Application of macrophage expressing anti-uPAR chimeric antigen receptor in preparation of anti-aging drugs

Abstract

The invention relates to an application of a macrophage expressing an anti-uPAR chimeric antigen receptor in preparation of an anti-aging drug, and discloses a new application of the macrophage (CAR-M) expressing the anti-uPAR chimeric antigen receptor in senescence diseases, and the mechanism is that uPAR positive cells are specifically recognized and apoptosis of the uPAR positive cells is induced by targeting senescence cell surface antigen uPAR. According to the uPAR-CAR-M, a humanized anti-uPAR single-chain antibody is used as a chimeric antigen receptor recognition region, so that the chimeric antigen receptor has high specificity, and the chimeric antigen receptor is used for promoting the functions of macrophages by using a CD8 alpha transmembrane structural domain, a CD28 costimulatory molecule and a Megf10 sequence, so that a better killing effect is given to the CAR-M. The problems of inflammatory microenvironment caused by abnormal aggregation of senescence cells, chronic tissue damage and senescence-related disease treatment disorder are solved, and the application value for treating senescence-related diseases (such as osteoarthritis and the like) expressing uPAR is important.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to an application of macrophages expressing anti-uPAR chimeric antigen receptors in the preparation of anti-aging drugs. Background Art

[0002] Chimeric antigen receptor modified T cells (CAR-T cells) are T cells modified by genetic engineering technology. They express tumor chimeric antigen receptors on their surface and can specifically recognize surface antigens of tumor cells. Currently, they are mainly used to treat a variety of malignant tumors through CAR-T cells. Among them, they have been used clinically in the field of hematological tumors, but their application in difficult solid tumors is still immature. However, many years of CAR-T research have found that eliminating specific cell populations can have a therapeutic effect, and CAR-T is designed specifically for such populations, so that researchers are no longer limited to the field of tumor treatment and begin to explore other refractory diseases caused by lesions of special cell populations, such as certain autoimmune diseases, myocardial fibrosis and other chronic diseases. Zhang B et al. successfully combined anti-fluorescein isothiocyanate (FITC) chimeric antigen receptor T cells (CAR-T cells) with FITC-labeled antigen peptide epitopes to eliminate self-reactive B cell subsets that recognize these antigens in patients with rheumatoid arthritis RA.

[0003] Given the success of CAR-T cell immunotherapy, the prospects for genetically engineered immune cells are brighter, which also means that the CAR-T strategy can be applied to other immune cells. Researchers at the University of Pennsylvania used anti-HER2 CAR-modified macrophages containing the intracellular domain of CD3-ζ. In two ectopic mouse models with solid tumor transplantation, a single injection of anti-HER2-CAR-M cells reduced tumor burden and prolonged mouse survival. Professor Jiang Xinyi's team from the School of Pharmacy of Shandong University generated CAR-MΦ cells by "in situ transformation" of macrophages, enabling them to specifically recognize and engulf glioma stem cells GSCs, and successfully applied them to postoperative immunotherapy in animal models of malignant gliomas. The Morrissey MA team designed a family of phagocytic chimeric antigen receptors (CAR-Ps), which can guide macrophages to engulf specific targets, such as cancer cells. CAR-Ps are composed of extracellular antibody fragments. After the extracellular segment is modified, it can guide CAR-P to target specific antigens.

[0004] Cellular senescence is a common phenomenon in the physiological or pathological processes of the body, characterized by cells showing stable cell cycle arrest and secreting a series of molecules to regulate the tissue microenvironment. Under pathological conditions, the abnormal aggregation of senescent cells will cause an inflammatory microenvironment, leading to chronic tissue damage, further aggravating the progression of many diseases. Correspondingly, removing senescent cells from damaged tissues can alleviate the corresponding pathological symptoms and even improve patient survival. Researchers have been committed to developing specific target drugs for senescent cells to selectively eliminate senescent cells. At present, there are also several small molecules that show the ability to lyse senescent cells, but most of them are not ideal and have strong side effects. Therefore, there is an urgent need to find new methods for removing senescent cells.

