-T has achieved great success in the treatment of leukemia derived from B cells, its relatively high recurrence rate and low efficiency in treating solid tumors are important challenges. Therefore, the development of a new generation of high-efficiency CAR-T is currently in urgent need in clinical practice. In addition to the third generation of CAR-T, there are other new CAR-T design strategies, that is, on the basis of the second generation of CAR-T, new independent CAR regulatory molecules are introduced to further enhance the function of CAR-T.

[0006] CAR-T prepared from patient's autologous blood cells targeting B cell surface molecules CD19 and CD20 has been relatively mature in the treatment of B cell leukemia, but although the response rate is high, there are a large number of recurrence phenomena, and in addition, the treatment efficiency for solid lymphoma is relatively low, which is related to the immunosuppressive microenvironment in solid tumors.

[0007] In solid tumors, there are various immune cells, tumor cells and stromal cells, which together form a tumor microenvironment. The tumor microenvironment is usually immunosuppressive and can inhibit the endogenous anti-tumor T cell response or adoptive T cells (such as CAR-T) at multiple levels, such as causing T cells to be exhausted, losing the function of killing tumors, and eventually T cells being eliminated. How to enhance the activation ability of CAR-T in solid tumors so that it can resist the immunosuppression in the tumor microenvironment is an important idea and direction for expanding CAR-T to the treatment of solid tumors.

[0008] And the current clinical use of CAR-T domain, there are still deficiencies in tumor killing ability and expansion ability, the curative effect is poor in controlling solid tumor / metastatic tumor, part of CAR-T adopts new type control molecule such as IL-12, 4-1BBL etc., these molecules affect CAR-T, also can produce non-specific activation to other non-CAR-T cells, there is potential possibility to cause immune side effect. SUMMARY

[0009] The present application aims at overcoming the defects in the prior art, and provides a chimeric antigen receptor comprising a costimulatory receptor and an application thereof, and provides a CAR-T cell constructed by a recombinant expression vector of the chimeric antigen receptor, for example, OX40 is an important costimulatory receptor, mainly expressed in activated CD4 and CD8 T cells, and shows multiple functions in the process of T cell activation, can promote the activation of T cells, express more effector molecules and reduce the expression of apoptosis-related genes, and the integration of costimulatory receptor signal in CAR-T has the potential function of enhancing effect.

[0010] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0011] The first object of the present application is to provide a chimeric antigen receptor comprising a costimulatory receptor, the structure of the chimeric antigen receptor is scFv(X)-(Y)CD3zeta-2A-(Z);wherein, X includes tumor targeting antibody or ligand, receptor capable of specific binding with tumor;Y is the intracellular region of a costimulatory receptor, and the costimulatory receptor is selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226;Z is a costimulatory receptor, and the costimulatory receptor is selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226.

[0012] In order to further optimize the above-mentioned chimeric antigen receptor, the technical measures adopted by the present application also include:

[0013] Further, the X is selected from anti-CD19 antibody, anti-CD20 antibody, EGFR antibody, HER2 antibody, EGFRVIII antibody, anti-PSMA antibody, anti-BCMA antibody, anti-CD22 antibody, anti-CD30 antibody. It can be understood that X can also be other proteins capable of specific binding with tumor.

[0014] Further, the X is an anti-CD20 antibody, the Y is 4-1BB, and the Z is selected from one of OX40, HVEM, ICOS, CD27, 4-1BB.

[0015] Further, the scFv(X)-(Y)CD3zeta is scFv-antihCD20-20BBZ, the sequence of which is shown as SEQ ID No. 1; the sequence of the OX40 is shown as SEQ ID No. 2; the sequence of the HVEM is shown as SEQ ID No. 3; the sequence of the ICOS is shown as SEQ ID No. 4; the sequence of the CD27 is shown as SEQ ID No. 5; the sequence of the 4-1BB is shown as SEQ ID No. 6; and the sequence of the 2A is shown as SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, or SEQ ID No. 10.

[0016] wherein each of the above sequences is specifically as follows:

[0017] SEQ ID No. 1:

[0018] QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR; SEQ ID No. 2:

[0019] MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI;

[0020] SEQ ID No. 3:

[0021] MEPPGDWGPPPWRSTPKTDVLRLVLYLTFLGAPCYAPALPSCKEDEYPVGSECCPKCSPGYRVKEACGELTGTVCEPCPPGTYIAHLNGLSKCLQCQMCDPAMGLRASRNCSRTENAVCGCSPGHFCIVQDGDHCAACRAYATSSPGQRVQKGGTESQDTLCQNCPPGTFSPNGTLEECQHQTKCSWLVTKAGAGTSSSHWVWWFLSGSLVIVIVCSTVGLIICVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNH;

[0022] SEQ ID No. 4:

[0023] MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL;

[0024] SEQ ID No. 5:

[0025] MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP;

[0026] SEQ ID No. 6: GSGATNFSLLKQAGDVEENPGP;

[0027] MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL;

[0028] SEQ ID No. 7: GSGATNFSLLKQAGDVEENPGP;

[0029] SEQ ID No. 8: GSGEGRGSLLTCGDVEENPGP;

[0030] SEQ ID No. 9: GSGQCTNYALLKLAGDVESNPGP;

[0031] SEQ ID No. 10: GSGVKQTLNFDLLKLAGDVESNPGP.

