Single-chain antibody targeting uPAR, chimeric antigen receptor and application of single-chain antibody and chimeric antigen receptor

Abstract

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to a single-chain antibody targeting uPAR, a chimeric antigen receptor and application of the single-chain antibody and the chimeric antigen receptor. Aiming at uPAR molecules, a hybridoma cell strain is obtained through antigen extracellular region optimization, mouse immunization and fusion, a mouse source single-chain antibody sequence with high affinity is obtained after sequencing, and the mouse source single-chain antibody sequence is constructed into a CAR-T cell targeting uPAR. In-vitro experiments show that the CAR-T cells of the targeted uPAR can effectively eliminate astrocytes and tumor cells of uPAR +, and in-vivo experiments show that the CAR-T cells of the targeted uPAR can effectively improve liver fibrosis of mice and can inhibit growth of liver cancer of the mice at the same time. The research provides a new thought for clinical treatment of liver fibrosis and other diseases, and provides a new target for CAR-T immunotherapy in treatment of cardiovascular diseases, even solid tumors and other diseases, so that the CAR-T immunotherapy has good practical application value.

Description

Technical Field

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

[0002] The information disclosed in the background art of the present invention is only intended to enhance the overall understanding of the present invention, and does not necessarily constitute an admission or imply in any form that this information forms the prior art already known to those of ordinary skill in the art.

[0003] Chronic liver disease is one of the diseases that seriously threaten human life safety, and about 2 million people die from liver diseases globally every year. Chronic liver diseases mainly include alcoholic fatty liver, non-alcoholic fatty liver, etc., and their occurrence is often accompanied by liver fibrosis. After liver fibrosis occurs, if not intervened, it will seriously develop into liver cirrhosis and finally lead to liver cancer. Chronic liver diseases, including liver fibrosis, are mainly caused by chronic hepatitis B virus infection, excessive drinking or irregular diet, etc. The characteristics of liver fibrosis are accompanied by the production of extracellular matrix proteins, including collagen, and the infiltration of some immune cells (mainly T cells) releases a large amount of pro-inflammatory factors such as TGF-β, IL-1 and IL-6, thereby causing damage to liver parenchymal cells. Among them, the pro-inflammatory factors released by immune cells will induce fibroblasts and myofibroblasts, etc. to release collagen, thereby causing liver fibrosis.

[0004] Liver fibrosis has potential harmfulness and explosiveness, and a considerable part of it will develop into liver cirrhosis in the following 15 - 20 years. Its main clinical complications include ascites, renal failure, hepatic encephalopathy and variceal bleeding, etc. Liver fibrosis is a pathological phenomenon that occurs after repeated liver injury and healing, accompanied by a large accumulation of extracellular matrix and replication of its types. HSCs are the main inducing cells for the production of extracellular matrix. In normal liver, HSCs maintain the metabolic balance and homeostasis of the liver, and HSCs are the main storage depot of vitamin A. When chronic injury occurs, HSCs will be activated and transformed into myofibroblasts. The activated HSCs gradually migrate to the liver injury site and accumulate in large numbers, releasing a large amount of extracellular matrix, including collagen, etc. to repair the damaged liver, thereby causing liver fibrosis. So far, although many potential therapeutic targets for liver fibrosis have been discovered, there has not yet been a standardized and effective treatment method for alleviating liver fibrosis. This is because continuous liver biopsy is required during the treatment process to accurately evaluate the changes of liver fibrosis, and long-term follow-up studies are needed, but humans have a low sensitivity to liver anti-fibrosis therapies and it is difficult to achieve the expected effect.