[0005] Urokinase plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol (GPI) anchored membrane protein, a member of the fibrinolytic system, which can promote the degradation of extracellular matrix during fibrinolysis, wound healing and tumorigenesis. At the same time, uPAR is a biomarker induced to express on the surface of senescent cells and is related to the occurrence and development of aging diseases. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention proposes the use of macrophages expressing anti-uPAR chimeric antigen receptors in the preparation of anti-aging drugs.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] Application of macrophages expressing anti-uPAR chimeric antigen receptor in the preparation of anti-aging drugs.

[0009] Furthermore, the drug induces apoptosis of uPAR-positive target cells by targeting the senescent cell surface antigen uPAR.

[0010] Furthermore, the drug includes a pharmaceutically acceptable carrier.

[0011] Furthermore, the method for constructing the macrophages expressing the anti-uPAR chimeric antigen receptor is: constructing an expression vector containing the chimeric antigen receptor gene, and then introducing the vector into the macrophages.

[0012] Furthermore, the introduction method is electroporation, or transduction, or transfection.

[0013] Furthermore, the expression vector is a viral vector, and the viral vector is a lentivirus, or an adenovirus, or a retrovirus.

[0014] Furthermore, the structure of the chimeric antigen receptor is: signal peptide, extracellular antigen receptor structure, hinge region, transmembrane domain, intracellular co-stimulatory signal region, and intracellular signal sequence.

[0015] Further,

[0016] The signal peptide is a CD8α signal peptide, and its amino acid sequence is SEQ ID NO.1;

[0017] The extracellular antigen receptor structure is a uPAR extracellular antigen receptor structure, and its amino acid sequence is SEQ ID NO.2;

[0018] The hinge region is the CD8α hinge region, and its amino acid sequence is SEQ ID NO.3;

[0019] The transmembrane domain is a CD8α transmembrane domain, and its amino acid sequence is SEQ ID NO.4;

[0020] The intracellular co-stimulatory signal region is the CD28 intracellular co-stimulatory signal region, and its amino acid sequence is SEQ ID NO.5;

[0021] The intracellular signal sequence is the Megf10 intracellular signal sequence, and its amino acid sequence is SEQ ID NO.6.

[0022] Furthermore,

[0023] The nucleotide sequence encoding the CD8α signal peptide is SEQ ID NO.7;

[0024] The nucleotide sequence encoding the uPAR extracellular antigen receptor structure is SEQ ID NO.8;

[0025] The nucleotide sequence encoding the CD8α hinge region is SEQ ID NO.9;

[0026] The nucleotide sequence encoding the CD8α transmembrane domain is SEQ ID NO.10;

[0027] The nucleotide sequence encoding the CD28 intracellular co-stimulatory signal region is SEQ ID NO.11;

[0028] The nucleotide sequence encoding the Megf10 intracellular signal sequence is SEQ ID NO.12.

[0029] The beneficial effects of the present invention are:

[0030] 1. The present invention relates to the use of macrophages expressing anti-uPAR chimeric antigen receptors in the preparation of anti-aging drugs, and discloses a new use of macrophages expressing anti-uPAR chimeric antigen receptors (CAR-M) in aging diseases. The mechanism is to specifically identify uPAR-positive cells and induce their apoptosis by targeting the surface antigen uPAR of senescent cells.

[0031] 2. The present invention discloses an application of macrophages expressing anti-uPAR chimeric antigen receptors in the preparation of anti-aging drugs. The CAR-M is a macrophage modified with a chimeric antigen receptor. The present invention uses a humanized anti-uPAR single-chain antibody as the chimeric antigen receptor recognition region to make the chimeric antigen receptor highly specific. The chimeric antigen receptor uses the CD8α transmembrane domain, CD28 co-stimulatory molecule and Megf10 sequence to promote the function of macrophages, giving CAR-M a better killing effect.