[0032] Further, 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.

[0033] A second object of the present application is to provide a recombinant expression vector of any of the above chimeric antigen receptors.

[0034] A third object of the present application is to provide a CAR-T cell constructed by the recombinant expression vector of any of the above chimeric antigen receptors.

[0035] A fourth object of the present application is to provide a preparation method of the above CAR-T cell, comprising the following steps:

[0036] Step one, construction of lentiviral vector and virus production;

[0037] The scFv(X)-(Y)CD3zeta, Z is added with 2A in the middle to form a fusion protein, lentiviral vectors are added at both ends, and co-transfection is performed with lentiviral packaging plasmids to obtain scFv(X)-(Y)CD3zeta-2A-(Z) virus.

[0038] Step two, preparation of scFv(X)-(Y)CD3zeta-2A-(Z) CAR-T cells;

[0039] After isolation and purification, human PBMCs are cultured, infected with the scFv(X)-(Y)CD3zeta-2A-(Z) virus obtained in step one, and cell expansion is performed under suitable conditions to prepare scFv(X)-(Y)CD3zeta-2A-(Z) CAR-T cells.

[0040] In order to further optimize the preparation method of the above CAR-T cell, the technical measures adopted by the present application further include:

[0041] Further, the specific steps of construction of the lentiviral vector and virus production include: scFv(X)-(Y)CD3zeta, Z is added with 2A in the middle by overlap PCR to form a fusion protein, and enzyme digestion sites are added at both ends to clone lentiviral vectors; endotoxin-free large-scale extraction is performed on the correct sequencing clone, co-transfection is performed with lentiviral packaging plasmids, the supernatant is collected at a predetermined time, the virus is concentrated by filtration and centrifugation, and scFv(X)-(Y)CD3zeta-2A-(Z) virus is obtained.

[0042] Still further, the specific steps of construction of the lentiviral vector and virus production are: scFv(X)-(Y)CD3zeta and OX40 are added with 2A sequences by overlap PCR, EcoRI and SalI enzyme digestion sites are added at both ends to clone pCDH-MSCVEF vectors, endotoxin-free large-scale extraction is performed on the correct sequencing clone, 293X is co-transfected with lentiviral packaging plasmids, the supernatant is collected after 48 and 72 hours, the virus is concentrated by centrifugation at 25000 RPM for 2 hours after 0.45uM filtration, and scFv(X)-(Y)CD3zeta-2A-(Z) virus is obtained.

[0043] Further, the specific steps of preparing the scFv(X)-(Y)CD3zeta-2A-(Z) CAR-T cell include: after the human PBMC is separated and purified, inoculating into a culture plate with suitable stimulation conditions, culturing for a predetermined time, infecting the scFv(X)-(Y)CD3zeta-2A-(Z) virus produced in step one, and expanding the cells according to suitable stimulation conditions. After 2 rounds of stimulation expansion, the obtained cells are the scFv(X)-(Y)CD3zeta-2A-(Z) CAR-T cells.

[0044] Further, the stimulation conditions for culturing the separated and purified human PBMC are anti-hCD3 and anti-hCD28, and the stimulation conditions for expanding the cells are using artificial antigen presenting cells or anti-hCD3 / 28 every 6 days.

[0045] Further, the specific steps of preparing the scFv(X)-(Y)CD3zeta-2A-(Z) CAR-T cell include: after the human PBMC is separated and purified, inoculating into a culture plate with suitable stimulation conditions, culturing for a predetermined time, infecting the scFv(X)-(Y)CD3zeta-2A-(Z) virus produced in step one, and expanding the cells according to suitable stimulation conditions. After 2 rounds of stimulation expansion, the obtained cells are the scFv(X)-(Y)CD3zeta-2A-(Z) CAR-T cells.

[0046] Further, the X is selected from anti-CD19 antibody, anti-CD20 antibody, EGFR antibody, HER2 antibody, and EGFRVIII antibody.

[0047] Further, the X is anti-CD20 antibody, the Y is 4-1BB, and the Z is selected from one of OX40, HVEM, ICOS, CD27, and 4-1BB.

[0048] Further, the scFv(X)-(Y)CD3zeta is scFv-antihCD20-20BBZ, the sequence of which is shown in SEQ ID No. 1; the sequence of the OX40 is shown in SEQ ID No. 2; the sequence of the HVEM is shown in SEQ ID No. 3; the sequence of the ICOS is shown in SEQ ID No. 4; the sequence of the CD27 is shown in SEQ ID No. 5; the sequence of the 4-1BB is shown in SEQ ID No. 6; and the sequence of the 2A is shown in SEQ ID No. 7.

[0049] Further, the lentivirus packaging plasmid in step one includes VSV-g, pMD Gag / Pol, RSV-REV, centrifugation uses Beckman ultracentrifuge and SW28 rotor.

