Chimeric antigen receptor constructed based on Siglec10, immune cell as well as preparation method and application of chimeric antigen receptor

Abstract

The invention discloses a chimeric antigen receptor constructed based on Siglec10, an immune cell and a preparation method and application of the chimeric antigen receptor and the immune cell, relates to the technical field of cellular immunity, and discloses a chimeric antigen receptor constructed through a targeted CD24-Siglec10 signal axis, and CAR-T, CAR-M and CAR-NK cells and the like constructed based on the receptor. By targeting a CD24-Siglec10 signal axis and integrating a natural receptor-ligand recognition mode and multi-signal domain collaborative design, the defects of antigen escape, TME inhibition and safety of a traditional CAR therapy in solid tumors are systematically solved, and an efficient, accurate and industrialized tumor immunotherapy new strategy 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 constructed based on Siglec10, immune cells, and preparation methods and applications thereof. Background Art

[0002] Chimeric antigen receptor (CAR) cell therapy, a core technology in tumor immunotherapy, has demonstrated significant efficacy in hematologic malignancies, but treatment for solid tumors still faces multiple bottlenecks. Different effector cell types, such as CAR-T, CAR-M (macrophages), and CAR-NK (natural killer cells), exhibit limitations in solid tumors due to their distinct biological characteristics, including antigen escape, resistance to the immunosuppressive microenvironment (TME), non-tumor-targeted toxicity, and insufficient cell infiltration. This is due to the fact that traditional CAR technology relies on scFv for antigen recognition. Its high-affinity binding mode not only increases the risk of off-target effects, but also, due to the significant conservation differences between scFvs across species (for example, mouse scFvs are prone to eliciting human anti-mouse antibody responses), preclinical models have difficulty predicting human safety. Furthermore, overactivation of traditional CAR signaling can induce T / NK cell exhaustion, reducing efficacy.

[0003] It can be seen that further research on CAR receptors is of great significance for CAR therapy. Summary of the Invention

[0004] The present invention discloses a chimeric antigen receptor constructed by targeting the CD24-Siglec10 signaling axis, 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 constructed based on Siglec10, comprising the following structure:

[0007] (a) an extracellular binding domain comprising the IgV domain of Siglec10, the amino acid sequence of which is shown in SEQ ID NO: 1;

[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 is: DGRFWIRVQESVMVPEGLCISVPCSFSYPRQDWTGSTPAYGYWFKAVTETTKGAPVATNHQSREVEMSTRGRFQLTGDPAKGNCSLVIRDAQMQDESQYFFRVE.

[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: 2, and the amino acid sequence of the TIR is shown in SEQ ID NO: 8. In the present invention, the sequence of SEQ ID NO: 2 is: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR; the sequence of SEQ ID NO: 8 is: NIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDG.

[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: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively. In the present invention, the sequence of SEQ ID NO: 3 is: MALPVTALLLPLALLLHAARP; the sequence of SEQ ID NO: 4 is: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD; and the sequence of SEQ ID NO: 5 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 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 CD24-highly expressed tumors, including but not limited to ovarian cancer, triple-negative breast cancer, liver cancer, pancreatic cancer or colorectal cancer.

[0028] Compared with existing technologies, this invention systematically addresses the antigen escape, TME suppression, and safety deficiencies of traditional CAR therapy in solid tumors by targeting the CD24-Siglec10 signaling axis, integrating natural receptor-ligand recognition patterns, and collaborative design of multiple signaling domains. It provides a new strategy for tumor immunotherapy that is efficient, precise, and industrializable for clinical use. The specific beneficial effects are as follows:

[0029] 1. High targeting and specific killing: CD24 is highly expressed on the surface of highly invasive solid tumors such as ovarian cancer, triple-negative breast cancer, and liver cancer, while it is lowly expressed in normal tissues or only located intracellularly. The CAR of the present invention directly binds to CD24 through the extracellular domain of Siglec10. In vitro experiments have shown that Siglec10-CAR-T / CAR-M has excellent killing efficiency against tumor cells with high CD24 expression, while the killing rate against cells with low CD24 expression is low, significantly reducing the risk of off-target effects. The natural binding mode of Siglec10 and CD24 avoids the nonspecific attacks caused by the high affinity of traditional scFv.

[0030] 2. Reversal of the immunosuppressive microenvironment (TME): The CD24-Siglec10 signaling axis inhibits macrophage phagocytic function by activating SHP-1 / 2 phosphatase.

[0031] 3. Safety has been optimized and it has low immunogenicity. Because the Siglec10 extracellular domain is derived from human protein, it effectively avoids the human anti-mouse antibody (HAMA) response induced by mouse scFv, thereby significantly improving safety.

