Preparation method of CAR-T cell targeting CD248

Abstract

The invention relates to the technical field of biological medicine, in particular to a preparation method of a CAR-T cell targeting CD248, the CAR-T cell targeting CD248 comprises a CAR targeting CD248, the CAR targeting CD248 sequentially comprises CD8 alpha, a biotin recognition structural domain, CD8 TM, CD28, CD137 and CD3 zeta from the N end to the C end, the amino acid sequence of the CAR targeting CD248 is shown as SEQ ID NO.1, and the coded DNA sequence of the CAR targeting CD248 is shown as SEQ ID NO.2. The invention further discloses a preparation method of the CAR-T cell targeting CD248. Biotin binding immune receptor T cells are designed for targeting CD248, myocardial fibrosis can be effectively inhibited, and the cardiac function is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for preparing CD248-targeted CAR-T cells. Background Art

[0002] Myocardial fibrosis is a key pathological manifestation of heart failure, and occurs in a variety of heart diseases that can lead to heart failure, such as acute myocardial infarction / ischemic cardiomyopathy, chemotherapy-related myocardial toxicity, hypertrophic cardiomyopathy, etc. Active and effective anti-fibrosis treatment has great clinical significance for improving cardiac function, but there is currently a lack of effective drugs to inhibit myocardial fibrosis in the clinic. Common beta-blockers and ACEI / ARB drugs can only indirectly or partially inhibit myocardial fibrosis, and cardiac function still inevitably deteriorates. The present invention performs single-cell RNA sequencing on cardiac interstitial cells after myocardial infarction in mice, screens out a special pathogenic fibroblast subpopulation F-Act, identifies its characteristic surface antigen CD248, and verifies that CD248-positive fibroblasts exist in a variety of other myocardial fibrosis models, such as doxorubicin-induced heart failure and hypertrophic cardiomyopathy caused by aortic arch stenosis. The present invention targets CD248 to design biotin-binding immune receptor T cells (BBIR-T), and targets and removes abnormally activated F-Act cells in the cardiac interstitium, which can effectively inhibit myocardial fibrosis and improve cardiac function. Summary of the invention

[0003] In view of the deficiencies of the prior art, the present invention proposes a method for preparing CAR-T cells targeting CD248.

[0004] The present invention is achieved through the following technical solutions: A CAR-T cell targeting CD248, which is a biotin-binding immune receptor T cell (BBIR-T) targeting CD248, includes a chimeric antigen receptor CAR.

[0005] Furthermore, the CAR comprises CD8α, biotin recognition domain, CD8 TM, CD28, CD137, and CD3ζ from N-terminus to C-terminus, and its amino acid sequence is shown in SEQ ID NO.1, and its encoded DNA sequence is shown in SEQ ID NO.2.

[0006] Furthermore, the CD8α comprises an amino acid sequence as shown in SEQ ID NO.3, and its encoded DNA sequence is shown in SEQ ID NO.4.

[0007] Furthermore, the biotin recognition domain includes an amino acid sequence as shown in SEQ ID NO.5, and its encoded DNA sequence is shown in SEQ ID NO.6.

[0008] Furthermore, the CD8 TM comprises an amino acid sequence as shown in SEQ ID NO.7, and its encoded DNA sequence is shown in SEQ ID NO.8.

[0009] Furthermore, the CD28 comprises an amino acid sequence as shown in SEQ ID NO.9, and its encoded DNA sequence is shown in SEQ ID NO.10.

[0010] Furthermore, the CD137 comprises an amino acid sequence as shown in SEQ ID NO.11, and its encoded DNA sequence is shown in SEQ ID NO.12.

[0011] Furthermore, the CD3ζ comprises an amino acid sequence as shown in SEQ ID NO.13, and its encoded DNA sequence is shown in SEQ ID NO.14.

[0012] The present invention also provides a biotinylated anti-CD248 engineered antibody targeting CD248.

[0013] Furthermore, the CAR-T cells can be combined with biotinylated anti-CD248 engineered antibodies.

[0014] Furthermore, the heavy chain of the CD248 engineered antibody comprises the amino acid sequence shown in SEQ ID NO.15, and the DNA sequence encoded by the heavy chain is shown in SEQ ID NO.16.

[0015] Furthermore, the light chain of the CD248 engineered antibody comprises the amino acid sequence shown in SEQ ID NO.17, and the DNA sequence encoded by the light chain is shown in SEQ ID NO.18.

[0016] Furthermore, the method for preparing the biotinylated anti-CD248 engineered antibody comprises the following steps: (a) 1 mL CD248 antibody (1 mg / mL) was dialyzed in 0.2 M sodium acetate buffer (pH 4.0) for 24 h, mixed with pepsin (1 mg / mL), pepsin:CD248 antibody = 1:20 (w:w), incubated in a 37°C water bath for 6 h, added with 250 μL 3M Tris-HCl (pH 9.0) to adjust the pH to 8.0, and dialyzed in 1× PBS (pH 8.0) at 4°C for 24 h to obtain a CD248 antibody digestion sample; (b) 700 μL of the CD248 antibody digestion sample described in step (a) was loaded onto a Sephadex G-100 chromatography column (column length 20 cm), and the flow-through was collected in separate tubes. The CD248 flow-through was mixed and concentrated to a final volume of 450 μL and a concentration of 0.2 mg / mL to obtain the CD248 antibody F(ab')2 fragment; (c) Using ThermoFisher EZ-Link TM The CD248 antibody F(ab')2 fragment was biotin-labeled using the sulfo-NHS-LC-Biotinylation Kit and dialyzed against 1×PBS (pH 8.0) at 4°C for 24 h to obtain the biotinylated anti-CD248 engineered antibody.

[0017] The present invention also provides a method for preparing the CD248-targeting CAR-T cells, comprising the following steps: (1) Preparation of peripheral blood mononuclear cells (PBMC): (11) Collect 10 mL of peripheral blood, mix thoroughly, transfer to two 50 mL centrifuge tubes, and centrifuge at 2000 rpm for 10 min to obtain the upper plasma layer and the lower blood cell sediment; (12) Resuspend the blood cell precipitate obtained in step (11) with 10 mL of physiological saline, and use a Pasteur pipette to fully disperse the cell clumps to obtain diluted blood. Take two new 50 mL centrifuge tubes, add 10 mL of lymphocyte separation solution to each tube, and slowly add the diluted blood along the tube wall to the upper layer of the separation solution. Centrifuge at 2000 rpm for 30 min; (13) After centrifugation in step (12), gently aspirate the middle white film layer with a Pasteur pipette, collect the mixture into a new 50 mL centrifuge tube, add physiological saline to 45 mL, mix thoroughly by inverting, centrifuge at 2000 rpm for 8 min, discard the supernatant, and obtain the cell pellet; (14) Add 45 mL of saline to the cell pellet of step (13) to resuspend the cells, centrifuge at 1500 rpm for 6 min; discard the supernatant, add 45 mL of saline to resuspend the cells, centrifuge at 1000 rpm for 6 min, discard the supernatant, and obtain peripheral blood mononuclear cells.

[0018] (2) T cell activation: (21) One day before PBMC extraction, dilute the anti-CD3 / CD28 antibody to 1 μg / mL with sterile PBS, add the diluted antibody to a hydrophobic tissue culture 24-well plate at 1 mL / well, seal with sealing film, and incubate at 4°C for 24 h to obtain a pre-coated 24-well plate; (22) Take out the 24-well plate pre-coated in step (21), discard the PBS containing the diluted antibody, and dry it in a clean bench; resuspend the peripheral blood mononuclear cells obtained in step (14) in RPMI-1640 medium and dilute to 10 6 cells / mL, and the diluted cells were added to the pre-coated 24-well plate, 1 mL / well, and cultured at 37°C and 5% CO2; (23) After culturing for 24 h in step (22), cytokines IL-2 100 IU / mL, IL-7 10 ng / mL, and IL-15 10 ng / mL were added and cultured for 24 h at 37°C and 5% CO2 to obtain activated T cells.