[0032] 3. The present invention discloses the use of anti-uPAR-CAR-M in the preparation of anti-aging drugs, which solves the problem of abnormal aggregation of senescent cells causing inflammatory microenvironment, leading to chronic tissue damage, and causing obstacles to the treatment of aging-related diseases. It has important application value in the treatment of aging-related diseases expressing uPAR (such as osteoarthritis, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the specific implementation of the present invention, the following will briefly introduce the drawings required for use in the specific implementation. In all the drawings, each element or part is not necessarily drawn according to the actual scale.

[0034] Figure 1 A is a schematic diagram of the structure of the chimeric antigen receptor (uPAR-CAR) targeting uPAR of the present invention; Figure 1 B is a schematic diagram of the structure of the chimeric antigen receptor of the present invention;

[0035] Figure 2 This is the plasmid map of uPAR-CAR-M in Example 1 of the present invention;

[0036] Figure 3 The results of restriction digestion and sequencing of the uPAR-CAR-M plasmid in Example 1 of the present invention;

[0037] Figure 4 This is a fluorescence detection diagram of the expression of THP-1 cells infected with lentivirus in Example 3 of the present invention;

[0038] Figure 5 This is the targeted recognition effect of uPAR CAR-M on uPAR positive cells in Example 4 of the present invention;

[0039] Figure 6This is the killing (apoptosis induction) effect of uPAR CAR-M on uPAR-positive cells in Example 4 of the present invention. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Technicians in this field can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0041] CD8α signal peptide amino acid sequence (SEQ ID NO.1):

[0042] MASPLTRFLSLNLLLLGESIILGSGEA

[0043] uPAR extracellular antigen receptor structural amino acid sequence (SEQ ID NO.2):

[0044] GVKLQQSGPEVVKPGASVKISCKASGYSFTNFYIHWVKQRPGQGLEWIGWIFHGSDNTEYNEKFKDKATLTADTSSSTAYMQLSSSLTSEDSAVYFCARWGPHWYFDVWGQGTTVT VSSGGGGSGGGGSGGGGSDIVLTQSPDITAASLGQKVTITCSASSSVSYMHWYQQKSGTSPKPWIFEISKLASGVPARFSGSGSGTSYSLTISSMEAEDAAIYYCQQWNYPFTFGGGTKLEIKR

[0045] CD8α hinge region amino acid sequence (SEQ ID NO.3):

[0046] KPQAPELRIFPKKMDAELGQKVDLVCEVLGSVSQGCSWLFQNSSSKLPQPTFVVYMASSHNKITWDEKLNSSKLFSAMRDTNNKYVLTLNKFSKENEGYYFCSVISNSVMYFSSVVPVLQKVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIY

[0047] CD8α transmembrane domain amino acid sequence (SEQ ID NO.4):

[0048] IWAPLAGICVALLLSLIITLI

[0049] The amino acid sequence of the intracellular co-stimulatory signal region of CD28 (SEQ ID NO.5):

[0050] RLKIQVRKAAIASREKADAVYTGLNTRSQETYETLKHEKPPQ

[0051] Megf10 intracellular signal sequence amino acid sequence (SEQ ID NO.6):

[0052] YRHKQKRKESSMPAVTYTPAMRVINADYTIAETLPHSNGGNANSHYFTNPSYHTLSQCATSPHVNNRDRMTIAKSKNNQLFVNLKNVNPGKRGTLVDCTGTLPADWKQGGYLNELGAFGLDRSYMGKSLKDLGK NSEYNSSTCSLSSSENPYATIKDPPALLPKSSECGYVEMKSPARRDSPYAEINNSTPANRNVYEVEPTVSVVQGVFSNSGHVTQDPYDLPKNSHIPCHYDLLPVRDSSSSPKREDGGGSNSTSSNSTSSSSSSSE

[0053] CD8α signal peptide nucleotide sequence (SEQ ID NO.7):

[0054] ATGGCCTCACCGTTGACCCGCTTTCTGTCGCTGAACCTGCTGCTGCTGGGTGAGTCGATTATCCTGGGGAGTGGAGAAGCT

[0055] uPAR extracellular region nucleotide sequence (SEQ ID NO.8):