[0050] A fifth object of the present application is to provide a preparation containing the CAR-T cell or containing the CAR-T cell prepared by the above preparation method; further, the preparation further includes a pharmaceutically acceptable diluent or excipient.

[0051] A sixth object of the present application is to provide a use of the above chimeric antigen receptor, the above CAR-T cell or the CAR-T cell prepared by the above preparation method in the preparation of a drug for treating or preventing tumors.

[0052] Further, 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, pancreatic blastoma, angioma, etc.

[0053] Compared with the prior art, the present application has the following beneficial effects:

[0054] The CAR-T cell of the present application significantly improves the tumor killing ability and expansion ability, and significantly improves the killing ability of solid / metastatic tumors; the CAR-T cell of the present application contains a costimulatory receptor (ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226, etc.) instead of a conventional ligand or secreted factor, which only acts on CAR-T cells, reducing the risk of causing immune side effects.

[0055] The present application first uses a costimulatory receptor for the construction of CAR-T, which significantly improves the activation ability and survival ability of CAR-T cells in tumors, controls the ability of solid / metastatic tumors, and improves the efficacy of CAR-T cells, thereby having more excellent anti-tumor efficacy. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 The schematic diagram of the structure of the chimeric antigen receptor (CAR) molecule containing the third signal receptor in each embodiment of the present application;

[0057] Figure 2 The schematic diagram of the virus titer measured after the BBZ-2A-OX40 virus infects 293 cells in an embodiment of the present application;

[0058] Figure 3A schematic diagram of virus titers measured after BBZ-2A-HVEM virus infection of 293 cells in an embodiment of the present application;

[0059] Figure 4 A schematic diagram of virus titers measured after BBZ-2A-ICOS virus infection of 293 cells in an embodiment of the present application;

[0060] Figure 5 A schematic diagram of virus titers measured after BBZ-2A-CD27 virus infection of 293 cells in an embodiment of the present application;

[0061] Figure 6 A schematic diagram of virus titers measured after BBZ-2A-4-1BB virus infection of 293 cells in an embodiment of the present application;

[0062] Figure 7 A schematic diagram of the results of BBZ CAR-T cell and BBZ-2A-OX40 CAR-T cell phenotype analysis in an embodiment of the present application;

[0063] Figure 8 A schematic diagram of the results of BBZ CAR-T cell and BBZ-2A-HVEM CAR-T cell phenotype analysis in an embodiment of the present application;

[0064] Figure 9 A schematic diagram of the results of BBZ CAR-T cell and BBZ-2A-ICOS CAR-T cell phenotype analysis in an embodiment of the present application;

[0065] Figure 10 A schematic diagram of the results of BBZ CAR-T cell and BBZ-2A-CD27 CAR-T cell phenotype analysis in an embodiment of the present application;

[0066] Figure 11 A schematic diagram of the results of BBZ CAR-T cell and BBZ-2A-4-1BB CAR-T cell phenotype analysis in an embodiment of the present application;

[0067] Figure 12 A schematic diagram of the expansion capacity of BBZ CAR-T cells and BBZ-2A-OX40 CAR-T cells in an embodiment of the present application;

[0068] Figure 13 A schematic diagram of the tumor killing capacity of BBZ CAR-T cells and BBZ-2A-OX40 CAR-T cells in an embodiment of the present application;

[0069] Figure 14Figure for anti-tumor ability of BBZ CAR-T cells and BBZ-2A-OX40 CAR-T cells in an embodiment of the present application;

[0070] Figure 15 Figure for in vivo survival ability of BBZ CAR-T cells and BBZ-2A-OX40 CAR-T cells in an embodiment of the present application. DETAILED DESCRIPTION

[0071] The present application provides a chimeric antigen receptor containing a costimulatory receptor, the structure of the chimeric antigen receptor is scFv(X)-(Y)CD3zeta-2A-(Z); wherein X is a tumor targeting antibody or other protein; Y is the intracellular region of a costimulatory receptor selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226; Z is a costimulatory receptor selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226. The present application also relates to a CAR-T cell constructed by a recombinant expression vector of any of the above chimeric antigen receptors and a preparation method thereof, a preparation containing the CAR-T cell and the application of the CAR-T cell.

[0072] The specific embodiments of the present application are further described below with reference to the accompanying drawings and examples. The following examples are only used to more clearly illustrate the technical solutions of the present application, and cannot be used to limit the protection scope of the present application.