[0019] (3) Lentiviral vector construction: (31) CAR sequence synthesis and lentiviral vector plasmid preparation were completed by Nanjing Novogene Biotechnology Co., Ltd., and the CAR gene sequence was inserted between the EcoRI and BamH1 restriction sites in the plasmid pCDH-EF1-MCS.

[0020] (4) Lentivirus packaging: (41) 293T cells were used as lentiviral packaging cells. The culture medium was high-glucose DMEM medium containing 10% FBS. 293T cells were passaged 24 h before the start of virus packaging. The cell density was 5 × 10 5 / mL, inoculated in a 100 mm cell culture dish with an inoculation volume of 12 mL, cultured at 37°C, 5% CO2 until the cell confluence rate reached 80%, and the cell culture medium was changed to Opti-MEM medium 2 h before transfection; (42) The lentiviral packaging system of Shanghai GeneCare was used for packaging. According to the instructions of the kit, the lentiviral vector plasmid, Lenti-Easy Packing Mix and Opti-MEM were mixed evenly at a dosage ratio of 16 μg:25 μL:1455 μL, and incubated at room temperature for 5 min to obtain the diluted packaging system; 60 μL of Lipofectamine 2000 reagent was mixed evenly with 1440 μL of Opti-MEM, and incubated at room temperature for 5 min to obtain the diluted Lipofectamine2000; (43) Mix the diluted packaging system and diluted Lipofectamine 2000 in a 1:1 volume ratio, gently invert to mix, and incubate at room temperature for 20 min to form a DNA-Lipofectamine 2000 transfection complex; (44) Add the transfection complex described in step (43) to the culture medium of the 293T cells cultured in step (41), gently shake the culture dish to mix, and continue to culture in a 37°C, 5% CO2 cell culture incubator. 8 h after transfection, discard the culture medium containing the transfection complex, add 10 mL PBS to wash the cells, discard the PBS, add 12 mL of fresh high-glucose DMEM medium containing 10% FBS, and continue to culture in a 37°C, 5% CO2 cell culture incubator for 48 h; (45) 48-72 h after transfection, collect the cell supernatant, centrifuge at 1500 rpm for 5 min, filter with a 0.22 µm filter membrane, add the filtrate to the filter cup, seal it, place the filter cup in the filtrate collection tube, and centrifuge at 4000 g for 15 min; separate the filter cup and the filtrate collection tube, invert the filter cup on the sample collection cup, centrifuge at 4°C and 1000 g for 2 min, and the sample collection cup contains the lentiviral concentrate, which is titered by fluorescence counting.

[0021] (5) Lentiviral infection of primary T lymphocytes: (51) Using the GeneCare Easy-T kit, Stimu-Buffer was added to 1× PBS at a ratio of 1:500 and mixed to obtain a mixed solution; 200 μL of the mixed solution was added to each well of a 24-well plate; the culture plate was tapped to make the mixed solution evenly cover the bottom of the culture plate, and then the plate was wrapped with sealing film on all sides and placed at 4°C; (52) After the culture plate in step (51) was placed for 24 h, the Stimu-Buffer solution was removed before use, and the plate was washed once with 500 μL / well PBS. The activated T cells cultured in step (23) were centrifuged at 300 g for 3 min, and the cells were resuspended in RPMI 1640 medium containing 10% FBS, 100-600 IU / mL IL-2, 100 U / mL penicillin, and 100 μg / mL streptomycin, and the concentration was adjusted to 0.7×10 6 cells / mL, added to the culture plate washed with PBS, and placed in a 37°C, CO2 incubator for 24 h; (53) Add TA at a ratio of 1:40 and follow the steps in step (51) to coat the culture dish before infection and place at 4°C for 24 h. Gently pipette the cells after culture in step (52), collect the cells into a 1.5 mL centrifuge tube, centrifuge at 300 g for 3 min, and resuspend the cells to 0.7 × 10 6 / mL, according to the formula virus amount = cell density × MOI / TU, add the corresponding volume of the lentiviral concentrate obtained in step (45) to obtain a cell-virus mixture; take out the TA-coated culture dish, wash the plate once with 500 μL / well PBS, add 400 μL of the cell-virus mixture, and culture at 37°C, 5% CO2; (54) After 24 h of culture in step (53), each well was gently refilled with the same volume of RPMI 1640 medium containing 10% FBS, 100-600 IU / mL IL-2, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cells were cultured at 37°C and 5% CO2 for 48 h. The cells were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and fresh RPMI1640 containing IL-2 (100-600 IU / ml) was added to resuspend the cells and transfer them for expansion culture. When the culture medium turned yellow or the cell density exceeded 2×10 6 When the cell density was 0.7-2×10 / mL, the cells were centrifuged at 300 g for 5 min to replace the medium and culture with fresh RPMI 1640 medium containing IL-2 (100-600 IU / ml) to maintain the cell density at 0.7-2×10 6 / mL, and obtained CAR-T cells targeting CD248.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses CD248 as a target for myocardial infarction fibrosis intervention, which can not only effectively inhibit excessive cardiac fibrosis, but also has high safety. The present invention prepares antibodies and CAR-T cells against CD248; wherein, the biotinylated anti-CD248 engineered antibody has a good affinity for CD248 protein; and CAR-T cells have good infiltration into the myocardial infarction injury area, which can effectively reduce the number of CD248+ fibroblasts in the ischemic area. Therefore, the biotinylated anti-CD248 engineered antibody and CAR-T cells of the present invention can significantly reduce cardiac interstitial fibrosis and improve cardiac contractile function. The present invention targets CD248 to design biotin-binding immune receptor T cells (BBIR-T), and targets the removal of abnormally activated F-Act cells in the cardiac interstitium, which can effectively inhibit myocardial fibrosis and improve cardiac function.