[0056] GGCGTGAAGCTGCAGCAGAGCGGCCCCGAGGTGGTGAAGCCCGGCGCCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACAGCTTCACCAACTTCTACATCCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTGGATCTTCCACGGCAGCGACAACACCGAGTACAACGAGAAGTTCAAGGACAAGGCCACCCTGACCGCCGACACCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCTGGGGCCCCCACTGGTACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGACATCGTGCTGACCCAGAGCCCCGACATCACCGCCGCCAGCCTGGGCCAGAAGGTGACCATCACCTGCAGCGCCAGCAGCAGCGTGAGCTACATGCACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCCCTGGATCTTCGAGATCAGCAAGCTGGCCAGCGGCGTGCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCATCTACTACTGCCAGCAGTGGAACTACCCCTTCACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGC

[0057] CD8α hinge region nucleotide sequence (SEQ ID NO.9):

[0058] AAGCCACAGGCACCCGAACTCCGAATCTTTCCAAAGAAAATGGACGCCGAACTTGGTCAGAAGGTGGACCTGGTATGTGAAGTGTTGGGGTCCGTTTCGCAAGGATGCTCTTGGCTCTTCCAGAACTCCAGCTCCAAACTCCCCCAGCCCACCTTCGTTGTCTATATGGCTTCATCCCACAACAAGATAACGTGGGACGAGAAGCTGAATTCGTCGAAACTGTTTTCTGCCATGAGGGACACGAATAATAAGTACGTTCTCACCCTGAACAAGTTCAGCAAGGAAAACGAAGGCTACTATTTCTGCTCAGTCATCAGCAACTCGGTGATGTACTTCAGTTCTGTCGTGCCAGTCCTTCAGAAAGTGAACTCTACTACTACCAAGCCAGTGCTGCGAACTCCCTCACCTGTGCACCCTACCGGGACATCTCAGCCCCAGAGACCAGAAGATTGTCGGCCCCGTGGCTCAGTGAAGGGGACCGGATTGGACTTCGCCTGTGATATTTAC

[0059] CD8α transmembrane region nucleotide sequence (SEQ ID NO.10):

[0060] ATCTGGGCACCCTTGGCCGGAATCTGCGTGGCCCTTCTGCTGTCCTTGATCATCAC TCTCATC

[0061] CD28 intracellular co-stimulatory signal region nucleotide sequence (SEQ ID NO.11):

[0062] CGACTCAAGATCCAGGTCCGAAAGGCAGCTATAGCCAGCCGTGAGAAAGCAGATGCTGTCTACACGGGCCTGAACACCCGGAGCCAGGAGACATATGAGACTCTGAAGCATGAGAAACCACCCCAG

[0063] Megf10 intracellular signal region nucleotide sequence (SEQ ID NO.12):

[0064] TACAGACACAAGCAGAAGAGGAAGGAATCAAGCATGCCGGCCGTGACCTACACCCCCGCCATGAGGGTCATCAATGCAGACTATACCATCGCAGAAACCCTGCCTCACAGCAATGGTGGAAATGCCAACAGCCACTACTTTACCAATCCCAGTTATCACACACTTAGCCAGTGTGCCACATCCCCTCATGTGAACAATAGGGACAGGATGACCATTGCAAAGTCAAAAAACAATCAGCTGTTTGTGAATCTTAAAAATGTGAATCCAGGGAAGAGAGGGACATTGGTGGACTGCACTGGGACATTGCCAGCTGACTGGAAGCAAGGAGGCTACCTCAATGAGCTTGGTGCTTTCGGGCTGGACAGAAGCTACATGGGAAAGTCCTTAAAAGATCTGGGGAAGAACTCTGAATATAATTCAAGCACTTGCTCCTTAAGCAGCTCTGAAAACCCATATGCCACCATTAAAGACCCGCCTGCACTCCTGCCTAAAAGCTCCGAGTGCGGCTACGTGGAGATGAAGTCGCCGGCGCGGAGAGACTCCCCATATGCAGAGATCAACAACTCAACTCCAGCCAACAGGAATGTCTATGAAGTCGAACCTACAGTGAGCGTTGTGCAAGGAGTATTCAGCAACAGCGGTCACGTCACCCAAGACCCATATGACCTTCCAAAGAACAGTCACATCCCTTGCCATTATGACCTGCTGCCAGTAAGGGACAGTTCATCCTCCCCAAAGAGAGAGGATGGTGGTGGCAGCAACAGCACCAGCAGCAACAGCACCAGCAGCAGCAGCAGCAGCAGTGAATGA