[0073] The chimeric antigen receptor (CAR) molecules containing costimulatory receptors involved in the following embodiments of the present application are BBZ-2A-OX40, BBZ-2A-HVEM, BBZ-2A-ICOS, BBZ-2A-CD27, and BBZ-2A-4-1BB, respectively, and the structures are as shown in Figure 1

[0074] Preparation of 20BBZ-2A-OX40 CAR-T cells

[0075] The preparation of 20BBZ-2A-OX40 CAR-T cells described in this embodiment includes the following steps:

[0076] 1. Construction of lentiviral vector pCDH-MSCVEF-20BBZ-2A-OX40 and virus production

[0077] ​The scFv-antihCD20-20BBZ (SEQ ID No. 1) and OX40 (SEQ ID No. 2) were cloned into pCDH-MSCVEF vector by overlap PCR with 2A (SEQ ID No. 7) sequence in the middle and EcoRI and Sail enzyme cutting sites at both ends. The endotoxin-free large-scale extraction of the correct sequencing clone was performed, and the 293X was co-transfected with lentivirus packaging plasmid (VSV-g, pMD Gag / Pol, RSV-REV). The supernatant was collected after 48 and 72 hours, filtered with 0.45 uM, and concentrated by Beckman ultracentrifuge and SW28 rotor at 25000 RPM for 2 hours. The virus was pCDH-MSCVEF-20BBZ-2A-OX40 virus (referred to as 20BBZ-2A-OX40 virus) for subsequent CAR-T cell production. The control pCDH-MSCVEF-20BBZ virus (referred to as 20BBZ virus) was also produced. The obtained virus was used to infect 293 cells, and the virus titer was measured, as shown in Figure 2 .

[0078] 2. Preparation of 20BBZ-2A-OX40 CAR-T cells and 20BBZ CAR-T cells

[0079] After the human PBMC was purified by Stemcell T cell separation kit, it was inoculated into anti-hCD3 and anti-hCD28 coated 96-well culture plate. After 2 days, 20BBZ virus and 20BBZ-2A-OX40 virus were infected according to MOI = 10-20. After 1 day, the liquid was changed for cell culture. According to every 6 days, artificial antigen presenting cells or anti-hCD3 / 28 were used for stimulation. After 2 rounds of stimulation, the obtained cells were 20BBZ CAR-T cells and 20BBZ-2A-OX40 CAR-T cells, which were used for subsequent experiments and phenotype analysis, and the results are shown in Figure 7 . As can be seen from the figure, the obtained cells are CAR positive.

[0080] Example 2- Preparation of 20BBZ-2A-HVEM CAR-T cells

[0081] The preparation of 20BBZ-2A-HVEM CAR-T cells described in this example includes the following steps:

[0082] 1. Construction of lentiviral vector pCDH-MSCVEF-20BBZ-2A-HVEM and virus production

[0083] The scFv-anti-hCD20-20BBZ (SEQ ID No. 1) and HVEM (SEQ ID No. 3) were cloned into pCDH-MSCVEF vector by overlap PCR with 2A (SEQ ID No. 8) sequence in the middle and EcoRI and Sail enzyme cutting sites at both ends. The endotoxin-free large clone was sequenced correctly, and co-transfected with lentivirus packaging plasmid (VSV-g, pMD Gag / Pol, RSV-REV) into 293X. The supernatant was collected after 48 and 72 hours, filtered with 0.45 uM, and concentrated by Beckman ultracentrifuge and SW28 rotor at 25000 RPM for 2 hours. The virus was pCDH-MSCVEF-20BBZ-2A-HVEM virus (referred to as 20BBZ-2A-HVEM virus), which was used for subsequent CAR-T cell production. At the same time, the control pCDH-MSCVEF-20BBZ virus (referred to as 20BBZ virus) was produced. The obtained virus was used to infect 293 cells, and the virus titer was measured, as shown in Figure 3 .

[0084] 2. Preparation of 20BBZ-2A-HVEM CAR-T cells and 20BBZ CAR-T cells

[0085] After the human PBMC was purified by Stemcell T cell separation kit, it was inoculated into anti-hCD3 and anti-hCD28 coated 96-well culture plate. After 2 days, 20BBZ virus and 20BBZ-2A-HVEM virus were infected according to MOI = 10-20, and the cells were cultured after 1 day of liquid change. According to every 6 days using artificial antigen presenting cells or anti-hCD3 / 28 stimulation, after 2 rounds of stimulation, the obtained cells were 20BBZ CAR-T cells and 20BBZ-2A-HVEM CAR-T cells, which were used for subsequent experiments and phenotype analysis, and the results are shown in Figure 8 . As can be seen from the figure, the obtained cells are CAR positive.

[0086] Example 3-Preparation of 20BBZ-2A-ICOS CAR-T cells

[0087] The preparation of 20BBZ-2A-ICOS CAR-T cells described in this example includes the following steps:

[0088] 1. Construction of lentiviral vector pCDH-MSCVEF-20BBZ-2A-ICOS and virus production

[0089] The scFv-antihCD20-20BBZ (SEQ ID No. 1) and ICOS (SEQ ID No. 4) were cloned into pCDH-MSCVEF vector by overlap PCR with 2A (SEQ ID No. 9) sequence in the middle and EcoRI and Sail enzyme cutting sites at both ends. The endotoxin-free large clone was sequenced correctly, and co-transfected with lentivirus packaging plasmid (VSV-g, pMD Gag / Pol, RSV-REV) into 293X. The supernatant was collected after 48 and 72 hours, filtered with 0.45 uM, and concentrated by Beckman ultracentrifuge and SW28 rotor at 25000 RPM for 2 hours. The virus was pCDH-MSCVEF-20BBZ-2A-ICOS virus (referred to as 20BBZ-2A-ICOS virus), which was used for subsequent CAR-T cell production. At the same time, the control pCDH-MSCVEF-20BBZ virus (referred to as 20BBZ virus) was produced. The obtained virus was used to infect 293 cells, and the virus titer was measured, as shown in Figure 4

