A single-chain antibody targeting uPAR, a chimeric antigen receptor and its application

Abstract

The present invention belongs to the field of biomedicine and molecular biology technology, and specifically relates to a single-chain antibody targeting uPAR, a chimeric antigen receptor and its application. The present invention targets the uPAR molecule, optimizes the extracellular region of the antigen, immunizes mice, and fuses to obtain a hybridoma cell line. After sequencing, a mouse single-chain antibody sequence with high affinity is obtained, and it is constructed into a CAR-T cell targeting uPAR. In vitro experiments show that CAR-T cells targeting uPAR can effectively eliminate uPAR+ stellate cells and tumor cells, and in vivo experiments show that uPAR CAR-T cells can effectively improve liver fibrosis in mice and inhibit the growth of liver cancer in mice. This study provides new ideas for the clinical treatment of diseases such as liver fibrosis, and provides a new target for CAR-T immunotherapy in the treatment of cardiovascular diseases and even solid tumors, and therefore has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and molecular biology, and specifically relates to a single-chain antibody targeting uPAR, a chimeric antigen receptor and applications thereof. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Chronic liver disease is a serious threat to human health, claiming approximately 2 million lives worldwide each year. Chronic liver disease primarily includes alcoholic fatty liver disease and non-alcoholic fatty liver disease. Its development is often accompanied by liver fibrosis. If left untreated, fibrosis can progress to cirrhosis and ultimately lead to liver cancer. Chronic liver disease, including liver fibrosis, is primarily caused by chronic hepatitis B virus infection, excessive alcohol consumption, or an irregular diet. Liver fibrosis is characterized by the production of extracellular matrix proteins, including collagen, and the infiltration of immune cells (primarily T cells) that release large amounts of proinflammatory cytokines such as TGF-β, IL-1, and IL-6, leading to damage to liver parenchymal cells. These proinflammatory cytokines induce collagen release from fibroblasts and myofibroblasts, leading to liver fibrosis.

[0004] Liver fibrosis is potentially harmful and explosive, with a significant proportion of patients developing cirrhosis over the next 15-20 years. Major clinical complications include ascites, renal failure, hepatic encephalopathy, and variceal bleeding. Liver fibrosis is a pathological phenomenon that occurs after repeated liver injury and healing, accompanied by the accumulation of a large amount of extracellular matrix and the proliferation of different types. HSCs are the primary inducing cells for extracellular matrix production. In the normal liver, HSCs maintain metabolic balance and homeostasis, and are the primary reservoir of vitamin A. Following chronic injury, HSCs are activated and transform into myofibroblasts. Activated HSCs gradually migrate to the site of liver injury and accumulate in large numbers. To repair the damaged liver, they release large amounts of extracellular matrix, including collagen, leading to liver fibrosis. To date, despite the discovery of numerous potential therapeutic targets for the treatment of liver fibrosis, no standardized and effective treatment for the alleviation of liver fibrosis has emerged. This is because serial liver biopsies are needed during treatment to accurately assess changes in liver fibrosis, and long-term follow-up studies are needed, but humans have low sensitivity to liver anti-fibrosis therapy, making it difficult to achieve the expected results.

[0005] Currently, the main clinical treatments for liver fibrosis rely on blocking drugs and various inhibitors, and a mature, safe, and effective immunotherapy approach has yet to be developed. Several studies have investigated targeting uPAR for the treatment of liver fibrosis, including a paper titled "Senolytic CAR-T cells reverse senescence-associated pathologies" published in Nature by Corina Amor's team. The team's uPAR-targeted CAR-T cells effectively eliminated senescent cells in mouse livers, thereby reversing liver fibrosis. Furthermore, a study published in the Journal of Hepatology in 2024 by Scott L.'s team demonstrated that activated and senescent HSCs in the livers of mice with liver fibrosis highly expressed uPAR. By targeting uPAR to eliminate senescent HSCs, liver fibrosis was effectively alleviated. Furthermore, studies have shown that uPAR plays a crucial role in extracellular matrix remodeling during fibrosis, mechanical injury, and tumor development. This is primarily due to the activation of urokinase, which further activates intracellular signaling pathways, leading to the release of matrix extracellularly. Therefore, targeting uPAR to eliminate activated HSCs is a feasible and effective way to alleviate and reverse liver fibrosis. However, there are relatively few antibodies targeting uPAR on the market, and most of them are large molecular weight recognition antibodies (150 kD). In the application of CAR-T, the effect of large molecular weight recognition antibodies is significantly inferior to scFv. There is an urgent need to develop a high-affinity single-chain antibody (scFv). Summary of the Invention