[0023] Sequence Listing: <SEQ ID NO.1, AA> : ; <SEQ ID NO.2, DNA> : <SEQ ID NO.3, AA> : MALPVTALLLPLALLLHAARP; <SEQ ID NO.4, DNA> : ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG; <SEQ ID NO.5, AA> : MVHATSPLLLLLLLSLALVAPGLSARKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEGGSGGSARKCSLTGKWTNDLGSNMTIGAVNSRGEFTGTYITAVTATSNEIKE SPLHGTQNTINKSGGSTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEGGSGGSARKCSLTGKWTNDLGSNMTIGAVNSRGEFTGTYITAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSE; <SEQ ID NO.6, DNA> : ATGGTGCACGCAACCTCCCCGCTGCTGCTGCTGCTGCTGCTCAGCCTGGCTCTGGTGGCTCCCGGCCTCTCTGCCAGGAAGAGGACCCAGCCCACCTTTGGCTTCACCGTCAATTGGAAGTTTTCAGAGTCCACCACTGTCTTCACGGGCCAGTGCTTCATAGACAGGAATGGGAAGGAGGTCCTGAAGACCATGTGGCTGCTGCGGTCAAGTGTTAATGACATTGGTGATGACTGGAAAGCTACCAGGGTCGGCATCAACATCTTCACTCGCCTGCGCACACAGAAGGAGGGAGGCTCCGGAGGCTCCGCCAGAAAGTGCTCGCTGACTGGGAAATGGACCAACGATCTGGGCTCCAACATGACCATCGGGGCTGTGAACAGCAGAGGTGAATTTACAGGCACCTACATCACAGCCGTAACAGCCACATCAAATGAGATCAAAGAGTCACCACTGCATGGGACACAAAACACCATCAACAAGTCCGGCGGATCCACCACTGTCTTCACGGGCCAGTGCTTCATAGACAGGAATGGGAAGGAGGTCCTGAAGACCATGTGGCTGCTGCGGTCAAGTGTTAATGACATTGGTGATGACTGGAAAGCTACCAGGGTCGGCATCAACATCTTCACTCGCCTGCGCACACAGAAGGAGGGAGGCTCCGGAGGCTCCGCCAGAAAGTGCTCGCTGACTGGGAAATGGACCAACGATCTGGGCTCCAACATGACCATCGGGGCTGTGAACAGCAGAGGTGAATTTACAGGCACCTACATCACAGCCGTAACAGCCACATCAAATGAGATCAAAGAGTCACCACTGCATGGGACACAAAACACCATCAACAAGAGGACCCAGCCCACCTTTGGCTTCACCGTCAATTGGAAGTTTTCAGAG <SEQ ID NO.7, AA>: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC; <SEQ ID NO.8, DNA>: ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC; <SEQ ID NO.9, AA>: FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS; <SEQ ID NO.10, DNA>: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC; <SEQ ID NO.11, AA>: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL; <SEQ ID NO.12, DNA>: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG <SEQ ID NO.13, AA>: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR; <SEQ ID NO.14, DNA>: AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC; <SEQ ID NO.15, AA>: EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLNLVATINSNGGNTYYPDSVKGRFTISRDNAKNTLYLLMSSLKSEDTAMYYCAREVYRYIDYWGRGTTLTVSS; <SEQ ID NO.16, DNA>: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGGCATGTCTTGGGTTCGCCAGACTCCAGACAAGAGGCTGAACTTGGTCGCAACCATTAATAGTAATGGTGGTAACACCTATTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCTAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGAGAAGTTTACCGATACATTGACTACTGGGGCCGAGGCACCACTCTCACAGTCTCCTCA; <SEQ ID NO.17, AA>: DIVMSQSPSSLAVSVGEKTSMSCKSSQSLLYTTNQKNYLAWYRQKPGQSPELLIYWASTRESGVPDRFTGSGSGTDFTLTITSVKAEDLAVYYCQQYYNYPPTFGGGTKLEIKR; <SEQ ID NO.18, DNA>: GACATTGTGATGTCCCAGTCCCCATCCTCCCTAGCTGTTTCAGTTGGAGAGAAGACTTCTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATACTACCAATCAGAAGAACTACTTGGCCTGGTACCGGCAGAAACCAGGGCAGTCTCCTGAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCACCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAACAATATTATAACTATCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAGATCAAACGG。 Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 The present invention shows the expression of CD248 after 1-4 injections of CAR-T cells targeting CD248 into the tail vein; Figure 2 The results of the effects of the CAR-T cells targeting CD248 on cardiac contractile dysfunction in mice; Figure 3 The results of the effects of the CAR-T cells targeting CD248 on the cardiac contraction and relaxation functions of mice; Figure 4 Eliminate CD248 for the CAR-T cells targeting CD248 of the present invention + Cellular effects; Figure 5 HE staining and Sirius red staining of the heart of the myocardial infarction mouse of the present invention; Figure 6 This is the result diagram of myocardial infarction area in mice of the present invention; Figure 7 The results of BNP, HW / BW, HW / TL and Lung Wet / Dry Ratio of myocardial infarction mice of the present invention are shown in FIG. Figure 8 This is a WGA staining picture of the heart of the myocardial infarction mouse of the present invention; Fig. 9 This is the result of the cross-sectional area of ​​myocardial fibers in mice with myocardial infarction according to the present invention. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments. It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention are purchased through commercial channels.

[0027] Example 1: A CAR-T cell targeting CD248 is a biotin-binding immune receptor T cell targeting CD248. The CAR comprises CD8α, a biotin recognition domain, CD8 TM, CD28, CD137, and CD3ζ from N-terminus to C-terminus, and its amino acid sequence is shown in SEQ ID NO.1, and its encoded DNA sequence is shown in SEQ ID NO.2.

[0028] The CD8α includes the amino acid sequence shown in SEQ ID NO.3, and the DNA sequence encoded by it is shown in SEQ ID NO.4; the biotin recognition domain includes the amino acid sequence shown in SEQ ID NO.5, and the DNA sequence encoded by it is shown in SEQ ID NO.6; the CD8 TM includes the amino acid sequence shown in SEQ ID NO.7, and the DNA sequence encoded by it is shown in SEQ ID NO.8; the CD28 includes the amino acid sequence shown in SEQ ID NO.9, and the DNA sequence encoded by it is shown in SEQ ID NO.10; the CD137 includes the amino acid sequence shown in SEQ ID NO.11, and the DNA sequence encoded by it is shown in SEQ ID NO.12; the CD3ζ includes the amino acid sequence shown in SEQ ID NO.13, and the DNA sequence encoded by it is shown in SEQ ID NO.14.

[0029] This embodiment also provides a biotinylated anti-CD248 engineered antibody targeting CD248, wherein the heavy chain of the CD248 engineered antibody includes the amino acid sequence shown in SEQ ID NO.15, and the DNA sequence encoded by it is shown in SEQ ID NO.16; the light chain of the CD248 engineered antibody includes the amino acid sequence shown in SEQ ID NO.17, and the DNA sequence encoded by it is shown in SEQ ID NO.18.

[0030] The method for preparing the biotinylated anti-CD248 engineered antibody comprises the following steps: (a) 1 mL CD248 antibody (1 mg / mL) was dialyzed in 0.2 M sodium acetate buffer (pH 4.0) for 24 h, mixed with pepsin (1 mg / mL), pepsin:CD248 antibody = 1:20 (w:w), incubated in a 37°C water bath for 6 h, added with 250 μL 3M Tris-HCl (pH 9.0) to adjust the pH to 8.0, and dialyzed in 1× PBS (pH 8.0) at 4°C for 24 h to obtain a CD248 antibody digestion sample; (b) 700 μL of the CD248 antibody digestion sample described in step (a) was loaded onto a Sephadex G-100 chromatography column (column length 20 cm), and the flow-through was collected in separate tubes. The CD248 flow-through was mixed and concentrated to a final volume of 450 μL and a concentration of 0.2 mg / mL to obtain the CD248 antibody F(ab')2 fragment; (c) Using ThermoFisher EZ-Link TMThe CD248 antibody F(ab')2 fragment was biotin-labeled using the sulfo-NHS-LC-Biotinylation Kit and dialyzed against 1×PBS (pH 8.0) at 4°C for 24 h to obtain the biotinylated anti-CD248 engineered antibody.

[0031] This embodiment also provides a method for preparing the CD248-targeted CAR-T cells, comprising the following steps: (1) Preparation of peripheral blood mononuclear cells (PBMC): (11) Collect 10 mL of peripheral blood, mix thoroughly, transfer to two 50 mL centrifuge tubes, and centrifuge at 2000 rpm for 10 min to obtain the upper plasma layer and the lower blood cell sediment; (12) Resuspend the blood cell precipitate obtained in step (11) with 10 mL of physiological saline, and use a Pasteur pipette to fully disperse the cell clumps to obtain diluted blood. Take two new 50 mL centrifuge tubes, add 10 mL of lymphocyte separation solution to each tube, and slowly add the diluted blood along the tube wall to the upper layer of the separation solution. Centrifuge at 2000 rpm for 30 min; (13) After centrifugation in step (12), gently aspirate the middle white film layer with a Pasteur pipette, collect the mixture into a new 50 mL centrifuge tube, add physiological saline to 45 mL, mix thoroughly by inverting, centrifuge at 2000 rpm for 8 min, discard the supernatant, and obtain the cell pellet; (14) Add 45 mL of saline to the cell pellet of step (13) to resuspend the cells, centrifuge at 1500 rpm for 6 min; discard the supernatant, add 45 mL of saline to resuspend the cells, centrifuge at 1000 rpm for 6 min, discard the supernatant, and obtain peripheral blood mononuclear cells.