[0005] At present, the main clinical treatment of liver fibrosis is the use of blocking drugs and various inhibitors, and mature, safe and effective immunotherapy has not yet been developed. There are currently many studies on the treatment of liver fibrosis with uPAR targeting, such as a research paper entitled "Senolytic CAR-T cells reverse senescence-associated pathologies" published by Corina Amor's team in the journal Nature. The CAR-T cells targeting uPAR constructed by the team can effectively remove senescent cells in the liver of mice, thereby reversing liver fibrosis. In addition, a study published in the Journal of Hepatology by Scott L.'s team in 2024 showed that activated and aged HSCs in the liver of mice with liver fibrosis highly expressed uPAR, and liver fibrosis could be effectively alleviated by constructing uPAR-targeted HSCs to eliminate senescent HSCs. In addition, studies have shown that in the process of fibrosis, mechanical injury and tumor development, uPAR plays an important role in the remodeling of the extracellular matrix, mainly because uPAR activates urokinase, further activates intracellular signaling pathways, and causes cells to release matrix to the extracellular space. Therefore, targeting uPAR to clear 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 (150kD). In the application of CAR-T, the effect of large molecular weight recognition antibodies is significantly inferior to scFv, and there is an urgent need to develop a high-affinity single-chain antibody (scFv). Summary of the invention

[0006] In view of the deficiencies of the above-mentioned prior art, the purpose of the present invention is to provide a single-chain antibody targeting uPAR, a chimeric antigen receptor and its application. Specifically, the present invention successfully prepares a single-chain antibody with high affinity for the uPAR molecule, and constructs a CAR and a mouse CAR-T cell that recognizes uPAR on the basis of the high-affinity scFv, and proves through in vitro and in vivo experiments that it has the effect of treating liver fibrosis and liver cancer. Based on the above research results, the present invention is completed.

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

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

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

[0010] (b) An amino acid sequence having at least 80% or more (such as 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity to any one of SEQ ID NOs. 5-10.

[0011] Furthermore, for the single-chain antibody targeting uPAR, the complementary determining regions include heavy-chain complementary determining regions and light-chain complementary determining regions;

[0012] Among them, the heavy-chain complementary determining regions include:

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

[0014] The light-chain complementary determining regions include:

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

[0016] Furthermore, the single-chain antibody can be a recombinant antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antigen-binding fragment of these antibodies, so as to expand its application scope, and no specific limitation is made here.

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

[0018] Furthermore, the heavy-chain antibody V H and the light-chain antibody V L can be directly connected or connected through a linker peptide; the linker peptide can specifically be (G4S)n, where n is a positive integer. For example, it can be any positive integer from 1 to 6. Preferably, n is 3, so as to be more conducive to the connection between the heavy-chain antibody V H and the light-chain antibody V L .

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

[0020] In the second aspect of the present invention, a chimeric antigen receptor targeting uPAR is provided, which at least includes an antigen-binding domain, and the antigen-binding domain includes the above single-chain antibody targeting uPAR.

[0021] Furthermore, the chimeric antigen receptor targeting uPAR is formed by tandemly connecting a signal peptide, an antigen-binding domain, a transmembrane region, a co-stimulatory signaling domain, and a signaling domain.

[0022] Among them, the signal peptide can be the CD8 signal peptide (Signal Peptide, SP), and its amino acid sequence is as shown in SEQ ID NO.1.

[0023] The transmembrane region can be the CD8 transmembrane region, and its amino acid sequence is as shown in SEQ ID NO.2.

[0024] The co-stimulatory signaling domain can be the 4-1BB co-stimulatory signaling domain, and its amino acid sequence is as shown in SEQ ID NO.3.

[0025] The signaling domain can be the CD3ζ signaling domain, and its amino acid sequence is as shown in SEQ ID NO.4.

[0026] In the third aspect of the present invention, a nucleic acid molecule is provided, and the nucleic acid molecule encodes the single-chain antibody targeting uPAR or the chimeric antigen receptor targeting uPAR.

[0027] In the fourth aspect of the present invention, a recombinant expression vector is provided, and the recombinant expression vector contains the above nucleic acid molecule.

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

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

[0030] In the present invention, the CAR-T cell can be obtained by infecting T cells with a lentivirus; the lentivirus is obtained by transfecting a lentiviral packaging cell with a recombinant lentiviral expression vector and then culturing the cells; the recombinant lentiviral expression vector is obtained by inserting the coding gene of the above chimeric antigen receptor into a lentiviral expression vector. In fact, those skilled in the art can prepare the above CAR-T cells according to the existing known technologies, and no specific limitation is made here.