[0006] In response to the shortcomings of the above-mentioned prior art, the present invention aims to provide a single-chain antibody targeting uPAR, a chimeric antigen receptor, and their applications. Specifically, the present invention successfully prepared a single-chain antibody with high affinity for the uPAR molecule, and based on this high-affinity scFv, constructed a CAR and murine CAR-T cells that recognize uPAR. In vitro and in vivo experiments have demonstrated that it has the effect of treating liver fibrosis and liver cancer. Based on the above research results, the present invention was completed.

[0007] In order to achieve the above technical objectives, the technical solutions provided by the present invention are as follows:

[0008] In a first aspect, the present invention provides a single-chain antibody targeting uPAR, wherein the single-chain antibody comprises a complementarity determining region (CDR); the amino acid sequence of the complementarity determining region is selected from:

[0009] (a) the amino acid sequence shown in any one of SEQ ID NOs. 5-10;

[0010] (b) an amino acid sequence that is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%) identical to any one of SEQ ID NOs. 5-10, respectively.

[0011] Furthermore, in the single-chain antibody targeting uPAR, the complementary determining regions comprise a heavy chain complementary determining region and a light chain complementary determining region;

[0012] Wherein, the heavy chain complementarity determining region comprises:

[0013] CDR1 with the amino acid sequence shown in SEQ ID NO.5, CDR2 with the amino acid sequence shown in SEQ ID NO.6, and CDR3 with the amino acid sequence shown in SEQ ID NO.7;

[0014] The light chain complementarity determining region comprises:

[0015] CDR1 with the amino acid sequence shown in SEQ ID NO.8, CDR2 with the amino acid sequence shown in SEQ ID NO.9, and CDR3 with the amino acid sequence shown in SEQ ID NO.10.

[0016] Furthermore, the single-chain antibody may be a recombinant antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antigen-binding fragment of these antibodies, thereby expanding its scope of application, which is not specifically limited here.

[0017] Furthermore, the single-chain antibody targeting uPAR comprises: a heavy chain antibody V as shown in SEQ ID NO.11 H The amino acid sequence of the light chain antibody V shown in SEQ ID NO.12 L The amino acid sequence of

[0018] Furthermore, the heavy chain antibody V H With light chain antibody V L The connection can be made directly or through a connecting peptide; the connecting peptide can be specifically (G4S)n, wherein n is a positive integer, for example, any positive integer between 1 and 6. Preferably, n is 3, which is more conducive to the heavy chain antibody V H With light chain antibody V L connection.

[0019] Therefore, the single-chain antibody can be composed of the above-mentioned light chain antibody, connecting peptide and heavy chain antibody in series.

[0020] The second aspect of the present invention provides a chimeric antigen receptor targeting uPAR, which comprises at least an antigen binding domain, wherein the antigen binding domain comprises the above-mentioned single-chain antibody targeting uPAR.

[0021] Furthermore, the chimeric antigen receptor targeting uPAR is composed of a signal peptide, an antigen binding domain, a transmembrane region, a co-stimulatory signal transduction domain and a signal transduction domain connected in series.

[0022] The signal peptide may be CD8 signal peptide (SP), the amino acid sequence of which is shown in SEQ ID NO.1.

[0023] The transmembrane region may be a CD8 transmembrane region, the amino acid sequence of which is shown in SEQ ID NO.2.

[0024] The co-stimulatory signal transduction domain may be a 4-1BB co-stimulatory signal transduction domain, the amino acid sequence of which is shown in SEQ ID NO.3.

[0025] The signal transduction domain may be a CD3ζ signal transduction domain, the amino acid sequence of which is shown in SEQ ID NO.4.

[0026] The third aspect of the present invention provides a nucleic acid molecule encoding the single-chain antibody targeting uPAR or the chimeric antigen receptor targeting uPAR.

[0027] The fourth aspect of the present invention provides a recombinant expression vector comprising the above-mentioned nucleic acid molecule.

