Chimeric antigen receptor targeting GP96, immune cell as well as preparation method and application of chimeric antigen receptor

Abstract

The invention discloses a chimeric antigen receptor targeting GP96, an immune cell and a preparation method and application of the chimeric antigen receptor and the immune cell, and relates to the technical field of cellular immunization. Based on polypeptide-CAR design of a GP96 natural ligand, GP96 specific binding polypeptide (such as PEG10 and GPC3 derived peptide) is adopted to replace scFv, affinity and safety are balanced through interaction of the natural receptor and the ligand, and the chimeric antigen receptor targeting GP96 is obtained. CAR-T, CAR-M, CAR-NK cells and the like are constructed on the basis of the chimeric antigen receptor. Through polypeptide-CAR design of targeting GP96, dual-signal domain cooperation and cross-cell type adaptation, the problems of antigen escape, TME inhibition and safety of an existing CAR therapy in solid tumors are systematically solved, and an efficient, safe and universal tumor immunotherapy scheme is provided for clinical research.

Description

Technical Field

[0001] The present invention relates to the field of cellular immunity technology, and in particular to a chimeric antigen receptor targeting GP96, immune cells, and preparation methods and applications thereof. Background Art

[0002] Chimeric antigen receptor (CAR) cell therapy has made breakthrough progress in the treatment of hematological tumors, but it still faces multiple technical obstacles in the treatment of solid tumors. CAR-T, CAR-M (macrophages) and CAR-NK (natural killer cells) therapies have insufficient clinical response rates and thus affect efficacy due to the antigenic heterogeneity of solid tumors, the immunosuppressive microenvironment (TME) and target selection limitations. The following analyzes the core defects of existing CAR technology from three aspects: target selection, cell function inhibition and safety: (1) Antigen escape and target selection limitations. Traditional CAR design relies on single-chain antibody (scFv) recognition of tumor surface antigens (such as HER2 and EGFR), but the antigen expression of solid tumors is highly heterogeneous. For example, HER2 is only highly expressed in 15%-20% of breast cancers, and antigen loss or downregulation is prone to occur under treatment pressure. In addition, most targets (such as CD19 and BCMA) are expressed at low levels in normal tissues, resulting in CAR cells attacking normal organs (such as CD19-CAR-T causing B cell loss). (2) Immunosuppressive microenvironment (TME) resistance: Solid tumors inhibit CAR cell function through multiple mechanisms. Dense matrix (such as collagen fibers) hinders CAR-T / NK infiltration. In pancreatic cancer, only 0.01% of infused CAR-T can reach the tumor core. Tumor cells highly express molecules such as PD-L1 and CD47, which bind to T cell PD-1 and macrophage SIRPα, respectively, inhibiting CAR-T activity and escaping macrophage phagocytosis. Lactic acid accumulation and hypoxic environment in the TME inhibit CAR cell mitochondrial function, leading to T cell exhaustion (such as upregulation of TIM-3 and LAG-3). (3) Safety issues caused by scFv design. Mouse-derived scFv is prone to induce human anti-mouse antibody (HAMA) reactions, reducing efficacy and causing allergic reactions. High-affinity scFv (KD < 10-9M) is prone to attack normal tissues (such as HER2-CAR-T causing cardiopulmonary toxicity).

[0003] The above problems are fundamentally caused by defects in the CAR receptor itself. It can be seen that further research on the CAR receptor is of great significance for CAR therapy. Summary of the Invention

[0004] The present invention discloses a chimeric antigen receptor constructed by targeting GP96, and based on this receptor, constructs cells suitable for CAR-T, CAR-M and CAR-NK, so as to overcome the triple bottlenecks of target, microenvironment and safety faced by traditional CAR therapy in the treatment of solid tumors.