[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] According to conventional practices, the drug can be formulated into dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc., for oral administration, topical application, suppositories, and sterile injection solutions.

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

[0034] The drug may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a microcapsule, liposome, nanoparticle or polymer and any combination thereof. The delivery vehicle of the pharmaceutically acceptable carrier may be a liposome, biocompatible polymer (including natural polymers and synthetic polymers), lipoprotein, polypeptide, polysaccharide, lipopolysaccharide, artificial virus envelope, inorganic (including metal) particles, and bacterial, phage, cosmid or plasmid vectors, etc., so as to broaden its application scope.

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

[0036] Furthermore, the drug can be administered into the body by known methods. For example, it can be delivered systemically via intravenous injection or locally injected into the tissue of interest. Such administration can be carried out via a single dose or multiple doses. Those skilled in the art understand that the actual dose to be administered in the present invention can vary to a large extent depending on various factors such as target cells, biological type or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, and so on.

[0037] Furthermore, the drug can be administered to humans or non-human mammals. The non-human mammals include mice, rats, guinea pigs, rabbits, dogs, monkeys, chimpanzees, etc., and are not specifically limited herein.

[0038] The present invention is verified through research. In vitro experiments of the uPAR-targeted CAR-T cells of the present invention show that the uPAR-targeted CAR-T cells can effectively eliminate uPAR+ stellate cells and tumor cells. In vivo experiments show that uPAR CAR-T cells can effectively improve liver fibrosis in mice and simultaneously inhibit the growth of liver cancer in mice. Therefore, the diseases mediated by high uPAR expression include fibrosis-mediated liver diseases and neoplastic diseases.

[0039] In the eighth aspect of the present invention, there is provided a detection product, which may comprise any one or more of the above-mentioned single-chain antibody, chimeric antigen receptor, CAR-T cell, drug, nucleic acid molecule, and recombinant expression vector.

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

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

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

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

[0044] The above technical solution targets the uPAR molecule. Through optimization of the extracellular region of the antigen, immunization of mice, and fusion to obtain a hybridoma cell line, a murine single-chain antibody sequence with relatively high affinity is obtained after sequencing, and it is constructed into uPAR-targeted CAR-T cells. In vitro experiments show that the uPAR-targeted CAR-T cells can effectively eliminate uPAR+ stellate cells and tumor cells. In vivo experiments show that uPAR CAR-T cells can effectively improve liver fibrosis in mice and simultaneously 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 new targets for CAR-T immunotherapy in the treatment of cardiovascular diseases and even solid tumors and other diseases. Therefore, it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The specification drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0046] Figure 1 Strategic diagram for immunizing mice of the present invention to obtain supernatants containing numerous antibody sequences

[0047] Among them: Five healthy C57 mice were immunized with the coating solution coated with the extracellular region antigen of uPAR. The immunization was carried out 5 times at intervals of about 15 days. During this period, the immunization effect was detected by orbital plexus venipuncture. On the third day after the 5th immunization, mouse spleen B cells were hybridized with myeloma cells. After screening, a positive monoclonal antibody cell line was obtained, and the antibody in the ascites was detected by ELISA to identify the optimal mouse cell line.

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

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

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

[0051] Among them: A is the diagram of mouse subcutaneous tumor modeling and uPAR CAR-T cell reinfusion strategy; B is the statistical chart of the change in the volume of mouse tumor cells, and the tumor volume was recorded every two days; C is the survival curve of mice. When the tumor volume of the mice reached 1000 mm 2 , the mice were declared dead.

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

[0053] Among them: A is the strategy of using CCL4 to establish a fibrosis model and reinfusing uPAR CAR-T cells; B is the detection of liver tissue fibrosis in mice by H.E.; C is the detection of liver tissue fibrosis in mice by Sirius red. Detailed implementation manners

[0054] It should be noted that the following detailed description is illustrative and is 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 of ordinary skill in the technical field 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 forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0056] The present invention will be further described in conjunction with specific examples below. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0057] The present invention will be further explained and illustrated by the following examples, but it does not constitute a limitation to the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The test methods without specific conditions indicated in the following examples are usually carried out under conventional conditions.