[0028] According to the present invention, the recombinant expression vector can be a viral vector, including a retroviral vector and a lentiviral vector; further preferably, it is a lentiviral vector, and the recombinant expression vector is a recombinant viral expression vector expressing the single-chain antibody or chimeric antigen receptor by inserting a nucleic acid molecule encoding the above-mentioned single-chain antibody or chimeric antigen receptor into a virus.

[0029] The fifth aspect of the present invention provides a CAR-T cell, which is a T lymphocyte modified by the chimeric antigen receptor targeting uPAR, thereby providing the necessary therapeutic means for the treatment of diseases mediated by high uPAR expression.

[0030] In the present invention, the CAR-T cells can be obtained by infecting T cells with a lentivirus; the lentivirus is obtained by transfecting a recombinant lentiviral expression vector into lentiviral packaging cells, followed by cell culture; the recombinant lentiviral expression vector is obtained by inserting the gene encoding the chimeric antigen receptor into a lentiviral expression vector. In practice, those skilled in the art can prepare the CAR-T cells using existing known techniques, and no specific limitations are imposed herein.

[0031] The seventh aspect of the present invention provides a drug for preventing and / or treating diseases mediated by high uPAR expression, which comprises any one or more of the following: the above-mentioned single-chain antibody, chimeric antigen receptor, CAR-T cell; or the above-mentioned nucleic acid molecule and recombinant expression vector.

[0032] The drug can be prepared into dosage forms such as powder, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc. for oral administration, external use, suppositories and sterile injection solutions according to conventional practices.

[0033] The drug may further include a pharmaceutically acceptable carrier, which may be a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, an excipient, a filler, a coagulant and a conditioning agent, a surfactant, a dispersant, or a defoaming agent.

[0034] The drug may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a microcapsule, liposome, nanoparticle, or polymer, or any combination thereof. The delivery vehicle of the pharmaceutically acceptable carrier may be a liposome, a biocompatible polymer (including natural and synthetic polymers), a lipoprotein, a polypeptide, a polysaccharide, a lipopolysaccharide, an artificial viral envelope, an inorganic (including metal) particle, or a bacterial, bacteriophage, cosmid, or plasmid vector, thereby broadening its application range.

[0035] The drug can also be used in combination with other drugs for preventing and / or treating diseases mediated by high uPAR expression. Other preventive and / or therapeutic compounds can be administered simultaneously with the main active ingredient, or administered simultaneously in the same composition.

[0036] Furthermore, the drug can be administered into the body in a known manner. For example, it can be delivered systemically by intravenous delivery or local injection into the tissue of interest. Such administration can be carried out via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dosage to be administered in the present invention can vary to a large extent depending on a variety of factors, such as the target cell, the type of organism or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0037] Furthermore, the subject of administration of the drug can be a human or a non-human mammal, and the non-human mammal includes mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc., which are not specifically limited here.

[0038] The present invention has demonstrated that uPAR-targeted CAR-T cells can effectively eliminate uPAR+ stellate cells and tumor cells in vitro, and in vivo experiments have shown that uPAR CAR-T cells can effectively improve liver fibrosis in mice and inhibit the growth of liver cancer in mice. Therefore, diseases mediated by uPAR overexpression include fibrosis-mediated liver diseases and tumor diseases.

[0039] The eighth aspect of the present invention provides a detection product, which may include: any one or more of the above-mentioned single-chain antibodies, chimeric antigen receptors, CAR-T cells, drugs, nucleic acid molecules and recombinant expression vectors.

[0040] The detection product can be a detection kit or a detection device, etc., which is not limited here.

[0041] Of course, as a detection kit or detection device, detection reagents such as buffer solution, cleaning solution, etc. are easily available and are not specifically limited here.

[0042] In the present invention, the detection product can be used to qualitatively or quantitatively detect the expression of uPAR, and can then be used for basic research on uPAR-related physiological or pathological changes or applied in actual clinical practice. Actual clinical applications include but are not limited to screening, (auxiliary) diagnosis, monitoring or prediction of the progression of diseases mediated by high uPAR expression, thereby broadening its scope of application.