[0090] 2. Preparation of 20BBZ-2A-ICOS CAR-T cells and 20BBZ CAR-T cells

[0091] After the human PBMC was purified by Stemcell T cell separation kit, it was inoculated into anti-hCD3 and anti-hCD28 coated 96-well culture plate. After 2 days, 20BBZ virus and 20BBZ-2A-ICOS virus were infected according to MOI = 10-20, and the cells were cultured after 1 day of liquid change. According to every 6 days using artificial antigen presenting cells or anti-hCD3 / 28 stimulation, after 2 rounds of stimulation, the obtained cells were 20BBZ CAR-T cells and 20BBZ-2A-ICOS CAR-T cells, which were used for subsequent experiments and phenotype analysis, and the results are shown in Figure 9

[0092] Example 4-Preparation of 20BBZ-2A-CD27 CAR-T cells

[0093] The preparation of 20BBZ-2A-CD27 CAR-T cells described in this example includes the following steps:

[0094] 1. Construction of lentiviral vector pCDH-MSCVEF-20BBZ-2A-CD27 and virus production

[0095] ​​The scFv-anti-hCD20-20BBZ (SEQ ID No. 1) and CD27 (SEQ ID No. 5) were cloned into pCDH-MSCVEF vector by overlap PCR with 2A (SEQ ID No. 10) sequence in the middle and EcoRI and Sail enzyme cutting sites at both ends. The endotoxin-free large clone was sequenced correctly, and co-transfected with lentivirus packaging plasmid (VSV-g, pMD Gag / Pol, RSV-REV) into 293X. The supernatant was collected after 48 and 72 hours, filtered with 0.45 uM, and concentrated by Beckman ultracentrifuge and SW28 rotor at 25000 RPM for 2 hours. The virus was pCDH-MSCVEF-20BBZ-2A-CD27 virus (referred to as 20BBZ-2A-CD27 virus), which was used for subsequent CAR-T cell production. At the same time, the control pCDH-MSCVEF-20BBZ virus (referred to as 20BBZ virus) was produced. The obtained virus was used to infect 293 cells, and the virus titer was measured, as shown in Figure 5 .

[0096] 2. Preparation of 20BBZ-2A-CD27 CAR-T cells and 20BBZ CAR-T cells

[0097] After the human PBMC was purified by Stemcell T cell separation kit, it was inoculated into anti-hCD3 and anti-hCD28 coated 96-well culture plate. After 2 days, 20BBZ virus and 20BBZ-2A-CD27 virus were infected according to MOI = 10-20, and the cells were cultured after 1 day of liquid change. According to every 6 days using artificial antigen presenting cells or anti-hCD3 / 28 stimulation, after 2 rounds of stimulation, the obtained cells were 20BBZ CAR-T cells and 20BBZ-2A-CD27 CAR-T cells, which were used for subsequent experiments and phenotype analysis, and the results are shown in Figure 10 . As can be seen from the figure, the obtained cells are CAR positive.

[0098] Example 5-Preparation of 20BBZ-2A-4-1BB CAR-T cells

[0099] The preparation of 20BBZ-2A-4-1BB CAR-T cells described in this example includes the following steps:

[0100] 1. Construction of lentiviral vector pCDH-MSCVEF-20BBZ-2A-4-1BB and virus production

[0101] The scFv-anti-hCD20-20BBZ (SEQ ID No. 1) and 4-1BB (SEQ ID No. 6) were cloned into pCDH-MSCVEF vector by overlap PCR with 2A (SEQ ID No. 7) sequence in the middle and EcoRI and Sail enzyme cutting sites at both ends. The endotoxin-free large-scale extraction of the correct sequencing clone was performed, and the lentivirus packaging plasmid (VSV-g, pMD Gag / Pol, RSV-REV) was co-transfected into 293X. The supernatant was collected after 48 and 72 hours, filtered with 0.45 uM, and concentrated by using a Beckman ultracentrifuge and SW28 rotor at 25000 RPM for 2 hours. The virus was pCDH-MSCVEF-20BBZ-2A-4-1BB virus (referred to as 20BBZ-2A-4-1BB virus) for subsequent CAR-T cell production. At the same time, the control pCDH-MSCVEF-20BBZ virus (referred to as 20BBZ virus) was produced. The obtained virus was used to infect 293 cells, and the virus titer was measured, as shown in Table 1. Figure 6