[0058] Example

[0059] Mouse immunization

[0060] For mouse uPAR immunization, the amino acid sequence of the extracellular region (uPAR ECD) is used, and the specific sequence is shown in SEQ ID NO.14. The mouse uPAR has 327 amino acid residues. Predicted by the TMHMM website, it shows that the 1-303 amino acid residues are the extracellular region, the 304-326 amino acid residues are the transmembrane region, and the 327 amino acid residue is the intracellular region. In addition, predicted by the Novopro website, the uPAR signal peptide is 1-23.

[0061] Among them, the uPAR amino acid sequence is shown in SEQ ID NO.15. The uPAR amino acid extracellular region sequence is shown in SEQ ID NO.16. Finally, the immunoprotein selected is the 24-300 amino acid residues of uPAR, and the immunoprotein sequence is shown in SEQ ID NO.17.

[0062] The uPAR 24 - 300 amino acid residue sequence was synthesized by Tsingke Biotechnology Company. Then, through cloning technology, the optimized DNA sequence was constructed into the PGEX-4T-1 vector using NdeI and HindIII as restriction sites, and transferred into Escherichia coli. After prokaryotic protein expression, the protein was purified multiple times, and then mice were immunized multiple times. After sequencing, multiple antibody sequences with higher affinity were obtained. The optimized uPAR scFv sequence, together with the CD8 transmembrane region, 4-1BB, and CD3ζ, was used to form the second-generation CAR and constructed into the lentiviral vector pCDH-EF1-MCS-T2A-copGFP.

[0063] Obtaining the uPAR extracellular region protein:

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

[0065] Screening of high-affinity uPAR scFv:

[0066] Blood sampling and detection: In this study, immunization was carried out according to the established immune protocol, and indicators such as titer determination were completed. Due to the large amount of detection data, only the data of the mice corresponding to the optimal cell line and antibody results obtained effectively and their related data were summarized and presented in this patent. The rest of the data was not included. After the first, second, and third blood samplings and detections, target mice with higher titers meeting the experimental requirements were selected for cell fusion. In this example, 1 mouse (No. 3) was preferably selected from 5 mice for fusion. The blood sampling and detection results are shown in Table 1.

[0067] Table 1 Blood sampling and detection results

[0068]

[0069] Fusion and cloning: Cell fusion was performed using SP2 / 0 mouse myeloma cells and spleen B cells of the selected mouse (No. 3). After fusion, through cultivation, observation, detection, and positive and negative control experiments, a batch of hybridoma cells meeting the experimental requirements was obtained, and they were continuously cultured and selected.

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

[0071] Table 2 Ascites detection results

[0072]

[0073] 3# Mouse Monoclonal Cell Line Selection and Sequencing: After the mice were fused, cloned once by limiting dilution method, and the supernatants were detected, a total of 10 cell lines meeting the project requirements were obtained (Table 3). Through comparison, one hybridoma with high titer and optimal affinity was sequenced to obtain the full-length uPAR antibody sequence. The variable region genes of the heavy chain / light chain of the antibody were obtained by PCR to obtain uPAR scFv.

[0074] Table 3 Record Table of Supernatants of 3# Mouse Hybridoma Cells

[0075]

[0076] Structure of CAR and Construction of CAR-T Cells:

[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 are from C57BL / 6J mice, which are purchased from Shanghai Model Organisms Center, Inc. After the mice are sacrificed by cervical dislocation and placed in 75% alcohol for about 5 minutes, the spleens of the mice are removed in a biosafety cabinet, ground to form a single cell suspension, and mouse T cells with a purity greater than 99% are obtained by sorting using a kit (EasySep Mouse T Cell Isolation Kit, STEMCELL). CD3 / CD28 activation magnetic beads from STEMCELL are added for activation, and RPMI1640 medium containing 10% fetal bovine serum, 50 IU / mL -L IL-7 (Proteintech) and 100 IU / mL -L IL-15 (Proteintech) is used for culture. After 48 hours, lentivirus with an MOI = 100 is used for infection, and the infection time is 6 hours. Thus, CAR-T cells with a positive rate of about 50% are obtained ( Figure 2Mouse uPAR CAR-T cells in group (C). Meanwhile, lentiviral empty vectors without the uPAR CAR sequence were used to package viruses to infect T cells as a control, named empty vector T cells. The lentiviruses used in this study employed the third-generation lentiviral packaging technology, which was packaged by our team through mature means and the titer was detected. To verify the cytotoxicity of the uPAR CAR-T cells in vitro, the experimental personnel constructed a cell line overexpressing the full-length mouse uPAR (1-327 amino acid residues). Using the human hepatic stellate cell line LX2 and the mouse hepatoma cell line Hepa1-6 as platforms, the uPAR-LX2 cell line and the uPAR-Hepa-6 cell line were successfully constructed ( Figure 2 B).

[0079] The results showed that: The present invention successfully constructed a second-generation CAR targeting the mouse uPAR antigen ( Figure 2 A), and this CAR was co-expressed with the GFP protein through the T2A sequence, and the GFP expression was used to show the CAR expression efficiency. By using lentiviruses to infect highly purified mouse T cells, CAR-T cells with a relatively high positive rate were obtained ( Figure 2 C).

[0080] From the above experiments and their results, the following conclusions can be drawn:

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

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

[0083] On the basis of successfully constructing CAR-T cells targeting the mouse uPAR antigen, the cytotoxicity of uPAR CAR-T cells was further verified. We co-cultured uPAR CAR-T cells with uPAR-LX2 cells and uPAR-Hepa1-6 cells respectively. The LX2 cells expressing mouse uPAR and the Hepa1-6 cells expressing uPAR were artificially constructed (2B). The effector-to-target ratio was set at 5:1 and 1:1, and the killing time was 12 h. The LDH kit (Promega) was used for detection.

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

[0085] From the above experiments and their results, the following conclusions can be drawn:

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

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

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

[0089] From the above experiments and their results, the following conclusions can be drawn:

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

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

[0092] C57 mice were intraperitoneally injected with CCl4 to establish a liver fibrosis model. After about 8 weeks, uPAR CAR-T cells were infused back via the tail vein. One week later, the livers of the mice were taken, and H.E. staining and Sirius red staining methods were used for detection, showing that compared with the mice in the non-model group and the empty vector-T cell infusion group, the degree of liver fibrosis in the mice in the uPAR CAR-T cell infusion group was significantly reduced ( Figure 4 B, C).

[0093] From the above experiments and their results, the following conclusions can be drawn:

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

[0095] Amino acid sequence information involved in the present invention:

[0096] The CD8 signal peptide (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 4-1BB intracellular region sequence 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-3 light chain CDR1 sequence is:

[0111] KSSQTAVSNVSSNILAA (SEQ ID NO.8)

[0112] The CDR2 sequence of the scFv-3 light chain is:

[0113] SASNLAS (SEQ ID NO.9)

[0114] The CDR3 sequence of the scFv-3 light chain 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] DIVMTQSPSSLPVSLGQKVTMNCKSSQTAVSNVSSNILAAWYQQKFTGLIGKLLVYSASNLASGVPSLIGGSGSGTNFTLTITSVQVEDLHQFDISTADYFCSASNWVFGGGTKLELK (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] MGLPRRLLLLLLLATTCVPASQGLQCMQCESNQSCLVEECALGQDLCRTTVLREWQDDRELEVVTRGCAHSEKTNRTMSYRMGSMIISLTETVCATNLCNRPRPGARGRAFPQGRYLECASCTSLDQSCERGREQSLQCRYPTEHCIEVVTLQSTERSLKDEDYTRGCGSLPGCPGTAGFHSNQTFHFLKCCNYTHCNGGPVLDLQSFPPNGFQCYSCEGNNTLGCSSEEASLINCRGPMNQCLVATGLDVLGNRSYTVRGCATASWCQGSHVADSFPTHLNVSVSCCHGSGCNSPTGGAPRP (SEQ ID NO.16)