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

[0033] 5. CAR therapy targeting CD24 has strong potential for clinical translation. It has shown significant therapeutic potential in highly prevalent solid tumors such as ovarian cancer and triple-negative breast cancer. Studies have shown that targeting CD24 can enhance immune clearance of tumor cells, thereby inhibiting tumor growth. Furthermore, combined use with chemotherapy, radiotherapy, or immune checkpoint inhibitors is expected to achieve synergistic effects, providing patients with a more comprehensive treatment plan. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the CD19-CAR-T and Siglec10-CAR-T structures.

[0035] Figure 2 The figure shows the expression analysis of CAR-T on the third day after CD19-CAR-T and Siglec10-CAR-T infected T cells, using non-transfected T cells as a control.

[0036] Figure 3 Schematic diagram of the structure of CD19-CAR-M and Siglec10-CAR-M.

[0037] Figure 4 The figure shows the expression analysis of CAR-M on the 4th day after CD19-CAR-M and Siglec10-CAR-M infected macrophages, with non-transfected M cells as a control.

[0038] Figure 5 The figure shows the comparison of CD24 expression in different tumor cells.

[0039] Figure 6 This is a comparison of the experimental results of CD19-CAR-T and Siglec10-CAR-T cells killing tumor cell lines with different CD24 positive expressions.

[0040] Figure 7 This is a comparison of the experimental results of CD19-CAR-M and Siglec10-CAR-M cells killing tumor cell lines with different CD24 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 to have the meaning 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, this example constructs Siglec10-CAR-T and Siglec10-CAR-M cells to explore the specific application of chimeric antigen receptors constructed based on Siglec10 in CAR cell therapy. The specific process is as follows:

[0047] 1. Vector Construction

[0048] (1) The construction process of Siglec10-CAR-T vector is as follows: based on the components of Siglec10 extracellular domain IgV region (shown in SEQ ID NO: 1), human CD8 signal peptide (shown in SEQ ID NO: 3), human CD8 hinge region (shown in SEQ ID NO: 4), human CD8 transmembrane region (shown in SEQ ID NO: 5), human 4-1BB intracellular region (shown in SEQ ID NO: 2), human CD3ζ intracellular region (shown in SEQ ID NO: 6) and T2A-mKate2 (shown in SEQ ID NO: 7), 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 (as shown in SEQ ID NO: 9 to 15), and the corresponding nucleotide sequence is obtained by artificial synthesis or PCR. Then, the nucleotide sequences corresponding to the CD8 signal peptide, Siglec10 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 the Siglec10-CAR-T vector.

[0049] Among them, the amino acid sequence of the human CD3ζ intracellular region SEQ ID NO: 6 is: VKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0050] T2A-mKate2 SEQ ID The amino acid sequence of NO: 7 is: MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKAVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQ DGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYCDLPSKLGHKLN.

[0051] SEQ ID The nucleotide sequence of NO: 9 (the extracellular domain IgV region of Siglec10) is: GATGGACGGTTCTGGATTCGCGTGCAAGAGAGCGTCATGGTGCCAGAAGGCCTGTGCATTTCCGTGCCTTGTAGCTTTAGCTATCCAAGACAAGATTGGACAGGCTCTACCCCAGCCTACGGCTACTGGTTCAAGGCTGTG ACCGAGACAACAAAGGGCGCTCCAGTCGCTACCAACCATCAGTCCAGGGAAGTCGAGATGTCCACTAGAGGTCGCTTCCAGCTGACTGGCGATCCAGCTAAGGGCAACTGCAGCCTGGTCATCAGAGACGCTCAGATGCAAGACGAAAGCCAGTATTTCTTCCGGGTGGAG.

[0052] The nucleotide sequence of SEQ ID NO: 10 (4-1BB) is: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGA.

[0053] The nucleotide sequence of SEQ ID NO: 11 (CD8 signal peptide) is: ATGGCCCTGCCCGTCACCGCTCTGCTGCTGCCCCTTGCTCTGCTTCTTCATGCAGCAAGGCCG.

[0054] The nucleotide sequence of SEQ ID NO: 12 (CD8 hinge region) is: ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCTAGCCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT.

[0055] The nucleotide sequence of SEQ ID NO: 13 (CD8 transmembrane region) is: ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC.