[0032] (2) T cell activation: (21) One day before PBMC extraction, dilute the anti-CD3 / CD28 antibody to 1 μg / mL with sterile PBS, add the diluted antibody to a hydrophobic tissue culture 24-well plate at 1 mL / well, seal with sealing film, and incubate at 4°C for 24 h to obtain a pre-coated 24-well plate; (22) Take out the 24-well plate pre-coated in step (21), discard the PBS containing the diluted antibody, and dry it in a clean bench; resuspend the peripheral blood mononuclear cells obtained in step (14) in RPMI-1640 medium and dilute to 10 6 cells / mL, and the diluted cells were added to the pre-coated 24-well plate, 1 mL / well, and cultured at 37°C and 5% CO2; (23) After culturing for 24 h in step (22), cytokines IL-2 100 IU / mL, IL-7 10 ng / mL, and IL-15 10 ng / mL were added and cultured for 24 h at 37°C and 5% CO2 to obtain activated T cells.

[0033] (3) Lentiviral vector construction: (31) CAR sequence synthesis and lentiviral vector plasmid preparation were completed by Nanjing Novogene Biotechnology Co., Ltd., and the CAR gene sequence was inserted between the EcoRI and BamH1 restriction sites in the plasmid pCDH-EF1-MCS.

[0034] (4) Lentivirus packaging: (41) 293T cells were used as lentiviral packaging cells. The culture medium was high-glucose DMEM medium containing 10% FBS. 293T cells were passaged 24 h before the start of virus packaging. The cell density was 5 × 10 5 / mL, inoculated in a 100 mm cell culture dish with an inoculation volume of 12 mL, cultured at 37°C, 5% CO2 until the cell confluence rate reached 80%, and the cell culture medium was changed to Opti-MEM medium 2 h before transfection; (42) The lentiviral packaging system of Shanghai GeneCare was used for packaging. According to the instructions of the kit, the lentiviral vector plasmid, Lenti-Easy Packing Mix and Opti-MEM were mixed evenly at a dosage ratio of 16 μg:25 μL:1455 μL, and incubated at room temperature for 5 min to obtain the diluted packaging system; 60 μL of Lipofectamine 2000 reagent was mixed evenly with 1440 μL of Opti-MEM, and incubated at room temperature for 5 min to obtain the diluted Lipofectamine2000; (43) Mix the diluted packaging system and diluted Lipofectamine 2000 in a 1:1 volume ratio, gently invert to mix, and incubate at room temperature for 20 min to form a DNA-Lipofectamine 2000 transfection complex; (44) Add the transfection complex described in step (43) to the culture medium of the 293T cells cultured in step (41), gently shake the culture dish to mix, and continue to culture in a 37°C, 5% CO2 cell culture incubator. 8 h after transfection, discard the culture medium containing the transfection complex, add 10 mL PBS to wash the cells, discard the PBS, add 12 mL of fresh high-glucose DMEM medium containing 10% FBS, and continue to culture in a 37°C, 5% CO2 cell culture incubator for 48 h; (45) 72 h after transfection, the cell supernatant was collected, centrifuged at 1500 rpm for 5 min, filtered through a 0.22 µm filter membrane, and the filtrate was added to a filter cup, sealed, and placed in a filtrate collection tube. The filter cup was centrifuged at 4000 g for 15 min. The filter cup and filtrate collection tube were separated, and the filter cup was inverted on the sample collection cup. The sample collection cup was centrifuged at 1000 g for 2 min at 4°C. The sample collection cup contained the lentiviral concentrate, and the titer was determined by fluorescence counting.

[0035] (5) Lentiviral infection of primary T lymphocytes: (51) Using the GeneCare Easy-T kit, Stimu-Buffer was added to 1× PBS at a ratio of 1:500 and mixed to obtain a mixed solution; 200 μL of the mixed solution was added to each well of a 24-well plate; the culture plate was tapped to make the mixed solution evenly cover the bottom of the culture plate, and then the plate was wrapped with sealing film on all sides and placed at 4°C; (52) After the culture plate in step (51) was placed for 24 h, the Stimu-Buffer solution was removed before use, and the plate was washed once with 500 μL / well PBS. The activated T cells cultured in step (23) were centrifuged at 300 g for 3 min, and the cells were resuspended in RPMI 1640 medium containing 10% FBS, 600 IU / mL IL-2, 100 U / mL penicillin, and 100 μg / mL streptomycin, and the concentration was adjusted to 0.7×10 6 cells / mL, added to the culture plate washed with PBS, and placed in a 37°C, CO2 incubator for 24 h; (53) Add TA at a ratio of 1:40 and follow the steps in step (51) to coat the culture dish before infection and place at 4°C for 24 h. Gently pipette the cells after culture in step (52), collect the cells into a 1.5 mL centrifuge tube, centrifuge at 300 g for 3 min, and resuspend the cells to 0.7 × 10 6 / mL, according to the formula virus amount = cell density × MOI / TU, add the corresponding volume of the lentiviral concentrate obtained in step (45) to obtain a cell-virus mixture; take out the TA-coated culture dish, wash the plate once with 500 μL / well PBS, add 400 μL of the cell-virus mixture, and culture at 37°C, 5% CO2; (54) After 24 h of culture in step (53), each well was gently supplemented with the same volume of RPMI 1640 medium containing 10% FBS, 600 IU / mL IL-2, 100 U / mL penicillin and 100 μg / mL streptomycin, and culture was continued at 37°C and 5% CO2. After 48 h, the cells were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and fresh RPMI 1640 medium containing IL-2 (600 IU / ml) was added to resuspend the cells, and the cells were transferred for expansion culture. After that, when the medium turned yellow, the cells were centrifuged at 300 g for 5 min to replace the medium for expansion culture, and the cells were cultured with fresh RPMI 1640 medium containing IL-2 (600 IU / ml) to maintain the cell density at 2×10 6 / mL, expanded culture for 12 days, and CAR-T cells targeting CD248 were obtained.

[0036] Example 2: A CAR-T cell targeting CD248, which is a biotin-binding immune receptor T cell targeting CD248. The CAR comprises CD8α, a biotin recognition domain, CD8 TM, CD28, CD137, and CD3ζ from N-terminus to C-terminus, and its amino acid sequence is shown in SEQ ID NO.1, and its encoded DNA sequence is shown in SEQ ID NO.2.

[0037] The CD8α includes the amino acid sequence shown in SEQ ID NO.3, and the DNA sequence encoded by it is shown in SEQ ID NO.4; the biotin recognition domain includes the amino acid sequence shown in SEQ ID NO.5, and the DNA sequence encoded by it is shown in SEQ ID NO.6; the CD8 TM includes the amino acid sequence shown in SEQ ID NO.7, and the DNA sequence encoded by it is shown in SEQ ID NO.8; the CD28 includes the amino acid sequence shown in SEQ ID NO.9, and the DNA sequence encoded by it is shown in SEQ ID NO.10; the CD137 includes the amino acid sequence shown in SEQ ID NO.11, and the DNA sequence encoded by it is shown in SEQ ID NO.12; the CD3ζ includes the amino acid sequence shown in SEQ ID NO.13, and the DNA sequence encoded by it is shown in SEQ ID NO.14.

[0038] This embodiment also provides a biotinylated anti-CD248 engineered antibody targeting CD248, wherein the heavy chain of the CD248 engineered antibody includes the amino acid sequence shown in SEQ ID NO.15, and the DNA sequence encoded by it is shown in SEQ ID NO.16; the light chain of the CD248 engineered antibody includes the amino acid sequence shown in SEQ ID NO.17, and the DNA sequence encoded by it is shown in SEQ ID NO.18.