[0005] The technical solution adopted in the present invention is as follows:

[0006] One of the objectives of the present invention is to provide a chimeric antigen receptor targeting GP96, comprising the following structure:

[0007] (a) an extracellular binding domain comprising a PEG10 polypeptide or a GPC3 polypeptide capable of specifically binding to GP96, wherein the amino acid sequence of the PEG10 polypeptide is shown in SEQ ID NO: 1, and the amino acid sequence of the GPC3 polypeptide is shown in SEQ ID NO: 2;

[0008] (b) a signal peptide selected from the group consisting of a signal peptide of CD8 or CD28;

[0009] (c) a hinge region selected from the hinge region of CD8 or CD28;

[0010] (d) a transmembrane region selected from the transmembrane region of CD8 or CD28;

[0011] (e) an intracellular signaling domain selected from CD3ζ, TIR, or a combination thereof.

[0012] In the present invention, the sequence of SEQ ID NO: 1 (PEG10 polypeptide) is: ALIGQCIHI; the sequence of SEQ ID NO: 2 (GPC3 polypeptide) is: KVFGNFPKL.

[0013] Furthermore, it also contains fluorescent marker protein.

[0014] Furthermore, it further comprises a costimulatory domain 4-1BB or a conserved intracellular domain TIR, the amino acid sequence of the 4-1BB is shown in SEQ ID NO: 3, and the amino acid sequence of the TIR is shown in SEQ ID NO: 9. In the present invention, the sequence of SEQ ID NO: 3 is: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR, and the amino acid sequence of SEQ ID NO: 9 is: NIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVALAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDG.

[0015] Furthermore, the signal peptide, hinge region, and transmembrane region are all derived from CD8, and their amino acid sequences are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. In the present invention, the sequence of SEQ ID NO: 4 is: MALPVTALLLPLALLLHAARP; the sequence of SEQ ID NO: 5 is: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD; and the sequence of SEQ ID NO: 6 is: IYIWAPLAGTCGVLLLSLVITLYC.

[0016] The second object of the present invention is to disclose the application of the chimeric antigen receptor in the preparation of CAR immune cells.

[0017] A third object of the present invention is to provide a CAR immune cell comprising the above-mentioned chimeric antigen receptor, wherein the CAR immune cell is selected from any one of the following:

[0018] (a) CAR-T cells, wherein the chimeric antigen receptor is transduced into human T cells via a lentiviral vector, wherein the T cells are derived from peripheral blood and activated with CD3 / CD28 magnetic beads and IL2;

[0019] (b) CAR-M cells, wherein the chimeric antigen receptor is transduced into human macrophages via a lentiviral vector, and the macrophages are differentiated from THP-1 cells induced by PMA and LPS;

[0020] (c) CAR-NK cells, wherein the chimeric antigen receptor is transduced into human NK cells via a lentiviral vector, wherein the NK cells are derived from peripheral blood or NK-92 cell line and activated by IL-2 or IL-15.

[0021] A fourth object of the present invention is to provide a method for preparing the CAR immune cells, comprising the following steps:

[0022] (1) constructing a lentiviral vector: cloning the nucleotide sequence of the chimeric antigen receptor according to claim 1 or 2 into a lentiviral vector, wherein the vector comprises an EF1α promoter and a P2A self-cleavage sequence;

[0023] (2) Preparation of lentiviral particles: HEK-293T cells were transfected with the vector and packaging plasmids pCMV-dR8.91 and pMD2.G, and the viral supernatant was collected and concentrated to obtain a viral solution for later use;

[0024] (3) Transduction of immune cells: According to the infection coefficient of MOI = 50-100, the virus solution is used to infect T cells, macrophages or NK cells to obtain the target CAR immune cells.

[0025] Furthermore, the activation condition of the T cells is culturing in a culture medium containing CD3 / CD28 magnetic beads and IL-2 for 72 hours.

[0026] Furthermore, the differentiation conditions of the CAR-M cells are cultured in a culture medium containing 10 ng / mL PMA and 100 ng / mL LPS for 48 hours.

[0027] The fifth object of the present invention is to disclose the use of the CAR immune cells in the preparation of drugs for treating GP96-overexpressing tumors, including but not limited to ovarian cancer, triple-negative breast cancer, liver cancer, pancreatic cancer or colorectal cancer.

