A chimeric antigen receptor targeting PDGFRβ, CAR-T cell and application thereof

Abstract

The present invention belongs to the field of biomedicine and molecular biology technology, and specifically relates to a chimeric antigen receptor targeting PDGFRβ, CAR-T cells and applications. Specifically, the present invention targets PDGFRβ molecules, obtains mouse single-chain antibody sequences through monoclonal antibody design and immunization of mice, constructs CAR-T cells targeting PDGFRβ, and confirms its in vitro killing of activated fibroblasts and therapeutic effects in hypertension or diabetes-related renal and cardiac fibrosis, providing a new method for clearing PDGFRβ in clinical treatment. + Myofibroblasts and fibroblasts provide a basis for the treatment of CKD diseases such as renal fibrosis, and provide new ideas for the clinical treatment of liver fibrosis, cardiovascular disease and various tumor diseases. Therefore, they have 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 chimeric antigen receptor targeting PDGFRβ, a CAR-T cell and an application thereof. Background Art

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

[0003] Fibrosis is a common pathological endpoint of chronic, progressive or severe acute diseases in almost all organs, and is also one of the bottlenecks in the treatment of various chronic diseases. Chronic Kidney Disease (CKD) is a global public health problem that seriously threatens human health. With the aging of the population, the increase in the prevalence of hypertension and diabetes, the global adult CKD prevalence is as high as more than 10%. It is expected that by 2040, CKD will become the fifth most common cause of death in the world, and by the end of this century, it will become the second leading cause of death in countries with a longer life expectancy. Renal fibrosis is an inevitable stage for chronic kidney disease caused by various reasons to develop into end-stage renal disease. The main clinical drugs for CKD, angiotensin-converting enzyme inhibitors and angiotensin receptor blockers, can slow down the decline of renal function, but cannot cure CKD and may have serious side effects. Studies on renal protective drugs such as TGF-β inhibitors, endothelin inhibitors, and renin inhibitors have all ended in failure. Although the hypoglycemic SGLT2 inhibitor canagliflozin has recently been used to treat CKD, the current treatment drug options are still very limited, and there is no method that can effectively prevent renal fibrosis and reverse the loss of renal function in CKD.

[0004] In recent years, chimeric antigen receptor (CAR)-modified T cells, namely CAR-T, have shown good anti-tumor effects in the treatment of malignant tumors such as leukemia, lymphoma, melanoma, and glioma. CAR-T immunotherapy is a technology that uses genetic engineering technology to transform T cells so that T cells can specifically recognize and kill target cells. A large number of studies have shown that CAR-T cells with 4-1BB as a co-stimulatory molecule have stronger persistence in the body, and the survival time in the body can exceed 3 months or even reach one year; while CAR-T cells with CD28 as a co-stimulatory molecule have a strong proliferation ability in the short term, but poor persistence, and the survival time in the body generally does not exceed one month. Recently, CAR-T cell therapy has been tried to treat non-tumor diseases such as autoimmune diseases and metabolic disorders, such as systemic lupus erythematosus, rheumatoid arthritis, and vascular diseases. Studies at the laboratory animal level have shown that CAR-T cells have a significant improvement in the treatment of liver fibrosis and myocardial fibrosis. However, the application of CAR-T cells in the treatment of chronic kidney disease and its related renal fibrosis is still blank.

[0005] CAR-T immune cells rely on genetically engineered recognition sequences, such as single-chain antibodies, ligands, receptors, and other antibodies that specifically recognize target cells, thereby effectively eliminating target cells. Currently, no suitable and effective CAR-T therapy targets targeting renal fibroblasts and myofibroblasts have been reported. Platelet-derived growth factor receptor β (PDGFRβ) belongs to the membrane molecule of the receptor tyrosine kinase family and is mainly expressed on the surface of fibroblasts, hepatic stellate cells, pericytes, myofibroblasts, and some tumor cells. PDGFRβ is a tyrosine kinase receptor for PDGF-B and PDGF-D. After activation, PDGFRβ induces downstream signaling, triggering cell proliferation, migration, and differentiation. Increased PDGFRβ signaling caused by activating mutations, gene translocations, or increased ligand abundance is associated with the pathogenesis of malignant tumors, atherosclerosis, and organ fibrosis. In kidney disease, blocking PDGFRβ signaling using specific PDGFRβ antibodies or nonspecific tyrosine kinase receptor inhibitors (such as imatinib) can improve renal injury in various kidney disease models. Neutralization of the PDGFRβ ligands PDGF-B or PDGF-D also has a protective effect in renal injury models. Currently, there are no studies on CAR-T immunotherapy targeting PDGFRβ. In CKD, myofibroblasts are important cells that promote renal fibrosis. They are mainly derived from pericytes around peritubular capillaries and perivascular fibroblasts. Compared with these source cells, myofibroblasts have higher expression of PDGFRβ on the surface.