[0065] Reagent source:

[0066] The transfection reagent is Lipo293 TM , with the product number C0521, purchased from Beyotime Biotechnology Co., Ltd.

[0067] Human monocytic THP-1 cell line: a suspension cell line purchased from Wuhan Pronocell Life Science Co., Ltd., the culture medium is 1640+10% FBS+0.05mMβ-mercaptoethanol+1% P / S.

[0068] 293T (human embryonic kidney cell) strain: adherent cells purchased from Wuhan Pronocell Life Science Co., Ltd., culture medium is DMEM+10% FBS+1% P / S.

[0069] PEG lentivirus purification reagent, catalog number P1201, was purchased from Chongqing Yingmao Shengye Biotechnology Co., Ltd.

[0070] The CAR-M of the present invention is a macrophage modified with a chimeric antigen receptor. The structural schematic diagram of CAR-M is as follows: Figure 1 As shown in A; the structure of the chimeric antigen receptor is as shown in Figure 1 As shown in B, it consists of a signal peptide (sequence is SEQ ID NO.1), an extracellular antigen receptor structure (sequence is SEQ ID NO.2), a hinge region (sequence is SEQ ID NO.3), a transmembrane domain (sequence is SEQ ID NO.4), an intracellular co-stimulatory signal region (sequence is SEQ ID NO.5), and an intracellular signal sequence (sequence is SEQ ID NO.6).

[0071] Example 1 Design and synthesis of CAR plasmid

[0072] The human CAR sequence was obtained by PCR amplification, including restriction sites, Kozak sequence, target gene CDS, and a GFP green fluorescent protein tag was added at the end; the specific nucleotide sequence was CD8α signal peptide (SEQ ID NO.7), uPA R extracellular antigen receptor structure (SEQ ID NO.8), CD8α hinge region (SEQ ID NO.9), CD8α transmembrane domain (SEQ ID NO.10), CD28 intracellular co-stimulatory signal region (SEQ ID NO.11), Megf10 intracellular signal sequence (SEQ ID NO.12). The target gene was constructed into the overexpression shuttle vector through XbaI-NheI. The shuttle vector map is shown in Figure 2 , with green fluorescence and Puro resistance, the target gene has been correctly cloned into the shuttle vector after sequencing verification. The results of restriction enzyme digestion and sequencing of uPAR-CAR-M plasmid are shown in Figure 3By cloning the sequence between the Xhol and NheI sites of the PLVX-puro plasmid, it can be seen that the size of the cloned product on the PCR gel is consistent with the size of the designed recombinant protein gene. In addition, the PCR product was sequenced and identified, and the results showed that there were no mismatches in all sequences. After ensuring that the designed plasmid sequence is correct, the bacterial solution of the positive clone is inoculated into about 50mL of LB medium containing 50mg / L Amp, 37℃, 200rmp, and shaken for about 18 hours. The bacteria were collected and the plasmid was extracted using an endotoxin-free plasmid mini-extraction kit.

[0073] Example 2 uPAR CAR lentivirus construction

[0074] The uPAR-CAR (chimeric antigen receptor targeting uPAR) lentivirus was packaged using the pLVX lentiviral vector packaging system.