[0102] 2. Preparation of 20BBZ-2A-4-1BB CAR-T cells and 20BBZ CAR-T cells

[0103] After the human PBMC was purified by Stemcell T cell separation kit, it was inoculated into anti-hCD3 and anti-hCD28 coated 96-well culture plates. After 2 days, 20BBZ virus and 20BBZ-2A-4-1BB virus were infected according to MOI = 10-20. After 1 day, the liquid was changed for cell culture. According to every 6 days, artificial antigen presenting cells or anti-hCD3 / 28 were used for stimulation. After 2 rounds of stimulation, the obtained cells were 20BBZ CAR-T cells and 20BBZ-2A-4-1BB CAR-T cells, which were used for subsequent experiments and phenotype analysis, and the results are shown in Table 2. Figure 11

[0104] Example 6 - Comparison of the expansion ability of 20BBZ CAR-T cells and 20BBZ-2A-OX40 CAR-T cells

[0105] The 20BBZ CAR-T cells and 20BBZ-2A-OX40 CAR-T cells obtained in step 2 of Example 1 were continuously cultured for 14 days, and stimulated once every 6 days with artificial antigen presenting cells. The cell count results are shown in Table 3. Figure 12

[0106] ​​​Example 7 - Comparison of tumor killing ability of 20BBZ CAR-T cells and 20BBZ-2A-OX40 CAR-T cells

[0107] The 20BBZ CAR-T cells and 20BBZ-2A-OX40 CAR-T cells prepared in step 2 of Example 1, the 20BBZ-2A-ICOS CAR-T cells prepared in step 2 of Example 3, and the 20BBZ-2A-CD27 CAR-T cells prepared in step 2 of Example 4 were inoculated into 96-well plates, and Raji tumor cells were added at a CAR-T:tumor cell ratio of 1:1, 1:2, and 1:4. After 24 and 48 hours, the survival ratio of tumor cells was compared, and the results are shown in Figure 13 As can be seen from the figure, the 20BBZ-2A-OX40 / ICOS / CD27 CAR-T cells have similar tumor killing ability to the 20BBZ CAR-T cells, and some of the CAR-T cells containing co-stimulatory receptors have stronger tumor killing ability.

[0108] Example 8 - Comparison of anti-tumor ability and in vivo survival ability of 20BBZ CAR-T cells and 20BBZ-2A-OX40 CAR-T cells

[0109] 10 6 Nalm-6 tumor cells were intravenously inoculated into B-NDG mice, and 10 7 20BBZ CAR-T cells and 20BBZ-2A-OX40 CAR-T cells were administered 6 days later. The survival rate of the mice was observed, and the content of tumor cells and CAR-T cells in the bone marrow of some mice was detected on day 7. The results are shown in Figure 14 and Figure 15 As can be seen from the figure, the 20BBZ-2A-OX40 CAR-T cells significantly prolonged the survival of the mice and expanded more in vivo compared to the 20BBZ CAR-T cells.

[0110] As can be seen from the above examples, the present application constructs a new CAR-T cell containing a co-stimulatory receptor, which significantly enhances the activation ability, survival ability, and expansion ability of the CAR-T cell in the tumor compared to the currently used CAR-T technology, and has more excellent anti-tumor efficacy.