[0056] The nucleotide sequence of SEQ ID NO: 14 (CD3ζ intracellular region) is: GTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC。

[0057] The nucleotide sequence of SEQ ID NO: 15 (T2A-MKATE2) is as follows: ATGAGCGAGCTGATTAAGGAGAACATGCACATGAAGCTGTACATGGAGGGCACCGTGAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACGAGGGCACCCAGACCATGAGAATCAAGGCGGTCGAGGGCGGCCCTCTCCCCTTCGCCTTCGACATCCTGGCTACCAGCTTCATGTACGGCAGCAAAACCTTCATCAACCACACCCAGGGCATCCCCGACTTCTTTAAGCAGTCCTTCCCCGAGGGCTTCACATGGGAGAGAGTCACCACATACGAAGACGGGGGCGTGCTGACCGCTACCCAGGACACCAGCCTCCAGGACGGCTGCCTCATCTACAACGTCAAGATCAGAGGGGTGAACTTCCCATCCAACGGCCCTGTGATGCAGAAGAAAACACTCGGCTGGGAGGCCTCCACCGAGACCCTGTACCCCGCTGACGGCGGCCTGGAAGGCAGAGCCGACATGGCCCTGAAGCTCGTGGGCGGGGGCCACCTGATCTGCAACTTGAAGACCACATACAGATCCAAGAAACCCGCTAAGAACCTCAAGATGCCCGGCGTCTACTATGTGGACAGAAGACTGGAAAGAATCAAGGAGGCCGACAAAGAGACCTACGTCGAGCAGCACGAGGTGGCTGTGGCCAGATACTGCGACCTCCCTAGCAAACTGGGGCACAAGCTGAAC。

[0058] (2) The construction process of Siglec10-CAR-M cell vector is as follows: based on the components of Siglec10 extracellular domain IgV region (shown in SEQ ID NO: 1), human CD8 signal peptide (shown in SEQ ID NO: 3), human CD8 hinge region (shown in SEQ ID NO: 4), human CD8 transmembrane region (shown in SEQ ID NO: 5), human CD3ζ intracellular region (shown in SEQ ID NO: 6), human TIR region (shown in SEQ ID NO: 8) and T2A-mKate2 (shown in SEQ ID NO: 7), a chimeric antigen receptor is constructed, and then the amino acid sequence of each fragment of the chimeric antigen receptor is reversed according to its amino acid sequence to reversely deduced its nucleotide sequence (as shown in SEQ ID NO: 9, 11 to 16), and the corresponding nucleotide sequence is obtained by artificial synthesis or PCR. Then, the nucleotide sequences corresponding to the CD8 signal peptide, Siglec10 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 the Siglec10-CAR-M vector.

[0059] Among them, the amino acid sequence of human TIR region SEQ ID NO: 8 is: NIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDG.

[0060] SEQ ID The nucleotide sequence of NO: 16 (human TIR region) is: AACATCTACGACGCTTCGTCATCTACAGCTCTCAGGACGAGGACTGGGTTCGAAACGAACTCGTGAAGAACCTGGAAGAGGGTGTACCACCATTCCAGCTGTGTCTGCACTATAGGGACTTCATTCCAGGTGTGGCTATTGCCGCTAACATCATCCATGAGGGTTTCCATAAGAGCCGGAAGGTCATCGTGGTGGTGTCTCAGCACTTCA TCCAGAGCAGATGGTGTATCTTCGAGTACGAGATTGCTCAGACCTGGCAGTTTCTGTCCAGTAGAGCTGGCATCTTCATCGTGCTGCAGAAGGTGGAAAAGACTCTGCTGAGGCAACAGGTCGAACTGTACAGACTGTTGAGCCGCAACACCTACCTCGAATGGGAAGATTCTGTGCTGGGTAGACACATCTTCTGGCGGAGACTGAGAAAGGCACTGTTGGATGGC.

[0061] 2. Lentivirus Preparation

[0062] (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.

[0063] (2) When the cell density reaches about 80%, transfection is performed. Vector (Siglec10-CAR-T vector or Siglec10-CAR-M vector) (20 μg): pCMU-dR8.91 (10 μg): pMD2.G (4 μg) = 5:2.5:1 are added to 2 mL of serum-free DMEM medium for each 15 cm dish. At the same time, 68 μL of lipo8000 is added, mixed, and added to the culture dish;

[0064] (3) Add 13 mL of DMEM medium containing 5% FBS to each 15 cm dish and shake gently;

[0065] (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;

[0066] (5) Take the supernatant of the culture medium in (4), centrifuge at 3500 rpm, 4°C for 15 min, and filter through a 0.45 μm filter membrane; 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;

[0067] (6) The viral solution was aliquoted and stored at -80°C for future use (including CAR-T and CAR-M viral solutions, hereinafter referred to as CAR-T or CAR-M lentiviral particles).