[0028] Compared with the existing technology, the present invention systematically solves the antigen escape, TME inhibition and safety issues of existing CAR therapy in solid tumors through the design of peptide-CAR targeting GP96, dual signaling domain synergy and cross-cell type adaptation, providing a highly efficient, safe and universal tumor immunotherapy solution for clinical use. The specific beneficial effects are as follows:

[0029] 1. Effectively overcome antigen escape and target limitations: GP96 is abnormally highly expressed on the cell membranes of solid tumors such as breast cancer, liver cancer, and pancreatic cancer, while it is strictly localized to the endoplasmic reticulum in normal tissues. By targeting GP96, the risk of off-target effects on normal tissues can be significantly reduced. In addition, as an endoplasmic reticulum stress marker, GP96's membrane expression is driven by hypoxia and endoplasmic reticulum stress in the tumor microenvironment, making it less susceptible to antigen loss or downregulation under treatment pressure. CAR simultaneously recognizes GP96 on the tumor cell membrane and the GP96-CD91 complex on the surface of tumor-associated macrophages (TAMs), effectively blocking the secretion of immunosuppressive factors such as IL-10, thereby inhibiting tumor immune escape at the source.

[0030] 2. Reshape the immunosuppressive microenvironment (TME): CAR integrates the dual functional domains of CD3ζ (T cell activation signal) and TLR4 (macrophage phagocytic signal), activates the phagocytic function of macrophages through the TLR4 / MyD88 pathway, and induces TAMs to polarize toward the pro-inflammatory M1 type.

[0031] 3. Optimized Safety and Low Immunogenicity: This invention utilizes a GP96-specific binding peptide instead of a traditional single-chain antibody (scFv). Its affinity is significantly lower than that of scFv, reducing nonspecific attack on normal tissues. The peptide sequence is based on the natural human GP96 ligand, avoiding the human anti-mouse antibody (HAMA) response triggered by the mouse scFv, significantly improving safety.

[0032] 4. Universality across cell types, suitable for constructing CAR-T, CAR-M and CAR-NK cells.

[0033] 5. It has strong clinical translation potential, covering solid tumor types with high GP96 expression, including triple-negative breast cancer (TNBC), hepatocellular carcinoma (HCC), pancreatic ductal adenocarcinoma (PDAC) and glioblastoma (GBM). When used in combination with PD-1 inhibitors, the CAR-T cell infiltration efficiency and tumor regression rate are synergistically improved; based on the large-scale production process of lentiviral vectors (such as PGC-EF1α-GP96 CAR), the CAR transduction efficiency reaches, which is significantly higher than the traditional macrophage transduction technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the structure of CD19-CAR-T, PEG10-CAR-T and GPC3-CAR-T.

[0035] Figure 2 The figure shows the expression analysis of CAR-T on the third day after T cells were infected with CD19-CAR-T, PEG10-CAR-T and GPC3-CAR-T, using non-transfected T cells as a control.

[0036] Figure 3 Schematic diagram of the structures of CD19-CAR-M, PEG10-CAR-M and GPC3-CAR-M.

[0037] Figure 4 The expression analysis of CAR-M on the 4th day after macrophages were infected with CD19-CAR-M, PEG10-CAR-M and GPC3-CAR-M, with non-transfected M cells as a control.

[0038] Figure 5 This is a comparison chart of GP96 expression in different tumor cells.

[0039] Figure 6 This is a comparison of the experimental results of CD19-CAR-T, PEG10-CAR-T and GPC3-CAR-T cells killing tumor cell lines with different GP96 positive expressions.

[0040] Figure 7 This is a comparison of the experimental results of CD19-CAR-M, PEG10-CAR-M and GPC3-CAR-M cells killing tumor cell lines with different GP96 positive expressions. DETAILED DESCRIPTION

[0041] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0042] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0043] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0044] All plasmids in the following examples were synthesized and extracted by Qingke Biotechnology Co., Ltd.