[0039] The method for preparing the biotinylated anti-CD248 engineered antibody comprises the following steps: (a) 1 mL CD248 antibody (1 mg / mL) was dialyzed in 0.2 M sodium acetate buffer (pH 4.0) for 24 h, mixed with pepsin (1 mg / mL), pepsin:CD248 antibody = 1:20 (w:w), incubated in a 37°C water bath for 6 h, added with 250 μL 3M Tris-HCl (pH 9.0) to adjust the pH to 8.0, and dialyzed in 1× PBS (pH 8.0) at 4°C for 24 h to obtain a CD248 antibody digestion sample; (b) 700 μL of the CD248 antibody digestion sample described in step (a) was loaded onto a Sephadex G-100 chromatography column (column length 20 cm), and the flow-through was collected in separate tubes. The CD248 flow-through was mixed and concentrated to a final volume of 450 μL and a concentration of 0.2 mg / mL to obtain the CD248 antibody F(ab')2 fragment; (c) Using ThermoFisher EZ-Link TM The CD248 antibody F(ab')2 fragment was biotin-labeled using the sulfo-NHS-LC-Biotinylation Kit and dialyzed against 1×PBS (pH 8.0) at 4°C for 24 h to obtain the biotinylated anti-CD248 engineered antibody.

[0040] This embodiment also provides a method for preparing the CD248-targeted CAR-T cells, comprising the following steps: (1) Preparation of peripheral blood mononuclear cells (PBMC): (11) Collect 10 mL of peripheral blood, mix thoroughly, transfer to two 50 mL centrifuge tubes, and centrifuge at 2000 rpm for 10 min to obtain the upper plasma layer and the lower blood cell sediment; (12) Resuspend the blood cell precipitate obtained in step (11) with 10 mL of physiological saline, and use a Pasteur pipette to fully disperse the cell clumps to obtain diluted blood. Take two new 50 mL centrifuge tubes, add 10 mL of lymphocyte separation solution to each tube, and slowly add the diluted blood along the tube wall to the upper layer of the separation solution. Centrifuge at 2000 rpm for 30 min; (13) After centrifugation in step (12), gently aspirate the middle white film layer with a Pasteur pipette, collect the mixture into a new 50 mL centrifuge tube, add physiological saline to 45 mL, mix thoroughly by inverting, centrifuge at 2000 rpm for 8 min, discard the supernatant, and obtain the cell pellet; (14) Add 45 mL of saline to the cell pellet of step (13) to resuspend the cells, centrifuge at 1500 rpm for 6 min; discard the supernatant, add 45 mL of saline to resuspend the cells, centrifuge at 1000 rpm for 6 min, discard the supernatant, and obtain peripheral blood mononuclear cells.

[0041] (2) T cell activation: (21) One day before PBMC extraction, dilute the anti-CD3 / CD28 antibody to 1 μg / mL with sterile PBS, add the diluted antibody to a hydrophobic tissue culture 24-well plate at 1 mL / well, seal with sealing film, and incubate at 4°C for 24 h to obtain a pre-coated 24-well plate; (22) Take out the 24-well plate pre-coated in step (21), discard the PBS containing the diluted antibody, and dry it in a clean bench; resuspend the peripheral blood mononuclear cells obtained in step (14) in RPMI-1640 medium and dilute to 10 6 cells / mL, and the diluted cells were added to the pre-coated 24-well plate, 1 mL / well, and cultured at 37°C and 5% CO2; (23) After culturing for 24 h in step (22), cytokines IL-2 100 IU / mL, IL-7 10 ng / mL, and IL-15 10 ng / mL were added and cultured for 24 h at 37°C and 5% CO2 to obtain activated T cells.

[0042] (3) Lentiviral vector construction: (31) CAR sequence synthesis and lentiviral vector plasmid preparation were completed by Nanjing Novogene Biotechnology Co., Ltd., and the CAR gene sequence was inserted between the EcoRI and BamH1 restriction sites in the plasmid pCDH-EF1-MCS.

[0043] (4) Lentivirus packaging: (41) 293T cells were used as lentiviral packaging cells. The culture medium was high-glucose DMEM medium containing 10% FBS. 293T cells were passaged 24 h before the start of virus packaging. The cell density was 5 × 10 5 / mL, inoculated in a 100 mm cell culture dish with an inoculation volume of 12 mL, cultured at 37°C, 5% CO2 until the cell confluence rate reached 80%, and the cell culture medium was changed to Opti-MEM medium 2 h before transfection; (42) The lentiviral packaging system of Shanghai GeneCare was used for packaging. According to the instructions of the kit, the lentiviral vector plasmid, Lenti-Easy Packing Mix and Opti-MEM were mixed evenly at a dosage ratio of 16 μg:25 μL:1455 μL, and incubated at room temperature for 5 min to obtain the diluted packaging system; 60 μL of Lipofectamine 2000 reagent was mixed evenly with 1440 μL of Opti-MEM, and incubated at room temperature for 5 min to obtain the diluted Lipofectamine2000; (43) Mix the diluted packaging system and diluted Lipofectamine 2000 in a 1:1 volume ratio, gently invert to mix, and incubate at room temperature for 20 min to form a DNA-Lipofectamine 2000 transfection complex; (44) Add the transfection complex described in step (43) to the culture medium of the 293T cells cultured in step (41), gently shake the culture dish to mix, and continue to culture in a 37°C, 5% CO2 cell culture incubator. 8 h after transfection, discard the culture medium containing the transfection complex, add 10 mL PBS to wash the cells, discard the PBS, add 12 mL of fresh high-glucose DMEM medium containing 10% FBS, and continue to culture in a 37°C, 5% CO2 cell culture incubator for 48 h; (45) 48 h after transfection, the cell supernatant was collected, centrifuged at 1500 rpm for 5 min, filtered through a 0.22 µm filter membrane, and the filtrate was added to a filter cup, sealed, and placed in a filtrate collection tube. The filter cup was centrifuged at 4000 g for 15 min. The filter cup and filtrate collection tube were separated, and the filter cup was inverted on the sample collection cup. The sample collection cup was centrifuged at 1000 g for 2 min at 4°C. The sample collection cup contained the lentiviral concentrate, and the titer was determined by fluorescence counting.

[0044] (5) Lentiviral infection of primary T lymphocytes: (51) Using the GeneCare Easy-T kit, Stimu-Buffer was added to 1× PBS at a ratio of 1:500 and mixed to obtain a mixed solution; 200 μL of the mixed solution was added to each well of a 24-well plate; the culture plate was tapped to make the mixed solution evenly cover the bottom of the culture plate, and then the plate was wrapped with sealing film on all sides and placed at 4°C; (52) After the culture plate in step (51) was placed for 24 h, the Stimu-Buffer solution was removed before use, and the plate was washed once with 500 μL / well PBS. The activated T cells cultured in step (23) were centrifuged at 300 g for 3 min, and the cells were resuspended in RPMI 1640 medium containing 10% FBS, 100 IU / mL IL-2, 100 U / mL penicillin, and 100 μg / mL streptomycin, and the concentration was adjusted to 0.7×10 6 cells / mL, added to the culture plate washed with PBS, and placed in a 37°C, CO2 incubator for 24 h; (53) Add TA at a ratio of 1:40 and follow the steps in step (51) to coat the culture dish before infection and place at 4°C for 24 h. Gently pipette the cells after culture in step (52), collect the cells into a 1.5 mL centrifuge tube, centrifuge at 300 g for 3 min, and resuspend the cells to 0.7 × 10 6 / mL, according to the formula virus amount = cell density × MOI / TU, add the corresponding volume of the lentiviral concentrate obtained in step (45) to obtain a cell-virus mixture; take out the TA-coated culture dish, wash the plate once with 500 μL / well PBS, add 400 μL of the cell-virus mixture, and culture at 37°C, 5% CO2; (54) After 24 h of culture in step (53), each well was gently supplemented with the same volume of RPMI 1640 medium containing 10% FBS, 100 IU / mL IL-2, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cells were cultured at 37°C and 5% CO2 for 48 h. The cells were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and fresh RPMI 1640 medium containing IL-2 (100 IU / ml) was added to resuspend the cells and transfer them for expansion culture. When the cell density in the culture medium exceeded 2×10 6 / mL, the cells were centrifuged at 300 g for 5 min, the medium was changed and the cells were expanded and cultured with fresh RPMI 1640 medium containing IL-2 (100 IU / ml) to maintain the cell density at 0.7×10 6 / mL, expanded culture for 12 days, and CAR-T cells targeting CD248 were obtained.