[0111] The specific embodiments of the present application are described in detail above, but they are only examples, and the present application is not limited to the specific embodiments described above. Any equivalent modifications and substitutions made by those skilled in the art to the present application are also within the scope of the present application. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present application should be included in the scope of the present application. SEQUENCE LIST <110> Shanghai Longyao Biotech Co., Ltd. <120> Chimeric antigen receptor comprising a costimulatory receptor and uses thereof <130> 0187-PA-002CN.DIV1 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 468 <212> PRT <213> Artificial Sequence <220> <223> scFv-antihCD20-20BBZ sequence <400> 1 Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Pro 115 120 125 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys 130 135 140 Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln 145 150 155 160 Thr Pro Gly Arg Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn 165 170 175 Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr 180 185 190 Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr 195 200 205 Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly 210 215 220 Gly Asp Trp Tyr Phe Asn Val Trp Gly Ala Gly Thr Thr Val Thr Val 225 230 235 240 Ser Ala Ala Ala Ala Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 245 250 255 Ala Pro Thr lie Ala Ser Gin Pro Leu Ser Leu Arg Pro Glu Ala Cys 260 265 270 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 275 280 285 Cys Asp lie Tyr lie Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 290 295 300 Leu Leu Ser Leu Val lie Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys 305 310 315 320 Leu Leu Tyr lie Phe Lys Gin Pro Phe Met Arg Pro Val Gin Thr Thr 325 330 335 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 340 345 350 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 355 360 365 Tyr Gin Gin Gly Gin Asn Gin Leu Tyr Asn Glu Leu Asn Leu Gly Arg 370 375 380 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 385 390 395 400 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gin Glu Gly Leu Tyr Asn 405 410 415 Glu Leu Gin Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu lie Gly Met 420 425 430 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gin Gly 435 440 445 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gin Ala 450 455 460 Leu Pro Pro Arg 465 <210> 2 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> Sequence for OX40 <400> 2 Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu 1 5 10 15 Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val 20 25 30 Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro 35 40 45 Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gin Asn Thr Val Cys 50 55 60 Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro 65 70 75 80 Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys 85 90 95 Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly 100 105 110 Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys 115 120 125 Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp 130 135 140 Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn 145 150 155 160 Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro 165 170 175 Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr 180 185 190 Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu 195 200 205 Val Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val 210 215 220 Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu 225 230 235 240 Arg Arg Asp Gin Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly 245 250 255 Gly Ser Phe Arg Thr Pro lie Gin Glu Glu Gin Ala Asp Ala His Ser 260 265 270 Thr Leu Ala Lys lie 275 <210> 3 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> Sequence of HVEM <400> 3 Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp Arg Ser Thr Pro 1 5 10 15 Lys Thr Asp Val Leu Arg Leu Val Leu Tyr Leu Thr Phe Leu Gly Ala 20 25 30 Pro Cys Tyr Ala Pro Ala Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro 35 40 45 Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys 50 55 60 Glu Ala Cys Gly Glu Leu Thr Gly Thr Val Cys Glu Pro Cys Pro Pro 65 70 75 80 Gly Thr Tyr Ile Ala His Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys 85 90 95 Gln Met Cys Asp Pro Ala Met Gly Leu Arg Ala Ser Arg Asn Cys Ser 100 105 110 Arg Thr Glu Asn Ala Val Cys Gly Cys Ser Pro Gly His Phe Cys Ile 115 120 125 Val Gln Asp Gly Asp His Cys Ala Ala Cys Arg Ala Tyr Ala Thr Ser 130 135 140 Ser Pro Gly Gln Arg Val Gln Lys Gly Gly Thr Glu Ser Gln Asp Thr 145 150 155 160 Leu Cys Gln Asn Cys Pro Pro Gly Thr Phe Ser Pro Asn Gly Thr Leu 165 170 175 Glu Glu Cys Gln His Gln Thr Lys Cys Ser Trp Leu Val Thr Lys Ala 180 185 190 Gly Ala Gly Thr Ser Ser Ser His Trp Val Trp Trp Phe Leu Ser Gly 195 200 205 Ser Leu Val Ile Val Ile Val Cys Ser Thr Val Gly Leu Ile Ile Cys 210 215 220 Val Lys Arg Arg Lys Pro Arg Gly Asp Val Val Lys Val Ile Val Ser 225 230 235 240 Val Gin Arg Lys Arg Gin Glu Ala Glu Gly Glu Ala Thr Val He Glu 245 250 255 Ala Leu Gin Ala Pro Pro Asp Val Thr Thr Val Ala Val Glu Glu Thr 260 265 270 He Pro Ser Phe Thr Gly Arg Ser Pro Asn His 275 280 <210> 4 <211> 199 <212> PRT <213> Artificial Sequence <220> <223> Sequence for ICOS <400> 4 Met Lys Ser Gly Leu Trp Tyr Phe Phe Leu Phe Cys Leu Arg He Lys 1 5 10 15 Val Leu Thr Gly Glu He Asn Gly Ser Ala Asn Tyr Glu Met Phe He 20 25 30 Phe His Asn Gly Gly Val Gin He Leu Cys Lys Tyr Pro Asp He Val 35 40 45 Gln Gin Phe Lys Met Gin Leu Leu Lys Gly Gly Gin He Leu Cys Asp 50 55 60 Leu Thr Lys Thr Lys Gly Ser Gly Asn Thr Val Ser He Lys Ser Leu 65 70 75 80 Lys Phe Cys His Ser Gin Leu Ser Asn Asn Ser Val Ser Phe Phe Leu 85 90 95 Tyr Asn Leu Asp His Ser His Ala Asn Tyr Tyr Phe Cys Asn Leu Ser 100 105 110 Ile Phe Asp Pro Pro Pro Phe Lys Val Thr Leu Thr Gly Gly Tyr Leu 115 120 125 His Ile Tyr Glu Ser Gin Leu Cys Cys Gin Leu Lys Phe Trp Leu Pro 130 135 140 Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu 145 150 155 160 Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro 165 170 175 Asn Gin Gin Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser 180 185 190 Arg Leu Thr Asp Val Thr Leu 195 <210> 5 <211> 260 <212> PRT <213> Artificial Sequence <220> <223> Sequence for CD27 <400> 5 Met Ala Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val 1 5 10 15 Gly Leu Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr 20 25 30 Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe 35 40 45 Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro 50 55 60 Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His 65 70 75 80 Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys 85 90 95 Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Cys Arg 100 105 110 Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu 115 120 125 Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His 130 135 140 Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met 145 150 155 160 Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr 165 170 175 His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Ile 180 185 190 Leu Val Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly Ala 195 200 205 Leu Phe Leu His Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser 210 215 220 Pro Val Glu Pro Ala Glu Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu 225 230 235 240 Glu Gly Ser Thr Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro 245 250 255 Ala Cys Ser Pro 260 <210> 6 <211> 255 <212> PRT <213> Artificial Sequence <220> <223> Sequence for 4-1BB <400> 6 Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu 1 5 10 15 Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro 20 25 30 Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gin He Cys Ser Pro Cys 35 40 45 Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gin Arg Thr Cys Asp He 50 55 60 Cys Arg Gin Cys Lys Gly Val Phe Arg Thr Arg Lys Gin Cys Ser Ser 65 70 75 80 Thr Ser Asn Ala Gin Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly 85 90 95 Ala Gly Cys Ser Met Cys Gin Gin Asp Cys Lys Gin Gly Gin Gin Leu 100 105 110 Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gin 115 120 125 Lys Arg Gly He Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys 130 135 140 Ser Val Leu Val Asn Gly Thr Lys Gin Arg Asp Val Val Cys Gly Pro 145 150 155 160 Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala 165 170 175 Pro Ala Arg Gin Pro Gly His Ser Pro Gin He He Ser Phe Phe Leu 180 185 190 Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu 195 200 205 Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 210 215 220 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 225 230 235 240 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 245 250 255 <210> 7 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Sequence for 2A <400> 7 Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 <210> 8 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Sequence for 2A <400> 8 Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu 1 5 10 15 Glu Asn Pro Gly Pro 20 <210> 9 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Sequence for 2A <400> 9 Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp 1 5 10 15 Val Glu Ser Asn Pro Gly Pro 20 <210> 10 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Sequence for 2A <400> 10 Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala 1 5 10 15 Gly Asp Val Glu Ser Asn Pro Gly Pro 20 25