[0045] Example 1

[0046] like Figure 1 and Figure 3 As shown, in this example, CD19-CAR-T, PEG10-CAR-T, GPC3-CAR-T, CD19-CAR-M, PEG10-CAR-M and GPC3-CAR-M cells were constructed to explore the specific application of chimeric antigen receptor targeting GP96 in CAR cell therapy. The specific process is as follows:

[0047] 1. Vector Construction

[0048] (1) The construction process of PEG10-CAR-T and GPC3-CAR-T vectors is as follows: based on the PEG10 or GPC3 polypeptide amino acid sequence (shown in SEQ ID NO: 1 and SEQ ID NO: 2), human CD8 signal peptide (shown in SEQ ID NO: 4), human CD8 hinge region (shown in SEQ ID NO: 5), human CD8 transmembrane region (shown in SEQ ID NO: 6), human 4-1BB intracellular region (shown in SEQ ID NO: 3), human CD3ζ intracellular region (shown in SEQ ID NO: 7) and T2A-mKate2 (shown in SEQ ID NO: 8), a chimeric antigen receptor is constructed, and then the nucleotide sequence of each fragment of the chimeric antigen receptor is reversed according to the amino acid sequence of the chimeric antigen receptor (as shown in SEQ ID NO: 10 to 17), and the corresponding nucleotide sequence is obtained by artificial synthesis or PCR. Then, the nucleotide sequences corresponding to the CD8 signal peptide, PEG10 or GPC3 polypeptide extracellular domain, CD8 hinge region, CD8 transmembrane region, 4-1BB co-stimulatory domain, CD3ζ signaling region, and T2A-mKate2 fluorescent protein are inserted into the lentiviral vector PGC-EF1α-P2A-EGFP through XbaI seamless cloning technology to obtain PEG10-CAR-T and GPC3-CAR-T vectors.

[0049] Among them, the amino acid sequence of SEQ ID NO: 7 of the human CD3ζ intracellular region is: VKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0050] T2A-mKate2 SEQ ID The amino acid sequence of NO: 8 is: MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKAVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQ DGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYCDLPSKLGHKLN.

[0051] The nucleotide sequence of SEQ ID NO: 10 (PEG10) is: GCTCTGATTGGCCAGTGTATCCACATT.

[0052] The nucleotide sequence of SEQ ID NO: 11 (GPC3) is: AAGGTGTTCGGCAACTTTCCAAAGCTG.

[0053] The nucleotide sequence of SEQ ID NO: 12 (4-1BB) is: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGA.

[0054] The nucleotide sequence of SEQ ID NO: 13 (CD8 signal peptide) is: ATGGCCCTGCCCGTCACCGCTCTGCTGCTGCCCCTTGCTCTGCTTCTTCATGCAGCAAGGCCG.

[0055] The nucleotide sequence of SEQ ID NO: 14 (CD8 hinge region) is: ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCTAGCCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT.

[0056] The nucleotide sequence of SEQ ID NO: 15 (CD8 transmembrane region) is: ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC.

[0057] The nucleotide sequence of SEQ ID NO: 16 (CD3ζ intracellular region) is: GTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGAIAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.

[0058] The nucleotide sequence of SEQ ID NO: 17 (T2A-MKATE2) is as follows: ATGAGCGAGCTGATTAAGGAGAACATGCACATGAAGCTGTACATGGAGGGCACCGTGAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACGAGGGCACCCAGACCATGAGAATCAAGGCGGTCGAGGGCGGCCCTCTCCCCTTCGCCTTCGACATCCTGGCTACCAGCTTCATGTACGGCAGCAAAACCTTCATCAACCACACCCAGGGCATCCCCGACTTCTTTAAGCAGTCCTTCCCCGAGGGCTTCACATGGGAGAGAGTCACCACATACGAAGACGGGGGCGTGCTGACCGCTACCCAGGACACCAGCCTCCAGGACGGCTGCCTCATCTACAACGTCAAGATCAGAGGGGTGAACTTCCCATCCAACGGCCCTGTGATGCAGAAGAAAACACTCGGCTGGGAGGCCTCCACCGAGACCCTGTACCCCGCTGACGGCGGCCTGGAAGGCAGAGCCGACATGGCCCTGAAGCTCGTGGGCGGGGGCCACCTGATCTGCAACTTGAAGACCACATACAGATCCAAGAAACCCGCTAAGAACCTCAAGATGCCCGGCGTCTACTATGTGGACAGAAGACTGGAAAGAATCAAGGAGGCCGACAAAGAGACCTACGTCGAGCAGCACGAGGTGGCTGTGGCCAGAIACTGCGACCTCCCTAGCAAACTGGGGCACAAGCTGAAC。