[0006] At the same time, CAR-T cells targeting PDGFRβ can inhibit renal fibrosis, prevent or even reverse the progression of CKD by clearing myofibroblasts and reducing their source cells. At the same time, CKD and cardiovascular disease (CVD) have common causes, such as diabetes and hypertension. Myofibroblasts in CVD hearts are derived from the expansion and differentiation of fibroblasts in damaged tissues. Therefore, CAR-T cells targeting PDGFRβ can simultaneously protect the fibrosis of the kidneys, heart and other affected organs caused by the same precursor disease. Summary of the invention

[0007] In view of the above-mentioned prior art, the purpose of the present invention is to provide a chimeric antigen receptor, CAR-T cell and application targeting PDGFRβ. Specifically, the present invention targets the PDGFRβ molecule, obtains the mouse single-chain antibody sequence by monoclonal antibody design and immunization of mice, constructs CAR-T cells targeting PDGFRβ, and confirms its therapeutic effect in killing activated fibroblasts in vitro and in hypertension or diabetes-related renal and cardiac fibrosis, providing a new target for CAR-T immunotherapy in the treatment of diseases such as CKD and CVD. Based on the above research results, the present invention is completed.

[0008] In order to achieve the above technical objectives, the technical solution provided by the present invention is as follows:

[0009] In a first aspect of the present invention, there is provided a chimeric antigen receptor targeting PDGFRβ, which comprises at least an antigen binding domain;

[0010] Wherein, the antigen binding domain is an anti-PDGFRβ antibody, and the anti-PDGFRβ antibody may be a single-chain antibody (scFv), which is composed of an antibody heavy chain and an antibody light chain;

[0011] The amino acid sequence of the antibody heavy chain (VH) is selected from:

[0012] a1) the amino acid sequence shown in SEQ ID NO.1; or,

[0013] a2) a polypeptide having the same function as the amino acid sequence shown in SEQ ID NO.1, wherein one or more amino acid residues are substituted and / or deleted and / or added;

[0014] The amino acid sequence of the antibody light chain (VL) is selected from:

[0015] b1) the amino acid sequence shown in SEQ ID NO.2; or

[0016] b2) A polypeptide having the same function as the amino acid sequence shown in SEQ ID NO. 2, wherein one or more amino acid residues are substituted and / or deleted and / or added.

[0017] In a2) and b2) above, the "one or several amino acids" are 1-10 amino acids, further 1-7 amino acids; more preferably, they are derivative polypeptides formed by substitution and / or deletion and / or addition of 1-3 amino acid residues.

[0018] Furthermore, the antibody heavy chain and the antibody light chain can be directly connected or connected through a linker; the linker is preferably selected from a polypeptide chain composed of alanine and / or serine and / or glycine, and the length of the linker is preferably 3 to 30 amino acids. Preferably, the linker is (GGGGS)n, where n is a positive integer, such as 1, 2, 3, 4, 5 or 6, and more preferably, n is 3.

[0019] Furthermore, the chimeric antigen receptor targeting PDGFRβ comprises a signal peptide, an antigen binding domain, a chimeric receptor hinge region, a co-stimulatory signal transduction domain and a signal transduction domain connected in series in sequence;

[0020] Further, the signal peptide is a CD8 SP signal peptide;

[0021] Further, the chimeric receptor hinge region is CD8 Hinge;

[0022] Further, the co-stimulatory signaling domain is a 4-1BB co-stimulatory signaling domain;

[0023] Further, the signal transduction domain is a CD3ζ signal transduction domain;

[0024] According to the present invention, the chimeric antigen receptor is composed of a CD8 SP signal peptide, an antigen binding domain (VH-(GGGGS)3-VL) that binds to the PDGFRβ antigen, a CD8 Hinge, a 4-1BB co-stimulatory signaling domain, and a CD3ζ signaling domain in series.

[0025] The second aspect of the present invention provides a nucleic acid molecule comprising nucleotides encoding the above-mentioned chimeric antigen receptor targeting PDGFRβ.

[0026] It should be noted that a person skilled in the art can easily mutate the nucleotide sequence encoding the chimeric antigen receptor targeting PDGFRβ of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the chimeric antigen receptor and have the same function.

[0027] Furthermore, the nucleic acid molecule sequentially includes a gene sequence encoding a CD8SP signal peptide, a gene sequence encoding an antigen binding domain that binds to the PDGFRβ antigen, a gene sequence encoding a CD8 Hinge, a gene sequence encoding a 4-1BB co-stimulatory signal transduction domain, and a gene sequence encoding a CD3ζ signal transduction domain.

[0028] The third aspect of the present invention provides a recombinant expression vector, which comprises the nucleic acid molecule described in the second aspect.