[0043] Beneficial technical effects of one or more of the above technical solutions:

[0044] The above technical solution targets the uPAR molecule. By optimizing the extracellular region of the antigen, immunizing mice, and fusing them to generate hybridoma cell lines, a high-affinity mouse single-chain antibody sequence was obtained after sequencing, which was then constructed into CAR-T cells targeting uPAR. In vitro experiments showed that CAR-T cells targeting uPAR can effectively eliminate uPAR+ stellate cells and tumor cells. In vivo experiments showed that uPAR CAR-T cells can effectively improve liver fibrosis in mice and inhibit the growth of liver cancer in mice. This research provides new ideas for the clinical treatment of diseases such as liver fibrosis and provides a new target for CAR-T immunotherapy in the treatment of cardiovascular diseases and even solid tumors, thus having great practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0046] Figure 1 Strategy diagram for obtaining supernatant containing numerous antibody sequences for immunizing mice according to the present invention

[0047] The study involved immunizing C57 mice with a coating solution containing uPAR extracellular domain antigens. Five healthy mice were immunized five times, approximately every 15 days. Blood samples were collected from the orbital venous plexus to assess immune responses. Three days after the fifth immunization, splenic B cells were hybridized with myeloma cells to screen for positive monoclonal antibody cell lines. Antibodies in ascites were then tested using ELISA to identify the optimal mouse cell line.

[0048] Figure 2 Construction and cytotoxicity detection of mouse CAR-T cells of the present invention

[0049] Among them: A is the structure and element diagram of uPAR CAR; B is the constructed cell line overexpressing the full-length mouse uPAR (1-337 amino acids), uPAR-LX2 and uPAR-Hepa1-6; C is the construction of mouse uPAR CAR-T cells, and the CAR-T cell positive rate was detected by flow cytometry; D is the LDH release assay for the cytotoxicity of uPAR CAR-T cells against uPAR-LX2 at effector-target ratios of 1:1 and 1:5; E is the LDH release assay for the cytotoxicity of uPAR CAR-T cells against uPAR-Hepa1-6 at effector-target ratios of 1:1 and 1:5.

[0050] Figure 3 In vivo anti-tumor experiment of uPAR CAR-T cells of the present invention

[0051] Among them: A is the mouse subcutaneous tumor model and uPAR CAR-T cell infusion strategy diagram; B is the statistics of mouse tumor cell volume changes, and the tumor volume was recorded every two days; C is the mouse survival curve, when the mouse tumor volume reached 1000 mm 2 , the mice were declared dead.

[0052] Figure 4 Effect of uPAR CAR-T cells on liver fibrosis

[0053] Among them: A is the use of CCL4 to create a fibrosis model and the uPAR CAR-T cell infusion strategy; B is the HE detection of mouse liver tissue fibrosis; C is the picrosirius red detection of mouse liver tissue fibrosis. DETAILED DESCRIPTION

[0054] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0055] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0056] The present invention will now be further described with reference to specific examples. The following examples are intended only to illustrate the present invention and are not intended to limit its contents. Experimental conditions not specified in the examples are generally based on conventional conditions or those recommended by the reagent company. Reagents and consumables used in the following examples are commercially available unless otherwise specified.

[0057] The present invention is further explained by the following examples, but is not intended to limit the present invention. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods in the following examples are generally carried out under conventional conditions.

[0058] Example

[0059] Mouse immunization

[0060] The amino acid sequence used for mouse UPAR immunization is the extracellular domain (uPAR ECD), as shown in SEQ ID NO. 14. Mouse uPAR has 327 amino acid residues. TMHMM prediction indicates that residues 1-303 are the extracellular domain, residues 304-326 are the transmembrane domain, and residue 327 is the intracellular domain. Furthermore, Novopro prediction indicates that the uPAR signal peptide is located at residues 1-23.

[0061] The uPAR amino acid sequence is shown in SEQ ID NO. 15. The uPAR extracellular region amino acid sequence is shown in SEQ ID NO. 16. The final immune protein selected is uPAR amino acid residues 24-300, and the immune protein sequence is shown in SEQ ID NO. 17.