[0045] Example 3: A CAR-T cell targeting CD248, which is a biotin-binding immune receptor T cell targeting CD248. The CAR comprises CD8α, a biotin recognition domain, CD8 TM, CD28, CD137, and CD3ζ from N-terminus to C-terminus, and its amino acid sequence is shown in SEQ ID NO.1, and its encoded DNA sequence is shown in SEQ ID NO.2.

[0046] The CD8α includes the amino acid sequence shown in SEQ ID NO.3, and the DNA sequence encoded by it is shown in SEQ ID NO.4; the biotin recognition domain includes the amino acid sequence shown in SEQ ID NO.5, and the DNA sequence encoded by it is shown in SEQ ID NO.6; the CD8 TM includes the amino acid sequence shown in SEQ ID NO.7, and the DNA sequence encoded by it is shown in SEQ ID NO.8; the CD28 includes the amino acid sequence shown in SEQ ID NO.9, and the DNA sequence encoded by it is shown in SEQ ID NO.10; the CD137 includes the amino acid sequence shown in SEQ ID NO.11, and the DNA sequence encoded by it is shown in SEQ ID NO.12; the CD3ζ includes the amino acid sequence shown in SEQ ID NO.13, and the DNA sequence encoded by it is shown in SEQ ID NO.14.

[0047] This embodiment also provides a biotinylated anti-CD248 engineered antibody targeting CD248, wherein the heavy chain of the CD248 engineered antibody includes the amino acid sequence shown in SEQ ID NO.15, and the DNA sequence encoded by it is shown in SEQ ID NO.16; the light chain of the CD248 engineered antibody includes the amino acid sequence shown in SEQ ID NO.17, and the DNA sequence encoded by it is shown in SEQ ID NO.18.

[0048] The method for preparing the biotinylated anti-CD248 engineered antibody comprises the following steps: (a) 1 mL CD248 antibody (1 mg / mL) was dialyzed in 0.2 M sodium acetate buffer (pH 4.0) for 24 h, mixed with pepsin (1 mg / mL), pepsin:CD248 antibody = 1:20 (w:w), incubated in a 37°C water bath for 6 h, added with 250 μL 3M Tris-HCl (pH 9.0) to adjust the pH to 8.0, and dialyzed in 1× PBS (pH 8.0) at 4°C for 24 h to obtain a CD248 antibody digestion sample; (b) 700 μL of the CD248 antibody digestion sample described in step (a) was loaded onto a Sephadex G-100 chromatography column (column length 20 cm), and the flow-through was collected in separate tubes. The CD248 flow-through was mixed and concentrated to a final volume of 450 μL and a concentration of 0.2 mg / mL to obtain the CD248 antibody F(ab')2 fragment; (c) Using ThermoFisher EZ-Link TM The CD248 antibody F(ab')2 fragment was biotin-labeled using the sulfo-NHS-LC-Biotinylation Kit and dialyzed against 1×PBS (pH 8.0) at 4°C for 24 h to obtain the biotinylated anti-CD248 engineered antibody.

[0049] This embodiment also provides a method for preparing the CD248-targeted CAR-T cells, comprising the following steps: (1) Preparation of peripheral blood mononuclear cells (PBMC): (11) Collect 10 mL of peripheral blood, mix thoroughly, transfer to two 50 mL centrifuge tubes, and centrifuge at 2000 rpm for 10 min to obtain the upper plasma layer and the lower blood cell sediment; (12) Resuspend the blood cell precipitate obtained in step (11) with 10 mL of physiological saline, and use a Pasteur pipette to fully disperse the cell clumps to obtain diluted blood. Take two new 50 mL centrifuge tubes, add 10 mL of lymphocyte separation solution to each tube, and slowly add the diluted blood along the tube wall to the upper layer of the separation solution. Centrifuge at 2000 rpm for 30 min; (13) After centrifugation in step (12), gently aspirate the middle white film layer with a Pasteur pipette, collect the mixture into a new 50 mL centrifuge tube, add physiological saline to 45 mL, mix thoroughly by inverting, centrifuge at 2000 rpm for 8 min, discard the supernatant, and obtain the cell pellet; (14) Add 45 mL of saline to the cell pellet of step (13) to resuspend the cells, centrifuge at 1500 rpm for 6 min; discard the supernatant, add 45 mL of saline to resuspend the cells, centrifuge at 1000 rpm for 6 min, discard the supernatant, and obtain peripheral blood mononuclear cells.

[0050] (2) T cell activation: (21) One day before PBMC extraction, dilute the anti-CD3 / CD28 antibody to 1 μg / mL with sterile PBS, add the diluted antibody to a hydrophobic tissue culture 24-well plate at 1 mL / well, seal with sealing film, and incubate at 4°C for 24 h to obtain a pre-coated 24-well plate; (22) Take out the 24-well plate pre-coated in step (21), discard the PBS containing the diluted antibody, and dry it in a clean bench; resuspend the peripheral blood mononuclear cells obtained in step (14) in RPMI-1640 medium and dilute to 10 6 cells / mL, and the diluted cells were added to the pre-coated 24-well plate, 1 mL / well, and cultured at 37°C and 5% CO2; (23) After culturing for 24 h in step (22), cytokines IL-2 100 IU / mL, IL-7 10 ng / mL, and IL-15 10 ng / mL were added and cultured for 24 h at 37°C and 5% CO2 to obtain activated T cells.

[0051] (3) Lentiviral vector construction: (31) CAR sequence synthesis and lentiviral vector plasmid preparation were completed by Nanjing Novogene Biotechnology Co., Ltd., and the CAR gene sequence was inserted between the EcoRI and BamH1 restriction sites in the plasmid pCDH-EF1-MCS.

[0052] (4) Lentivirus packaging: (41) 293T cells were used as lentiviral packaging cells. The culture medium was high-glucose DMEM medium containing 10% FBS. 293T cells were passaged 24 h before the start of virus packaging. The cell density was 5 × 10 5 / mL, inoculated in a 100 mm cell culture dish with an inoculation volume of 12 mL, cultured at 37°C, 5% CO2 until the cell confluence rate reached 80%, and the cell culture medium was changed to Opti-MEM medium 2 h before transfection; (42) The lentiviral packaging system of Shanghai GeneCare was used for packaging. According to the instructions of the kit, the lentiviral vector plasmid, Lenti-Easy Packing Mix and Opti-MEM were mixed evenly at a dosage ratio of 16 μg:25 μL:1455 μL, and incubated at room temperature for 5 min to obtain the diluted packaging system; 60 μL of Lipofectamine 2000 reagent was mixed evenly with 1440 μL of Opti-MEM, and incubated at room temperature for 5 min to obtain the diluted Lipofectamine2000; (43) Mix the diluted packaging system and diluted Lipofectamine 2000 in a 1:1 volume ratio, gently invert to mix, and incubate at room temperature for 20 min to form a DNA-Lipofectamine 2000 transfection complex; (44) Add the transfection complex described in step (43) to the culture medium of the 293T cells cultured in step (41), gently shake the culture dish to mix, and continue to culture in a 37°C, 5% CO2 cell culture incubator. 8 h after transfection, discard the culture medium containing the transfection complex, add 10 mL PBS to wash the cells, discard the PBS, add 12 mL of fresh high-glucose DMEM medium containing 10% FBS, and continue to culture in a 37°C, 5% CO2 cell culture incubator for 48 h; (45) 60 h after transfection, the cell supernatant was collected, centrifuged at 1500 rpm for 5 min, filtered through a 0.22 µm filter membrane, and the filtrate was added to a filter cup, sealed, and placed in a filtrate collection tube. The filter cup was centrifuged at 4000 g for 15 min. The filter cup and filtrate collection tube were separated, and the filter cup was inverted on the sample collection cup. The sample collection cup was centrifuged at 1000 g for 2 min at 4°C. The sample collection cup contained the lentiviral concentrate, and the titer was determined by fluorescence counting.