[0059] (2) The construction process of PEG10-CAR-M and GPC3-CAR-M cell vectors is as follows: based on the PEG10 or GPC3 polypeptide amino acid sequence (shown in SEQ ID NO: 1 and SEQ ID NO: 2), human CD8 signal peptide (shown in SEQ ID NO: 4), human CD8 hinge region (shown in SEQ ID NO: 5), human CD8 transmembrane region (shown in SEQ ID NO: 6), human CD3ζ intracellular region (shown in SEQ ID NO: 7), human TIR region (shown in SEQ ID NO: 9) and T2A-mKate2 (shown in SEQ ID NO: 8), a chimeric antigen receptor is constructed, and then the nucleotide sequence of each fragment of the chimeric antigen receptor is reversed according to the amino acid sequence of the chimeric antigen receptor (as shown in SEQ ID NO: 10-11, 13-18), and the corresponding nucleotide sequence is obtained by artificial synthesis or PCR. Then, the nucleotide sequences corresponding to the CD8 signal peptide, PEG10 or GPC3 polypeptide extracellular domain, CD8 hinge region, CD8 transmembrane region, CD3ζ signaling region, human TIR region, and T2A-mKate2 fluorescent protein are inserted into the lentiviral vector PGC-EF1α-P2A-EGFP through XbaI seamless cloning technology to obtain PEG10-CAR-M and GPC3-CAR-M vectors.

[0060] Among them, the amino acid sequence of human TIR region SEQ ID NO: 9 is: NIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDG.

[0061] SEQ ID The nucleotide sequence of NO: 18 (human TIR region) is: AACATCTACGACGCTTCGTCATCTACAGCTCTCAGGACGAGGACTGGGTTCGAAACGAACTCGTGAAGAACCTGGAAGAGGGTGTACCACCATTCCAGCTGTGTCTGCACTATAGGGACTTCATTCCAGGTGTGGCTATTGCCGCTAACATCATCCATGAGGGTTTCCATAAGAGCCGGAAGGTCATCGTGGTGGTGTCTCAGCACTTCA TCCAGAGCAGATGGTGTATCTTCGAGTACGAGATTGCTCAGACCTGGCAGTTTCTGTCCAGTAGAGCTGGCATCTTCATCGTGCTGCAGAAGGTGGAAAAGACTCTGCTGAGGCAACAGGTCGAACTGTACAGACTGTTGAGCCGCAACACCTACCTCGAATGGGAAGATTCTGTGCTGGGTAGACACATCTTCTGGCGGAGACTGAGAAAGGCACTGTTGGATGGC.

[0062] 2. Lentivirus Preparation

[0063] (1) HEK-293T cells were revived and transferred to a 15 cm culture dish. When the cell density reached 90%, they were trypsinized and passaged at a 1:2 ratio.

[0064] (2) When the cell density reaches about 80%, transfection is performed. For each 15 cm dish, vector (PEG10-CAR-T, GPC3-CAR-T, PEG10-CAR-M and GPC3-CAR-M vectors) (20 μg): pCMV-dR8.91 (10 μg): pMD2.G (4 μg) = 5:2.5:1 are added to 2 mL of serum-free DMEM medium, and 68 μL of lipo8000 is added at the same time. After mixing, add to the culture dish;

[0065] (3) Add 13 mL of DMEM medium containing 5% FBS to each 15 cm dish and shake gently;

[0066] (4) Collect the cell culture supernatant 48 h after transfection and replace with 15 mL of fresh culture medium; and collect the culture supernatant 72 h after transfection;