[0075] After the HEK-293T cells were adjusted to an exponential growth state, the 293T cells were digested with trypsin, centrifuged at 1000 rpm for 4 min, the supernatant was gently aspirated, 1 mL of complete medium was added and the cells were gently blown to make a single cell suspension, and then 15 mL of PBS was added and blown again to mix; centrifuged at 800 rpm for 4 min, the supernatant was gently aspirated, 1 mL of complete medium was added and the cells were gently blown to make a single cell suspension, and then 10 mL of complete medium was added to mix, with a density of 4-5×10 6 / dish. The next day, the cells reached 80-90% density and were replaced with serum-free medium 1 h before transfection. TM (Biyuntian: C0521) transfection reagent requirements, mix plasmid and transfection reagent, and perform cell transfection; 18 to 24 hours after transfection, carefully aspirate the cell culture medium, discard it in a triangular flask, and then seal it for high temperature and high pressure sterilization. The culture medium is replaced here to provide a medium with sufficient nutrition for virus production, and transfection reagents, plasmids, etc. can also be removed. 24 hours after the liquid is replaced, the cell culture supernatant is aspirated into a 50mL centrifuge tube, and fresh culture medium is added at the same time. At 48 hours, the cell culture supernatant is aspirated into a 50mL centrifuge tube and the cells are discarded. The supernatant is filtered with a 0.45μm sterile filter and transferred to a new centrifuge tube. According to the PEG lentivirus purification reagent (Yinmao Shengye: P1201), the lentivirus is concentrated and purified. Finally, the lentivirus precipitate is dissolved in DMEM, aliquoted at 100uL / tube, and stored at -80℃.

[0076] Example 3 Infection-induced uPAR CAR-M

[0077] The day before infection, THP-1 cells were inoculated in 12-well plates at a density of 4×10 5 / well. According to MOI=100, the lentivirus concentrate containing 8ug / ml of polybrene prepared in Example 2 was added to the culture medium and incubated in a CO2-containing incubator for 30 minutes, followed by centrifugation in a 32°C, 500g centrifuge for 90 minutes. Then, it was placed in a CO2-containing incubator and cultured for 24 hours before being replaced with 1640 complete culture medium containing 10% FBS for continued culture. After 24 hours of infection, the cells were killed with a culture medium containing a final concentration of Puro of 0.5μg / mL for 2 days, and then cultured with a culture medium without Puro. At the same time, 48-72 hours after lentivirus infection, the infection ratio was observed by fluorescence microscopy. The results of lentivirus infection are shown in the figure. Figure 4 As shown in the figure, it can be seen that after infection, about 96% of THP-1 cells express uPAR CAR molecules. PMA (100 ng / ml) was added to the infected THP-1 cells for stimulation for 72 hours, and the 1640 complete culture medium was replaced and cultured for 24 hours to induce uPAR CAR-THP-1 to become uPAR CAR-M.

[0078] Example 4 Functional verification of uPAR CAR-M to study the targeting recognition and killing (inducing apoptosis) effects of uPAR CAR-M on uPAR-positive target cells

[0079] The experiment was divided into two groups: the control group was the NC-M group, which was macrophages expressing only GFP; the experimental group was the uPAR-CAR-M group. The fluorescence intensity was collected and analyzed using an Olympus laser confocal microscope to perform targeted recognition and killing (inducing apoptosis) experiments. First, 293T cells were cultured at 1×10 5 Each well was inoculated into a 12-well cell culture plate, with 3 replicates per group. Transfection was performed when the inoculated cells reached 70-90% confluence. TM Dilute Lipofectamine in culture medium TM 3000 reagent (2 tubes) and mix thoroughly. Use Opti-MEM TM Dilute DNA in culture medium to prepare DNA master mix, then add P3000 TM Reagents and mix thoroughly. TMDiluted DNA (1:1 ratio) was added to the 3000 reagent, incubated at room temperature for 10-15 minutes, DNA-lipid complexes were added to the cells, and the cells were incubated at 37°C for 2 days. Fluorescence microscopy showed that uPAR-positive 293T cells successfully expressed RFP red fluorescence. Then, equal amounts of NC-M and uPAR-CAR-M cells were added, and the cells were incubated in a 37-degree carbon dioxide incubator for 24 hours and analyzed by fluorescence microscopy. Among them, GFP-positive cells (green fluorescence) represent CAR-M, RFP-positive cells (red fluorescence) represent 293T cells expressing uPAR, and GFP and RFP double positive represent uPAR-CAR-M cells that can recognize uPAR-positive target cells.