[0053] (5) Lentiviral infection of primary T lymphocytes: (51) Using the GeneCare Easy-T kit, Stimu-Buffer was added to 1× PBS at a ratio of 1:500 and mixed to obtain a mixed solution; 200 μL of the mixed solution was added to each well of a 24-well plate; the culture plate was tapped to make the mixed solution evenly cover the bottom of the culture plate, and then the plate was wrapped with sealing film on all sides and placed at 4°C; (52) After the culture plate in step (51) was placed for 24 h, the Stimu-Buffer solution was removed before use, and the plate was washed once with 500 μL / well PBS. The activated T cells cultured in step (23) were centrifuged at 300 g for 3 min, and the cells were resuspended in RPMI 1640 medium containing 10% FBS, 200 IU / mL IL-2, 100 U / mL penicillin, and 100 μg / mL streptomycin, and the concentration was adjusted to 0.7×10 6 cells / mL, added to the culture plate washed with PBS, and placed in a 37°C, CO2 incubator for 24 h; (53) Add TA at a ratio of 1:40 and follow the steps in step (51) to coat the culture dish before infection and place at 4°C for 24 h. Gently pipette the cells after culture in step (52), collect the cells into a 1.5 mL centrifuge tube, centrifuge at 300 g for 3 min, and resuspend the cells to 0.7 × 10 6 / mL, according to the formula virus amount = cell density × MOI / TU, add the corresponding volume of the lentiviral concentrate obtained in step (45) to obtain a cell-virus mixture; take out the TA-coated culture dish, wash the plate once with 500 μL / well PBS, add 400 μL of the cell-virus mixture, and culture at 37°C, 5% CO2; (54) After 24 h of culture in step (53), each well was gently supplemented with the same volume of RPMI 1640 medium containing 10% FBS, 200 IU / mL IL-2, 100 U / mL penicillin and 100 μg / mL streptomycin, and culture was continued at 37°C and 5% CO2. After 48 h, the cells were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and fresh RPMI 1640 medium containing IL-2 (200 IU / ml) was added to resuspend the cells, and the cells were transferred for expansion culture. After that, when the culture medium turned yellow, the cells were centrifuged at 300 g for 5 min to replace the medium for expansion culture, and cultured with fresh RPMI 1640 medium containing IL-2 (200 IU / ml) to maintain the cell density at 1×10 6 / mL, expanded culture for 12 days, and CAR-T cells targeting CD248 were obtained.

[0054] The only difference between the comparative example and Example 1 is that the CAR described in Example 1 is replaced by a CAR targeting CD19, and from the N-terminus to the C-terminus, CD8α, anti-CD19 (FMC63) scFv, CD8 TM, CD28, CD137, and CD3ζ are sequentially included to obtain anti-CD19 CAR-T cells.

[0055] Experimental Example 1: Constructing a mouse myocardial infarction (MI) model. The construction method is consistent with patent document CN202110041450.5. After MI modeling, biotinylated anti-CD248 engineered antibody (BF) was injected at a dose of 30 mg / kg. Starting from 14 days after MI, 10 BF was injected into the tail vein every 3 days. 6 The CAR-T cells targeting CD248 (BBIR-T) obtained in Example 1 were used to detect the CD248 in the myocardium by flow cytometry. + Cells, the results are as follows Figure 1 shown.

[0056] Figure 1 The results showed that biotinylated anti-CD248 engineered antibody (BF) can target CD248 and has a good affinity for CD248 protein; CAR-T cells can bind to BF and reduce the number of CD248+ fibroblasts. There was no significant difference between 3 and 4 injections of CAR-T cells, and it was significantly better than 1 and 2 injections. Therefore, the treatment plan of 3 injections was determined.

[0057] Experimental Example 2: MI+BF, MI+BF+Activated T, MI+BBIR-T, MI+BF+BBIR-T, and MI+BF+anti-CD19 CAR-T groups were set up. First, the mice were subjected to cardiac ultrasound (UCG). After myocardial infarction modeling, except for the MI+BBIR-T group, the mice were injected with biotinylated anti-CD248 engineered antibody (BF) at a dose of 30 mg / kg. Cardiac ultrasound was performed again 7 and 14 days after modeling. 14, 17, and 20 days after modeling, the MI+BF+Activated T, MI+BBIR-T, MI+BF+BBIR-T, and MI+BF+anti-CD19 CAR-T groups were injected with 10 6 Activated T (T cells not transfected with lentivirus after activation), Example 2 BBIR-T, Example 2 BBIR-T, anti-CD19 CAR-T, MI+BF group was injected with equal volume of normal saline, and cardiac ultrasound was performed again 28 days after modeling to record left ventricular ejection fraction (LVEF, %), left ventricular shortening fraction (LVFS, %), left ventricular end-systolic anterior wall thickness (LVAWs, mm) and left ventricular end-diastolic anterior wall thickness (LVAWd, mm). The results are shown in Figure 2 shown.

[0058] Figure 2 The results showed that BBIR-T treatment improved cardiac systolic dysfunction and left ventricular remodeling in mice 28 days after modeling, as manifested by significant upregulation of LVEF, LVFS, LVAWs and LVAWd, indicating that BF can target CD248, BBIR-T can bind to BF, and BBIR-T treatment can effectively delay the deterioration of cardiac function.

[0059] Experimental Example 3: The mice were grouped and treated according to the method of Experimental Example 2. The injected BBIR-T was obtained in Example 3. A sham operation group (Sham) was set up at the same time. The construction method of the sham operation group was consistent with the patent document CN202110041450.5. After 28 days of modeling, UCG and left heart catheterization were performed to measure left ventricular pressure (LVP, mmHg), left ventricular end-diastolic pressure (LVEDP, mmHg), maximum rate of change of left ventricular pressure (LV dP / dt maximum, mmHg / s), and maximum rate of change of left ventricular pressure (LV dP / dtminimum, mmHg / s). The results are as follows: Figure 3 shown.

[0060] Figure 3The results showed that compared with the sham operation group, the LVP and LV dP / dt maximum of the myocardial infarction model group decreased significantly, and the LVEDP and LV dP / dt minimum increased significantly. BBIR-T treatment can improve myocardial dysfunction, as shown by a significant increase in LV dP / dt maximum and a significant decrease in LVEDP and LV dP / dt minimum. This indicates that BBIR-T treatment can effectively improve the systolic and diastolic function of the mouse heart.

[0061] Experimental Example 4: The groups were treated according to the method of Experimental Example 3. The injected BBIR-T was obtained in Example 1. The proportion of CD248 positive cells in the myocardium of mice in each treatment group was detected by flow cytometry 28 days after modeling. The results were as follows: Figure 4 shown. Figure 4 The results showed that BBIR-T cell treatment could eliminate at least 60% of CD248-positive cells in the myocardium.