[0062] The uPAR 24-300 amino acid residue sequence was synthesized by Qingke Biotechnology Co., Ltd., and the optimized DNA sequence was then constructed into the PGEX-4T-1 vector using NdeI and HindIII restriction sites through cloning technology. The sequence was then transformed into Escherichia coli and expressed in prokaryotes. The protein was purified multiple times and then used to immunize mice multiple times. After sequencing, a variety of antibody sequences with high affinity were obtained. The better uPAR scFv sequence was combined with the CD8 transmembrane region, 4-1BB, and CD3ζ to form a second-generation CAR, which was constructed into the lentiviral vector pCDH-EF1-MCS-T2A-copGFP.

[0063] Acquisition of uPAR extracellular domain protein:

[0064] The pMAL-c5x vector carrying a partial sequence of the uPAR extracellular domain was transferred into BL21 competent bacteria (Escherichia coli) for protein expression. The obtained protein was analyzed by SDS-PAGE gel electrophoresis. The protein molecular weight was estimated to be approximately 30 kDa based on the number of amino acid residues. The induced and activated bacteria were transferred to LB medium containing ampicillin resistance and the purified protein was obtained after culture.

[0065] Screening of high-affinity uPAR scFv:

[0066] Blood sampling and testing: This study followed a mature immunization process and completed titer determination and other indicator tests. Due to the large amount of test data, this study only summarized and summarized the mice and related data corresponding to the effective acquisition of the optimal cell line and antibody results. The remaining data is not reflected in this patent. After the first, second, and third blood sampling tests, the target mice with high titers that met the experimental requirements were selected for fusion. In this example, one mouse, mouse 3#, was selected from five mice for fusion. The blood sampling test results are shown in Table 1.

[0067] Table 1 Blood test results

[0068]

[0069] Fusion and cloning: SP2 / 0 mouse myeloma cells were used for cell fusion with spleen B cells of the preferred mouse (3#). After fusion, a batch of hybridoma cells that met the experimental requirements were obtained through culture, observation, detection and positive and negative control tests, and they were further cultured and selected.

[0070] Ascites and antibody testing: Ascites (antibodies) were prepared for all cell lines obtained in this study. After testing various indicators, a total of 10 ascites samples that met the project requirements were obtained. The test data are shown in Table 2.

[0071] Table 2 Ascites test results

[0072]

[0073] 3# Mouse strain identification and sequencing: After mouse fusion, primary cloning by limiting dilution, and supernatant testing, a total of 10 cell lines that met the project requirements were obtained (Table 3). By comparison, the hybridoma with the highest titer and best affinity was sequenced to obtain the full-length uPAR antibody sequence. The antibody heavy chain / light chain variable region genes were obtained by PCR, and the uPAR scFv was obtained.

[0074] Table 3 3# mouse hybridoma cell supernatant record

[0075]

[0076] CAR structure and CAR-T cell construction:

[0077] The present invention adopts the traditional second-generation CAR structure. The specific molecular structure sequence of CAR is: CD8 SP-VL-(G4S)3-VH-CD8TM-41BB-CD3ζ ( Figure 2 A).

[0078] Obtaining mouse T cells and constructing CAR-T cells: The T cells used in the present invention were derived from C57BL / 6J mice purchased from Shanghai Nanmo Biotechnology Co., Ltd. The mice were killed by cervical dislocation and immersed in 75% alcohol for about 5 minutes. The spleen of the mice was removed in a biosafety cabinet and ground to form a single cell suspension. Mouse T cells with a purity greater than 99% were obtained after sorting using a kit (EasySep Mouse T Cell Isolation Kit, STEMCELL). CD3 / CD28 activation magnetic beads from STEMCELL were added for activation, and the cells were cultured using a 50 IU mL -L IL-7 (Proteintech) and 100 IU mL -L IL-15 (Proteintech) was cultured in RPMI1640 medium. After 48 h, the cells were infected with lentivirus at an MOI of 100 for 6 h, and the positive rate was about 50% ( Figure 2C) of mouse uPAR CAR-T cells. At the same time, T cells obtained by infecting the virus with an empty lentiviral vector without uPAR CAR sequence were used as a control and named empty T cells. The lentivirus used in this study used the third-generation lentiviral packaging technology, which was packaged by our team through mature means and titered. In order to verify the cytotoxicity of the uPAR CAR-T cells in vitro, the experimenters constructed a cell line that overexpressed the full length of mouse uPAR (1-327 amino acid residues). Using the human hepatic stellate cell line LX2 and the mouse liver cancer cell line Hepa1-6 as platforms, they successfully constructed the uPAR-LX2 cell line and the uPAR-Hepa-6 cell line ( Figure 2 B).