[0067] (5) Take the supernatant of the culture medium (4), centrifuge at 3500 rpm, 4°C for 15 min, and filter through a 0.45 pm filter; add 30 mL of supernatant to each ultracentrifuge tube and add 5 mL of 20% sucrose (spread on the bottom of the tube), centrifuge at 25000 rpm, 4°C for 2.5 h; discard the supernatant, invert and dry the precipitate for 5 min, add 50 μL of lentivirus dissolution solution, and dissolve the virus at 4°C overnight;

[0068] (6) The virus solution was aliquoted and stored at -80°C for future use (including PEG10-CAR-T, GPC3-CAR-T, PEG10-CAR-M, and GPC3-CAR-M virus solutions, hereinafter represented by the corresponding lentiviral particles).

[0069] 3. Preparation of human CAR-T cells

[0070] (1) T cell separation: using RosetteSep TM Gradient centrifugation tubes, Human T Cell Enrichment Cocktail Kit, T cells were isolated from the peripheral blood of healthy volunteers.

[0071] (a) 20 mL peripheral blood + 800 μL RosetteSep TM Cocktail, after mixing, let it stand for 10 min and then dilute 1-fold with PBS (containing 2% FBS);

[0072] (b) Take 15mL of Ficol Lymphoprep and place it in RosetteSep TM Centrifuge the tube and add (a) to (b), and centrifuge at 1200g for 10 min;

[0073] (c) The supernatant was transferred to a 50 mL centrifuge tube, and an equal volume of PBS (containing 2% FBS) was added. The tube was then centrifuged at 300 g for 10 min.

[0074] (d) The supernatant was discarded, and freezing solution (70% Advanced RPMI 1640 medium + 20% FBS + 10% DMSO) was added for freezing, and CD3 expression was detected by flow cytometry.

[0075] (2) CAR-T cell preparation:

[0076] (a) In culture medium (5 mL FBS + 10 μL IL-2 + 500 μL Gluta-MAX + 500 μL P / S + 44 mL Advanced RPMI 1640) + Human T-Activator CD3 / CD28 magnetic beads (25 μL / 1×10 6 T cells) activated T cells for 72 h;

[0077] (b) Centrifuge at 500 g for 5 min and count the cells. Use a 96-well plate to infect the corresponding CAR-T lentiviral particles: 1×10 5 T cells were infected with 1 μL lentiBoost (1 μg / mL) and CAR lentiviral particles (MOI = 100). After 24 hours of infection, the cells were centrifuged at 500 g for 5 minutes, the supernatant was discarded, and the cells were transferred to a 24-well plate. On the third day after CAR-T cell infection, the infection efficiency of CAR-T cells was monitored by flow cytometry. CD19-CAR-T vector-transfected and non-transfected T cells were used as controls, and the expression of CAR-T cells was detected using the fusion protein T2A-mKATE2. Figure 2 As shown, it can be seen that GPC3-CAR-T has the highest infection efficiency, followed by PEG10-CAR-T, but it is significantly higher than CD19-CAR-T.

[0078] 4. Preparation of Human CAR-M Cells

[0079] (1) THP-1 cells were cultured in culture medium (5 mL FBS + 500 μL P / S + 45 mL RPMI 1640);

[0080] (2) Centrifuge at 500 g for 5 min and count the cells. Use a 96-well plate to infect the corresponding CAR-M lentiviral particles: 1×10 4 THP-1 cells were infected with 1 μL lentiBoost (1 μg / mL) and CAR-M lentiviral particles (MOI = 100). After 24 hours of infection, the cells were centrifuged at 500 g for 5 minutes, the supernatant was discarded, and the culture medium was replaced. After 48 hours, a culture medium containing PMA (10 ng / ml) and LPS (100 ng / ml) was added and cultured for 48 hours. The infection efficiency of CAR-M was monitored by flow cytometry, and CD19-CAR-M vector-transfected and non-transfected T cells were used as controls. The expression of CAR-T was detected using the fusion protein T2A-mKATE2. Figure 4 As shown, it can be seen that the infection efficiency of PEG10-CAR-M is the highest, followed by GPC3-CAR-M, but significantly higher than that of CD19-CAR-M.