[0062] Experimental Example 5: The mice were divided into groups and injected with BBIR-T obtained in Example 2. The mouse hearts were stained with HE and Sirius red. The results were as follows: Figure 5 As shown in the figure, the left ventricular wall thickness (LV thickness, mm), myocardial infarction size (Infarct size, mm 2 ), the percentage of myocardial infarction area to the total left ventricular myocardial area (Infarct size / LV,%), the area of ​​myocardial infarction junction zone (PIZ size, mm 2 ), the percentage of myocardial infarction zone area to the total left ventricular myocardial area (PIZ size, mm 2 / LV, %. The results are as follows Figure 6 As shown, Figure 5-Figure 6 The results showed that CD248 CAR-T cell treatment could inhibit myocardial fibrosis, reduce infarct size, and suppress ventricular remodeling.

[0063] Experimental Example 6: The mice were divided into groups and treated. The BBIR-T injected was obtained in Example 3. The brain natriuretic peptide (BNP, ng / mL), heart weight to body weight percentage (HW / BW, %), heart weight to tibia length ratio (HW / TL, g / cm) and lung wet / dry ratio of the mice were measured. The results are as follows: Figure 7 As shown; WGA staining was performed, and the results were as shown Figure 8 The cross-sectional area of ​​myocardial fibers (CSA, μm 2 ), the result is as follows Fig. 9 shown.

[0064] Figure 7-Figure 9The results showed that BBIR-T cells could inhibit cardiomyocyte hypertrophy.

[0065] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0066] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A CAR-T cell targeting CD248, characterized in that: The invention comprises a chimeric antigen receptor CAR, wherein the CAR comprises CD8α, a biotin recognition domain, CD8 TM, CD28, CD137, and CD3ζ in sequence from the N-terminus to the C-terminus, and the amino acid sequence thereof is shown in SEQ ID NO.1, and the DNA sequence encoded therein is shown in SEQ ID NO.2; the CD8α comprises the amino acid sequence shown in SEQ ID NO.3, and the DNA sequence encoded therein is shown in SEQ ID NO.4; the biotin recognition domain comprises the amino acid sequence shown in SEQ ID NO.5, and the DNA sequence encoded therein is shown in SEQ ID NO.6; the CD8 TM comprises the amino acid sequence shown in SEQ ID NO.7, and the DNA sequence encoded therein is shown in SEQ ID NO.8; the CD28 comprises the amino acid sequence shown in SEQ ID NO.9, and the DNA sequence encoded therein is shown in SEQ ID NO.10; the CD137 comprises the amino acid sequence shown in SEQ ID NO.11, and the DNA sequence encoded therein is shown in SEQ ID NO.12; the CD3ζ comprises the amino acid sequence shown in SEQ ID NO.13, and the DNA sequence encoded therein is shown in SEQ ID Shown in NO.

14.

2. The CD248-targeting CAR-T cell according to claim 1, characterized in that: The CAR-T cells can be combined with biotinylated anti-CD248 engineered antibodies.

3. The CD248-targeting CAR-T cell according to claim 2, characterized in that: The heavy chain of the biotinylated anti-CD248 engineered antibody comprises the amino acid sequence shown in SEQ ID NO.15, and the DNA sequence encoded by the heavy chain is shown in SEQ ID NO.16; The light chain thereof comprises the amino acid sequence shown in SEQ ID NO.17, and the DNA sequence encoded by the light chain thereof is shown in SEQ ID NO.

18.

4. The CD248-targeting CAR-T cell according to claim 3, characterized in that: The method for preparing the biotinylated anti-CD248 engineered antibody comprises the following steps: (a) CD248 antibody was dialyzed in sodium acetate buffer, mixed with pepsin, pepsin:CD248 antibody = 1:20 (w:w), Tris-HCl was added to adjust the pH, and dialyzed to obtain a CD248 antibody digestion sample; (b) taking the CD248 antibody digestion sample described in step (a), applying the sample to chromatography, collecting the flow-through in separate tubes, mixing and concentrating the CD248 flow-through to obtain the CD248 antibody F(ab')2 fragment; (c) The CD248 antibody F(ab')2 fragment was labeled with biotin and dialyzed to obtain the biotinylated anti-CD248 engineered antibody.

5. The method for preparing CD248-targeted CAR-T cells according to claim 1, characterized in that: The following steps are involved: (1) Preparation of peripheral blood mononuclear cells: (11) Collect peripheral blood, mix thoroughly, and centrifuge to obtain blood cell pellet; (12) resuspending the blood cell pellet obtained in step (11) to obtain diluted blood, slowly adding the diluted blood to the upper layer of the separation solution along the tube wall, and centrifuging; (13) After centrifugation in step (12), the middle buffy coat layer is aspirated, supplemented with physiological saline, centrifuged, and the supernatant is discarded to obtain a cell pellet; (14) Wash the cell pellet obtained in step (13), centrifuge, and discard the supernatant to obtain peripheral blood mononuclear cells. 6.(2) T cell activation: (21) diluting the anti-CD3 / CD28 antibody with PBS, adding it to the culture plate, and letting it stand to obtain a pre-coated plate; (22) taking the pre-coated well plate of step (21), adding the peripheral blood mononuclear cells obtained in step (14) into the pre-coated well plate for culture; (23) After culturing for 24 h in step (22), cytokines IL-2, IL-7, and IL-15 were added to obtain activated T cells. 7.(3) Lentiviral vector construction: (31) CAR sequence synthesis and lentiviral vector plasmid preparation were performed, and the CAR gene sequence was inserted between the EcoRI and BamH1 restriction sites in the plasmid pCDH-EF1-MCS. 8.(4) Lentivirus packaging: (41) 293T cells were passaged and the cell density was adjusted to 5×10 5 / mL, culture until the cell fusion rate is 80%; (42) Mix the lentiviral vector plasmid, Lenti-Easy Packing Mix, and Opti-MEM, and incubate to obtain a diluted packaging system; Mix Lipofectamine 2000 reagent and Opti-MEM evenly, incubate, and obtain diluted Lipofectamine 2000; (43) Mixing the diluted packaging system and the diluted Lipofectamine 2000, and incubating to form a DNA-Lipofectamine 2000 transfection complex; (44) adding the transfection complex described in step (43) to the culture medium of the 293T cells cultured in step (41), discarding the culture medium containing the transfection complex after transfection, adding fresh culture medium, and continuing to culture; (45) 48–72 h after transfection, the cell supernatant was collected, centrifuged, filtered, and the sample was collected to obtain the lentiviral concentrate, and the titer was determined by fluorescence counting. 9.(5) Lentiviral infection of primary T lymphocytes: (51) Stimu-Buffer was added to 1× PBS and mixed to obtain a mixed solution. The mixed solution was added to each well of a plate and placed at 4°C. (52) After leaving the plate in step (51) for 24 h, remove the Stimu-Buffer solution before use, wash the plate with PBS, centrifuge the activated T cells obtained by culture in step (23), resuspend the cells in a culture medium containing FBS, IL-2, penicillin and streptomycin, and add the cells to the culture plate washed with PBS for culture; (53) Operate TA according to step (51) to coat the culture dish before infection and place at 4°C; resuspend the cells cultured in step (52) with Trans B (TB), add the lentivirus concentrate obtained in step (45) to obtain a cell-virus mixture; take out the TA-coated culture dish, wash the plate with PBS, and add the cell-virus mixture for culture; (54) After 24 h of culture in step (53), each well was gently replenished with an equal volume of culture medium containing FBS, IL-2, penicillin and streptomycin and culture was continued. After 48 h, the supernatant was discarded by centrifugation, and fresh culture medium containing IL-2 was added to resuspend the cells and transfer and expand the culture to obtain CD248-targeted CAR-T cells.

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