[0079] The results showed that the present invention successfully constructed a second-generation CAR targeting mouse uPAR antigen ( Figure 2 A), the CAR and GFP protein were expressed in parallel through the T2A sequence, and the GFP expression showed the CAR expression efficiency. By using lentivirus to infect high-purity mouse T cells, CAR-T cells with a high positive rate were obtained ( Figure 2 C).

[0080] From the above experiments and their results, we can draw the following conclusions:

[0081] Through molecular cloning experiments, a traditional second-generation CAR targeting mouse uPAR antigen was successfully constructed and verified by sequencing. After obtaining mouse spleen-derived T cells, mouse CAR-T cells were successfully constructed. At the same time, uPAR-LX-2 and uPAR-Hepa1-6 cell lines overexpressing the full length of mouse uPAR were successfully constructed, laying the foundation for subsequent functional exploration of CAR-T cells and the smooth progress of in vitro and in vivo experiments.

[0082] Mouse uPAR CAR-T cells have a strong ability to kill uPAR+ cells in vitro:

[0083] Based on the successful construction of CAR-T cells targeting mouse uPAR antigen, we further validated the cytotoxicity of uPAR CAR-T cells. We co-cultured uPAR CAR-T cells with uPAR-LX2 cells and uPAR-Hepa1-6 cells, respectively. LX2 cells expressing mouse uPAR and Hepa1-6 cells expressing uPAR were artificially constructed (2B). The effector-target ratio was set at 5:1 and 1:1, respectively. The killing time was 12 hours, and the LDH assay was performed using an LDH kit (Promega).

[0084] The results showed that the killing efficiency of target cells by empty-T cells was very low, while the target cell death rate in the uPAR CAR-T cell group was relatively high ( Figure 2D, E).

[0085] From the above experiments and their results, we can draw the following conclusions:

[0086] uPAR CAR-T cells have a strong killing ability against uPAR+ cells in vitro, and the killing efficiency gradually increases with the increase of the effector-target ratio.

[0087] uPAR CAR-T cells inhibit tumor growth in vivo and prolong survival:

[0088] To explore the anti-tumor effect of uPAR CAR-T cells in vivo, the researchers used severely immunodeficient mice (B-NSG) (purchased from Biocytogen) to establish a model. After subcutaneous injection of mouse liver cancer cells expressing uPAR, uPAR CAR-T cells were infused back into the mice. Figure 3 A). The results showed that uPAR CAR-T cells effectively inhibited the growth of tumor cells. Compared with uPAR CAR-T cells, the tumor growth trend of mice in the empty-T cell group was similar to that of the PBS-transfused group, and the growth rate was faster ( Figure 3 B). In terms of mouse survival, the uPAR CAR-T cell-infused group significantly prolonged the survival of mice, with a median survival of 33.14 days, while the median survival of mice in the PBS group and the empty T cell group was 17.86 days and 18.57 days, respectively ( Figure 3 C).

[0089] From the above experiments and their results, we can draw the following conclusions:

[0090] uPAR CAR-T cells have strong anti-tumor function in vivo, significantly inhibiting the growth of tumor cells and prolonging the survival of mice, indicating that the uPAR scFv we screened can effectively recognize and bind to uPAR antigens after being expressed as protein in vivo.

[0091] uPAR CAR-T cells can effectively alleviate CCL4-induced liver fibrosis:

[0092] CCl4 was injected intraperitoneally into C57 mice to create a liver fibrosis model. After about 8 weeks, uPAR CAR-T cells were reinfused into the tail vein. One week later, the livers of the mice were harvested and examined using HE staining and picrosirius red staining. The results showed that the degree of liver fibrosis in the uPAR CAR-T cell-infused group was significantly reduced compared with the un-modeled mice group and the empty-T cell-infused group ( Figure 4 B, C).

[0093] From the above experiments and their results, we can draw the following conclusions:

[0094] uPAR CAR-T cells can alleviate liver fibrosis in mice by clearing activated HSCs in the liver.