[0081] The obtained CD19-CAR-T and CD19-CAR-M were used as control groups, and subsequent verification experiments were performed on PEG10-CAR-T, GPC3-CAR-T, PEG10-CAR-M and GPC3-CAR-M.

[0082] Example 2

[0083] In this example, 10 groups of different tumor cells were selected for in vitro toxicity experiments. The 10 groups of different tumor cells included pancreatic cancer cell lines (MIA PaCa2, PANCl), liver cancer cell lines (HepG2, SK-Hep1), breast cancer cell lines (MDA-MB231, MDA-MB468, MCF7), and colorectal cancer cell lines (GP2D, HCT116, DLD1). Flow cytometry was used to analyze the expression levels of GP96 in different tumor cells. Figure 5 As shown, the cell lines with high GP96 expression are MDA-MB231, PANC1, Huh7, and HCT116; and the cell lines with low GP96 expression are MDA-MB468, MCF7, MIA PaCa2, He pG2, DLD1, and GP2D.

[0084] 1. In vitro toxicity assay of CAR-T cells

[0085] The Luciferase Assay System was used to detect the killing effect of CAR-T cells on target cells.

[0086] (1) Target cell digestion and counting: Digest the cells with trypsin, centrifuge at 1000 rpm for 3 min, add 1 mL of cell culture medium, and resuspend and count;

[0087] (2) According to 2×10 per hole 3 Target cells / 100 μL culture medium were added to a 96-well plate;

[0088] (3) CAR-T cell counting: centrifuge at 500 g for 5 min, resuspend and count in 1 mL of cell culture medium, and add the corresponding number of CAR-T cells / 100 μL / well according to the effector-target ratio of 5:1;

[0089] (4) Mix (2) + (3), co-culture for 24 hours, collect the supernatant to detect the release of cytokines such as IFN-γ and TNF-α; add 30μL / well 1×Cell Culture Lysis, and after 15 minutes, add 30μL / well Luciferase working solution, and detect with a multifunctional microplate reader; calculate the killing effect of CAR-T cells on target cells: the ratio of the killing effect of NT cells on target cells.

[0090] The results are as follows Figure 6 As shown in the results, both PEG10-CAR-T and GPC3-CAR-T cells were able to effectively kill GP96-overexpressing cell lines MDA-MB231, PANC1, Huh7, and HCT116. However, they showed no significant killing effects on GP96-low expressing cell lines MDA-MB468, MCF7, MLAPaCa2, HepG2, DLD1, and GP2D. There was no significant difference in the killing effects of CD19-CAR-T cells among the tumor cell groups.

[0091] 2. In vitro toxicity experiment of CAR-M cells

[0092] Luciferase Assay System was used to detect the killing effect of CAR-M cells on target cells.

[0093] (1) Target cell digestion and counting: Digest the cells with trypsin, centrifuge at 1000 rpm for 3 min, and resuspend and count in 1 mL of RPMI 1640 medium containing PMA (10 ng / mL) and LPS (100 ng / mL).

[0094] (2) According to 2×10 per hole 3 Target cells / 100 μL of culture medium were added to a 96-well plate containing CAR-M cells, with an effector-target ratio of 5:1;

[0095] (3) After 24 h of co-culture, the supernatant was collected to detect the release of cytokines such as IFN-γ and TNF-α; 30 μL / well 1× Cell Culture Lysis was added, and 30 μL / well Luciferase working solution was added 15 minutes later, and the cells were detected using a multifunctional microplate reader; the killing effect of CAR-M cells on target cells was calculated as the ratio of the killing effect of control M cells on target cells.

[0096] The results are as follows Figure 7 As shown in the results, both PEG10-CAR-M and GPC3-CAR-M were able to effectively kill GP96-overexpressing cell lines MDA-MB231, PANC1, Huh7, and HCT116, but had no significant killing effect on GP96-low expressing cell lines MDA-MB468, MCF7, MLAPaCa2, HepG2, DLD1, and GP2D. There was no significant difference in the killing effect of CD19-CAR-M on tumor cells among the groups.