[0080] uPAR CAR-M's targeted recognition of uPAR-positive cells Figure 5 As shown, DAPI represents the cell nucleus, uPAR represents uPAR+ 293T target cells, and GFP represents NC-M and uPAR-CAR-M. It can be seen from the figure that the control group NC-M has no obvious phagocytic effect on uPAR+ cells, but uPAR-CAR-M can phagocytize uPAR-positive target cells, indicating that CAR-M targeting uPAR has a targeted recognition effect on uPAR-positive target cells.

[0081] uPAR CAR-M induces apoptosis of uPAR-positive cells Figure 6 As shown, uPAR represents uPAR+ 293T target cells, and Tunel represents apoptotic cells. It can be seen from the figure that after the uPAR+ cells were treated with the culture supernatant of uPAR-CAR-M for 48 hours, Tunel staining was performed. The Tunel positivity rate in the uPAR-CAR-M group was higher than that in the NC-M group, indicating that uPAR-CAR-M can induce apoptosis of uPAR-positive target cells.

[0082] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments. On the basis of the present invention, some modifications or improvements can be made thereto, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. Use of macrophages expressing anti-uPAR chimeric antigen receptor in the preparation of anti-aging drugs.

2. The use according to claim 1, characterized in that: The drug induces apoptosis of uPAR-positive target cells by targeting the senescent cell surface antigen uPAR.

3. The use according to claim 1, characterized in that: The drug includes a pharmaceutically acceptable carrier.

4. The use according to claim 2 or 3, characterized in that: The method for constructing the macrophage expressing the anti-uPAR chimeric antigen receptor is as follows: constructing an expression vector containing the chimeric antigen receptor gene, and then introducing the vector into the macrophage.

5. The use according to claim 4, characterized in that: The introduction method is electroporation, transduction, or transfection.

6. The use according to claim 4, characterized in that: The expression vector is a viral vector, and the viral vector is a lentivirus, or an adenovirus, or a retrovirus.

7. The use according to any one of claims 1 to 6, characterized in that: The structure of the chimeric antigen receptor is: signal peptide, extracellular antigen receptor structure, hinge region, transmembrane domain, intracellular costimulatory signal region, and intracellular signal sequence.

8. The use according to claim 7, characterized in that: The signal peptide is a CD8α signal peptide, and its amino acid sequence is SEQ ID NO.1; The extracellular antigen receptor structure is a uPAR extracellular antigen receptor structure, and its amino acid sequence is SEQ ID NO.2; The hinge region is the CD8α hinge region, and its amino acid sequence is SEQ ID NO.3; The transmembrane domain is a CD8α transmembrane domain, and its amino acid sequence is SEQ ID NO.4; The intracellular co-stimulatory signal region is the CD28 intracellular co-stimulatory signal region, and its amino acid sequence is SEQ ID NO.5; The intracellular signal sequence is the Megf10 intracellular signal sequence, and its amino acid sequence is SEQ ID NO.

6.

9. The use according to claim 8, characterized in that: The nucleotide sequence encoding the CD8α signal peptide is SEQ ID NO.7; The nucleotide sequence encoding the uPAR extracellular antigen receptor structure is SEQ ID NO.8; The nucleotide sequence encoding the CD8α hinge region is SEQ ID NO.9; The nucleotide sequence encoding the CD8α transmembrane domain is SEQ ID NO.10; The nucleotide sequence encoding the CD28 intracellular co-stimulatory signal region is SEQ ID NO.11; The nucleotide sequence encoding the Megf10 intracellular signal sequence is SEQ ID NO.12.

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