[0095] The amino acid sequence information involved in the present invention is:

[0096] The CD8 signal peptide (SP) sequence is:

[0097] MASPLTRFLSLNLLLLGESIILGSGEA (SEQ ID NO.1)

[0098] The CD8 transmembrane region sequence is:

[0099] TTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHRSR (SEQ ID NO.2)

[0100] The sequence of the 4-1BB intracellular region is:

[0101] KWIRKKFPHIFKQPFKKTTGAAQEEDACSCRCPQEEEGGGGGYEL (SEQ ID NO.3)

[0102] The CD3ζ chain sequence is:

[0103] FSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR (SEQ ID NO.4)

[0104] The scFv-1 heavy chain CDR1 sequence is:

[0105] NYIMN (SEQ ID NO. 5)

[0106] The scFv-1 heavy chain CDR2 sequence is:

[0107] DIYKVNNRFSNEKFKT (SEQ ID NO.6)

[0108] The scFv-1 heavy chain CDR3 sequence is:

[0109] HIHYSGYTDYDPSLKS(SEQ ID NO.7)

[0110] The scFv-1 light chain CDR1 sequence is:

[0111] KSSQTAVSNVSSNILAA (SEQ ID NO.8)

[0112] The scFv-1 light chain CDR2 sequence is:

[0113] SASNLAS (SEQ ID NO. 9)

[0114] The scFv-1 light chain CDR3 sequence is:

[0115] HQFDIST (SEQ ID NO. 10)

[0116] The heavy chain sequence is:

[0117] QVQLQQSGAELVKPGTSVKMSCITSGYTFTNYIMNWVKQRPKRGLEWIGDIYKVNNRFSNEKFKTRATLSVDTSISTAYMNLSSLTQEDSAVYFCARHIHYSGYTDYDPSLKSWGAGTQVTVSS (SEQ ID NO.11)

[0118] The light chain sequence is:

[0119] DIVMTQSPSSLPVSLGQKVTMNCKSSQTAVSNVSSNILAAWYQQKFTGLIGKLLVYSASNLASGVPSLIGGSSGTNFTLTITSVQVEDLHQFDISTADYFCSASNWVFGGGTKLELK (SEQ ID NO.12)

[0120] The G4S linker sequence is:

[0121] GGGGSGGGGSGGGGS (SEQ ID NO.13)

[0122] The uPAR ECD sequence is:

[0123] FLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIALVDGRGTAFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGISSGRSQNHLYTHMTHFLKQCFSL (SEQ ID NO.14)

[0124] The amino acid sequence of uPAR is:

[0125] MGLPRRLLLLLLLATTCVPASQGLQCMQCESNQSCLVEECALGQDLCRTTVLREWQDDRELEVVTRGCAHSEKTNRTMSYRMGSMIISLTETVCATNLCNRPRPGARGRAFPQGRYLECASCTSLDQSCERGREQSLQCRYPTEHCIEVVTLQSTERSLKDEDYTRGCGSLPGCPGTAGFHSNQTFHFLKCCNYTHCNGGPVLDLQSFPPNGFQCYSCEGNNTLGCSSEEASLINCRGPMNQCLVATGLDVLGNRSYTVRGCATASWCQGSHVADSFPTHLNVSVSCCHGSGCNSPTGGAPRPGPAQLSLIASLLLTLGLWGVLLWT (SEQ ID NO.15)

[0126] The extracellular region amino acid sequence of uPAR is:

[0127] MGLPRRLLLLLLLATTCVPASQGLQCMQCESNQSCLVEECALGQDLCRTTVLREWQDDRELEVVTRGCAHSEKTNRTMSYRMGSMIISLTETVCATNLCNRPRPGARGRAFPQGRYLECASCTSLDQSCERGREQSLQCRYPTEHCIEVVTLQ STERSLKDEDYTRGCGSLPGCPGTAGFHSNQTFHFLKCCNYTHCNGGPVLDLQSFPPNGFQCYSCEGNNTLGCSSEEASLINCRGPMNQCLVATGLDVLGNRSYTVRGCATASWCQGSHVADSFPTHLNVSVSCCHGSGCNSPTGGAPRP (SEQ ID NO.16)

[0128] The uPAR amino acid residue sequence 24-300 is:

[0129] LQCMQCESNQSCLVEECALGQDLCRTTVLREWQDDRELEVVTRGCAHSEKTNRTMSYRMGSMIISLTETVCATNLCNRPRPGARGRAFPQGRYLECASCTSLDQSCERGREQSLQCRYPTEHCIEVVTLQSTERSLKDED YTRGCGSLPGCPGTAGFHSNQTFHFLKCCNYTHCNGGPVLDLQSFPPNGFQCYSCEGNNTLGCSSEEASLINCRGPMNQCLVATGLDVLGNRSYTVRGCATASWCQGSHVADSFPTHLNVSVSCCHGSGCNSPTGG (SEQ ID NO.17)

[0130] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A single-chain antibody targeting uPAR, characterized in that It comprises complementarity determining regions, comprising a heavy chain complementarity determining region and a light chain complementarity determining region; Wherein, the heavy chain complementarity determining region comprises: CDR1 with the amino acid sequence shown in SEQ ID NO.5, CDR2 with the amino acid sequence shown in SEQ ID NO.6, and CDR3 with the amino acid sequence shown in SEQ ID NO.7; The light chain complementarity determining region comprises: CDR1 with the amino acid sequence shown in SEQ ID NO.8, CDR2 with the amino acid sequence shown in SEQ ID NO.9, and CDR3 with the amino acid sequence shown in SEQ ID NO.

10.

2. The single-chain antibody according to claim 1, wherein The single-chain antibody targeting uPAR comprises: a heavy chain antibody V as shown in SEQ ID NO.11 H The amino acid sequence of the light chain antibody V shown in SEQ ID NO.12 L amino acid sequence.

3. The single-chain antibody according to claim 2, wherein The heavy chain antibody V H With light chain antibody V L Directly connected or connected through a connecting peptide; the connecting peptide is (G4S)n, wherein n is a positive integer.

4. The single-chain antibody according to claim 3, wherein The n is 3.

5. A chimeric antigen receptor targeting uPAR, characterized in that It comprises at least an antigen-binding domain, wherein the antigen-binding domain comprises the single-chain antibody according to any one of claims 1 to 4.

6. The chimeric antigen receptor according to claim 5, wherein The chimeric antigen receptor targeting uPAR is composed of a signal peptide, an antigen binding domain, a transmembrane region, a co-stimulatory signal transduction domain and a signal transduction domain in series; The signal peptide is a CD8 signal peptide, and its amino acid sequence is shown in SEQ ID NO.1; The transmembrane region is the CD8 transmembrane region, and its amino acid sequence is shown in SEQ ID NO.2; The costimulatory signal transduction domain is a 4-1BB costimulatory signal transduction domain, the amino acid sequence of which is shown in SEQ ID NO.3; The signal transduction domain is the CD3ζ signal transduction domain, and its amino acid sequence is shown in SEQ ID NO.

4. 7 . A nucleic acid molecule encoding the single-chain antibody targeting uPAR according to any one of claims 1 to 4 or encoding the chimeric antigen receptor targeting uPAR according to claim 5 or 6.

8. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid molecule of claim 7.

9. A CAR-T cell, characterized in that: The CAR-T cell is a T lymphocyte modified by the chimeric antigen receptor targeting uPAR according to claim 5 or 6.

10. A drug for preventing and / or treating diseases mediated by overexpression of uPAR, characterized in that: It comprises any one or more of the single-chain antibody according to any one of claims 1 to 4, the chimeric antigen receptor according to claim 5 or 6, the nucleic acid molecule according to claim 7, the recombinant expression vector according to claim 8, and the CAR-T cell according to claim 9.

11. The drug according to claim 10, characterized in that The drug further comprises a pharmaceutically acceptable carrier; The diseases mediated by high uPAR expression include fibrosis-mediated liver diseases and tumor diseases.

12. A detection product, characterized in that: The detection product comprises any one or more of the single-chain antibody according to any one of claims 1 to 4, the chimeric antigen receptor according to claim 5 or 6, the nucleic acid molecule according to claim 7, the recombinant expression vector according to claim 8, the CAR-T cell according to claim 9, and the drug according to any one of claims 10 to 11.

13. The detection product according to claim 12, wherein: The detection product is a detection kit or a detection device; the detection product can be used to qualitatively or quantitatively detect the expression of uPAR.

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