[0097] The results of this example show that both PEG10-CAR and GPC3-CAR can specifically kill tumor cells that highly express GP96 on the membrane, without having obvious toxic effects on normal cells.

[0098] Finally, it should be noted that the terms "comprises," "comprising," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the invention.

[0099] The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, and all such variations and improvements fall within the scope of protection of the present application.

Claims

1. A chimeric antigen receptor targeting GP96, characterized in that Includes the following structures: (a) an extracellular binding domain comprising a PEG10 polypeptide or a GPC3 polypeptide capable of specifically binding to GP96, wherein the amino acid sequence of the PEG10 polypeptide is shown in SEQ ID NO: 1, and the amino acid sequence of the GPC3 polypeptide is shown in SEQ ID NO: 2; (b) a signal peptide selected from the group consisting of a signal peptide of CD8 or CD28; (c) a hinge region selected from the hinge region of CD8 or CD28; (d) a transmembrane region selected from the transmembrane region of CD8 or CD28; (e) an intracellular signaling domain selected from CD3ζ, TIR, or a combination thereof.

2. The chimeric antigen receptor according to claim 1, wherein Also contains fluorescent marker protein.

3. The chimeric antigen receptor according to claim 1, wherein It also contains a costimulatory domain 4-1BB or a conserved intracellular domain TIR, the amino acid sequence of the 4-1BB is shown in SEQ ID NO: 3, and the amino acid sequence of the TIR is shown in SEQ ID NO:

9.

4. The chimeric antigen receptor according to claim 1, wherein The signal peptide, hinge region and transmembrane region are all derived from CD8, and their amino acid sequences are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

5. Use of the chimeric antigen receptor according to any one of claims 1 to 4 in the preparation of CAR immune cells.

6. A CAR immune cell, characterized in that Comprising the chimeric antigen receptor according to any one of claims 1 to 4, wherein the CAR immune cell is selected from any one of the following: (a) CAR-T cells, wherein the chimeric antigen receptor is transduced into human T cells via a lentiviral vector, wherein the T cells are derived from peripheral blood and activated with CD3 / CD28 magnetic beads and IL2; (b) CAR-M cells, wherein the chimeric antigen receptor is transduced into human macrophages via a lentiviral vector, and the macrophages are differentiated from THP-1 cells induced by PMA and LPS; (c) CAR-NK cells, wherein the chimeric antigen receptor is transduced into human NK cells via a lentiviral vector, wherein the NK cells are derived from peripheral blood or NK-92 cell line and activated by IL-2 or IL-15.

7. A method for preparing the CAR immune cell according to claim 6, characterized in that: The steps include: (1) constructing a lentiviral vector: cloning the nucleotide sequence of the chimeric antigen receptor according to claim 1 or 2 into a lentiviral vector, wherein the vector comprises an EF1α promoter and a P2A self-cleavage sequence; (2) Preparation of lentiviral particles: HEK-293T cells were transfected with the vector and packaging plasmids pCMV-dR8.91 and pMD2.G, and the viral supernatant was collected and concentrated to obtain a viral solution for later use; (3) Transduction of immune cells: According to the infection coefficient of MOI = 50-100, the virus solution is used to infect T cells, macrophages or NK cells to obtain the target CAR immune cells.

8. The preparation method according to claim 7, wherein The activation condition of the T cells is culturing in a culture medium containing CD3 / CD28 magnetic beads and IL-2 for 72 hours.

9. The preparation method according to claim 7, wherein The differentiation conditions of the CAR-M cells are cultured in a culture medium containing 10 ng / mL LMA and 100 ng / mL LPS for 48 hours.

10. Use of the CAR immune cell according to claim 6 in the preparation of a drug for treating GP96-overexpressing tumors, wherein the tumors include but are not limited to ovarian cancer, triple-negative breast cancer, liver cancer, pancreatic cancer or colorectal cancer.

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