Claims

1. A chimeric antigen receptor comprising a co-stimulatory receptor, characterized in that, The structure of the chimeric antigen receptor is scFv(X)-(Y)CD3zeta-2A-(Z); X includes tumor-targeting antibodies or ligands and receptors that can specifically bind to tumors; Y represents the intracellular region of the co-stimulatory receptor, which is 4-1BB; Z is a co-stimulatory receptor, the co-stimulatory receptor is OX40, the sequence of OX40 is shown in SEQ ID No. 2, the sequence of 4-1BB is shown in SEQ ID No. 6, and the sequence of 2A is shown in SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 or SEQ ID No.

10.

2. A chimeric antigen receptor comprising a co-stimulatory receptor according to claim 1, characterized in that, X is selected from anti-CD19 antibody, anti-CD20 antibody, EGFR antibody, HER2 antibody, EGFRVIII antibody, anti-PSMA antibody, anti-BCMA antibody, anti-CD22 antibody, and anti-CD30 antibody.

3. A chimeric antigen receptor comprising a co-stimulatory receptor according to claim 1, characterized in that, X is an anti-CD20 antibody.

4. A chimeric antigen receptor comprising a co-stimulatory receptor according to claim 3, characterized in that, The scFv(X)-(Y)CD3zeta is scFv-antihCD20-20BBZ, and its sequence is shown in SEQ ID No.

1.

5. A CAR-T cell constructed from a recombinant expression vector of a chimeric antigen receptor as described in any one of claims 1 to 4.

6. The method for preparing CAR-T cells according to claim 5, characterized in that, Includes the following steps: Step 1: Construction of lentiviral vectors and virus production; 2A was added to the middle of scFv(X)-(Y)CD3zeta,Z to form a gene encoding the fusion protein. Lentiviral vectors were added to both ends and co-transfected with lentiviral packaging plasmids to obtain scFv(X)-(Y)CD3zeta-2A-(Z) virus. Step 2: Preparation of scFv(X)-(Y)CD3zeta-2A-(Z) CAR-T cells; After being isolated and purified, human PBMCs were cultured and infected with the scFv(X)-(Y)CD3zeta-2A-(Z) virus obtained in step one. Cell expansion was carried out under suitable conditions to prepare scFv(X)-(Y)CD3zeta-2A-(Z) CAR-T cells.

7. The method for preparing CAR-T cells according to claim 6, characterized in that, The specific steps for constructing the lentiviral vector and producing the virus include: The gene encoding the fusion protein scFv(X)-(Y)CD3zeta,Z was formed by adding 2A to the middle of the scFv(X)-(Y)CD3zeta,Z using overlap PCR, and restriction enzyme sites were added to both ends to clone the lentiviral vector. The endotoxin-free clone with correct sequencing was co-transfected with the lentiviral packaging plasmid, and the supernatant was collected at a predetermined time. The virus was filtered, centrifuged and concentrated to obtain scFv(X)-(Y)CD3zeta-2A-(Z) virus.

8. The method for preparing CAR-T cells according to claim 6, characterized in that, The specific steps for preparing scFv(X)-(Y)CD3zeta-2A-(Z) CAR-T cells include: separating and purifying human PBMCs, inoculating them into a culture plate with suitable stimulation conditions, culturing them for a predetermined time, infecting them with the scFv(X)-(Y)CD3zeta-2A-(Z) virus produced in step one, expanding the cells according to suitable stimulation conditions, and after two rounds of stimulation and expansion, the obtained cells are scFv(X)-(Y)CD3zeta-2A-(Z) CAR-T cells.

9. A formulation containing CAR-T cells as described in claim 5.

10. The chimeric antigen receptor as described in any one of claims 1 to 4, and the CAR-T cell as described in claim 5, in the preparation of drugs for the treatment or prevention of lymphoma.

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