[0068] 3. Preparation of human CAR-T cells

[0069] (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.

[0070] (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);

[0071] (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;

[0072] (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.

[0073] (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.

[0074] (2) CAR-T cell preparation:

[0075] (a) In culture medium (5 mL FBS + 10 μL IL-2 + 500 VL 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;

[0076] (b) Centrifuge at 500 g for 5 min and count the cells. Infect 96-well plates with 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 the infection efficiency of Siglec10-CAR-T is high.

[0077] 4. Preparation of Human CAR-M Cells

[0078] (1) THP-1 cells were cultured in culture medium (5 mL FBS + 500 μL P / S + 45 mL RPMI 1640);

[0079] (2) Centrifuge at 500 g for 5 min and count the cells. Infect 96-well plates with 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, the cells were cultured with medium containing PMA (10 ng / mL) and LPS (100 ng / mL) 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 Siglec10-CAR-M is high.

[0080] Subsequent verification experiments were performed on the obtained CD19-CAR-T, Siglec10-CAR-T, CD19-CAR-M and Siglec10-CAR-M.

[0081] Example 2

[0082] In this example, 10 groups of different tumor cells were selected to conduct in vitro toxicity experiments on the CD19-CAR-T, Siglec10-CAR-T, CD19-CAR-M and Siglec10-CAR-M obtained in Example 1. The 10 groups of different tumor cells included pancreatic cancer cell lines (MIA PaCa2, PANC1), 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 CD24 in different tumor cells. Figure 5 As shown, the cell lines with high CD24 expression include MDA-MB468, MCF7, PANC1, HepG2, and GP2D; the cell lines with low CD24 expression include MDA-MB231, MIA PaCa2, SK-Hep1, DLD1, and HCT116.

[0083] 1. In vitro toxicity assay of CAR-T cells

[0084] The Luciferase Assay System was used to detect the killing effect of CAR-T cells on target cells.

[0085] (1) Target cell digestion and counting: Digest the cells with trypsin, centrifuge at 1000 rpm for 3 min, resuspend in 1 mL of T cell culture medium, and count;

[0086] (2) According to 2×10 per hole 3 Target cells / 100 μL culture medium were added to a 96-well plate;

[0087] (3) CAR-T cell counting: centrifuge at 500 g for 5 min, resuspend and count in 1 mL of T cell culture medium, and add the corresponding number of CAR-T cells / 100 μL / well according to the effector-target ratio of 5:1;

[0088] (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.

[0089] The results are as follows Figure 6As shown, Siglec10-CAR-T cells effectively killed CD24-overexpressing cell lines MDA-MB468, MCF7, PANC1, HepG2, and GP2D, but showed no significant killing effect on CD24-low expressing cell lines MDA-MB231, MIA PaCa2, SK-Hep1, DLD1, and HCT116. CD19-CAR-T cells showed no significant difference in their killing effect on tumor cells among the groups.

[0090] 2. In vitro toxicity experiment of CAR-M cells

[0091] Luciferase Assay System was used to detect the killing effect of CAR-M cells on target cells.

[0092] (1) Target cell digestion and counting: cells were digested with trypsin, centrifuged at 1000 rpm for 3 min, and resuspended in 1 mL of RPMI 1640 medium containing PMA (10 ng / ml) and LPS (100 ng / ml) for counting;

[0093] (2) According to 2×10 per hole 3 Target cells / 100 μL culture medium were added to a 96-well plate containing CAR-M cells, with an effector-target ratio of 5:1:

[0094] (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.

[0095] The results are as follows Figure 7 As shown, Siglec10-CAR-M effectively kills CD24-overexpressing cell lines MDA-MB468, MCF7, PANC1, HepG2, and GP2D, but has no significant killing effect on CD24-low expressing cell lines MDA-MB231, MIA PaCa2, SK-Hep1, DLD1, and HCT116. There was no significant difference in the killing effect of CD19-CAR-M on tumor cells among the groups.

[0096] The results of this example show that Siglec10-CAR can specifically kill tumor cells that highly express CD24 on the membrane without having obvious toxic effects on normal cells.

[0097] 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.

[0098] 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 constructed based on Siglec10, characterized in that Includes the following structures: (a) an extracellular binding domain comprising the IgV domain of Siglec10, the amino acid sequence of which is shown in SEQ ID NO: 1; (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: 2, and the amino acid sequence of the TIR is shown in SEQ ID NO:

8.

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: 3, SEQ ID NO: 4 and SEQ ID NO: 5, 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, 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 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 cells according to claim 6 in the preparation of a drug for treating CD24-highly expressed 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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