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

[0129] LQCMQCESNQSCLVEECALGQDLCRTTVLREWQDDRELEVVTRGCAHSEKTNRTMSYRMGSMIISLTETVCATNLCNRPRPGARGRAFPQGRYLECASCTSLDQSCERGREQSLQCRYPTEHCIEVVTLQSTERSLKDEDYTRGCGSLPGCPGTAGFHSNQTFHFLKCCNYTHCNGGPVLDLQSFPPNGFQCYSCEGNNTLGCSSEEASLINCRGPMNQCLVATGLDVLGNRSYTVRGCATASWCQGSHVADSFPTHLNVSVSCCHGSGCNSPTGG (SEQ ID NO.17)

[0130] The above - mentioned embodiments are only for illustrating the technical concept and features of the present invention. The purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it accordingly, and it cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A single-chain antibody targeting uPAR, characterized in that, It includes complementary determining regions, and the amino acid sequences of the complementary determining regions are selected from: (a) The amino acid sequences shown in any one of SEQ ID NOs. 5-10; (b) Amino acid sequences that are at least 80% identical to any one of the amino acid sequences shown in SEQ ID NOs. 5-10, respectively.

2. The single-chain antibody according to claim 1, characterized in that The single-chain antibody targeting uPAR, and the complementary determining regions include heavy-chain complementary determining regions and light-chain complementary determining regions; Among them, the heavy-chain complementary determining regions include: 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 complementary determining regions include: 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; Further, the single-chain antibody targeting uPAR comprises: the heavy-chain antibody V with the amino acid sequence shown in SEQ ID NO. 11, and the light-chain antibody V with the amino acid sequence shown in SEQ ID NO. 12; H L ​​ Further, the heavy-chain antibody V H is directly linked to or linked through a linker peptide to the light-chain antibody V L ; the linker peptide is (G4S)n, where n is a positive integer, and further, n is 3.

3. A chimeric antigen receptor targeting uPAR, characterized in that, It at least includes an antigen-binding domain, and the antigen-binding domain includes the single-chain antibody described in claim 1 or 2; Furthermore, the chimeric antigen receptor targeting uPAR is formed by serially connecting a signal peptide, an antigen-binding domain, a transmembrane region, a co-stimulatory signaling domain, and a signaling domain; The signal peptide is the 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 co-stimulatory signaling domain is the 4-1BB co-stimulatory signaling domain, and its amino acid sequence is shown in SEQ ID NO. 3; The signaling domain is the CD3ζ signaling domain, and its amino acid sequence is shown in SEQ ID NO.

4.

4. A nucleic acid molecule encoding the single-chain antibody targeting uPAR described in any one of claims 1-2 or encoding the chimeric antigen receptor targeting uPAR described in claim 3.

5. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid molecule described in claim 4.

6. A CAR-T cell, characterized in that, The CAR-T cell is a T lymphocyte modified by the chimeric antigen receptor targeting uPAR described in claim 3.

7. A drug for preventing and / or treating diseases mediated by high uPAR expression, characterized in that, It includes any one or more of the single-chain antibody described in claim 1 or 2, the chimeric antigen receptor described in claim 3, the nucleic acid molecule described in claim 4, the recombinant expression vector described in claim 5, and the CAR-T cell described in claim 6.

8. The medicament according to claim 7, characterized in that, The drug further includes a pharmaceutically acceptable carrier; The diseases mediated by high uPAR expression include fibrosis-mediated liver diseases and neoplastic diseases.

9. A detection product, characterized in that, The detection product includes any one or more of the single-chain antibody described in claim 1 or 2, the chimeric antigen receptor described in claim 3, the nucleic acid molecule described in claim 4, the recombinant expression vector described in claim 5, the CAR-T cell described in claim 6, and the drug described in any one of claims 7-8.

10. The detection product according to claim 9, wherein The detection product is a detection kit or a detection device; the detection product can be used for qualitative or quantitative detection of the expression of uPAR.

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