[0029] According to the present invention, the recombinant expression vector is a viral vector, and the viral vector may be a retroviral vector or a lentiviral vector; further preferably, it is a lentiviral vector, and the recombinant expression vector is a recombinant viral vector expressing the chimeric antigen receptor by inserting the nucleic acid molecule of the chimeric antigen receptor into a virus.

[0030] The fourth aspect of the present invention provides a CAR-T cell, which is a T lymphocyte modified by the chimeric antigen receptor targeting PDGFRβ. The present invention provides a new treatment method for CAR-T treatment of CKD and CVD and other related diseases with high expression of PDGFRβ.

[0031] Specifically, the CAR-T cells can be prepared by the following methods, such as infecting T cells with lentivirus; the lentivirus is obtained by transfecting lentivirus packaging cells with a recombinant lentiviral vector and then culturing the cells; the lentiviral vector is obtained by inserting the coding gene of the above-mentioned chimeric antigen receptor into a lentiviral vector.

[0032] The fifth aspect of the present invention provides the use of the above-mentioned chimeric antigen receptor, nucleic acid molecule, recombinant expression vector, and CAR-T cell in the preparation of drugs for preventing and / or treating diseases related to high PDGFRβ expression.

[0033] Specifically, the diseases associated with high PDGFRβ expression include, but are not limited to, kidney disease, liver disease, cardiovascular disease and tumor diseases, such as chronic kidney disease mediated by diabetes and / or hypertension, etc., which are not specifically limited here.

[0034] A sixth aspect of the present invention provides a product for preventing and / or treating diseases related to high PDGFRβ expression, wherein the active ingredient of the product may be the above-mentioned chimeric antigen receptor or the above-mentioned CAR-T cell.

[0035] Specifically, the diseases associated with high PDGFRβ expression include, but are not limited to, kidney disease, liver disease, cardiovascular disease and tumor diseases, such as chronic kidney disease mediated by diabetes and / or hypertension, etc., which are not specifically limited here.

[0036] The product may be a drug; when the product is a drug, the drug may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier 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.

[0037] The drug may also include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a microcapsule, a liposome, a nanoparticle or a polymer and any combination thereof. The delivery vehicle of the pharmaceutically acceptable carrier may be a liposome, a biocompatible polymer (including a natural polymer and a synthetic polymer), a lipoprotein, a polypeptide, a polysaccharide, a lipopolysaccharide, an artificial viral envelope, an inorganic (including metal) particle, and a bacteria, a bacteriophage, a clay or a plasmid vector.

[0038] The drug can also be used in combination with other drugs for preventing and / or treating diseases related to high PDGFRβ expression. Other preventive and / or therapeutic compounds can be administered simultaneously with the main active ingredient, or even administered simultaneously in the same composition.

[0039] The medicine of the present invention can be applied to the body by known means. For example, it can be delivered to the tissue of interest by intravenous systemic delivery or local injection. It can be applied optionally via intravenous, percutaneous, intranasal, mucosal or other delivery methods. Such application can be carried out via single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be applied in the present invention can vary depending on various factors to a great extent, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0040] The seventh aspect of the present invention provides a method for preventing and / or treating diseases related to high expression of PDGFRβ, which comprises administering an effective amount of the above-mentioned chimeric antigen receptor, CAR-T cell or product to a subject.

[0041] Beneficial technical effects of one or more of the above technical solutions:

[0042] The above technical scheme uses PDGFRβ as a target, thereby successfully preparing an antibody targeting PDGFRβ and constructing a second-generation CAR plasmid vector. By isolating T cells and programming them with a lentiviral vector encoding CAR, a T cell expressing a chimeric antigen receptor (CAR) targeting PDGFRβ is obtained. Experiments have shown that PDGFRβCAR-T cells can effectively eliminate PDGFRβ in vitro. + Target cells, in vivo experiments were carried out by constructing a variety of mouse CKD models, including UUO models, diabetes and hypertension-related chronic kidney disease (CKD) models, etc. After the CAR-T cells were reinfused into the tail vein, it was found that compared with the untreated group, the degree of renal fibrosis in mice was significantly reduced, and the expression levels of fibrosis-related proteins such as Vimentin, Fibronectin, Collagen I, and α-SMA decreased. In vivo experiments showed that PDGFRβCAR-T cells can effectively prevent and treat mouse CKD, etc. This invention provides an antibody that recognizes and binds to the mouse PDGFRβ antigen with high affinity. The CAR-T cells constructed based on this antibody showed good therapeutic effects in mice. This study provides a method for clearing PDGFRβ in clinical treatment. + Myofibroblasts and fibroblasts provide a basis for the treatment of CKD diseases such as renal fibrosis, and provide new ideas for the clinical treatment of liver fibrosis, cardiovascular disease and various tumor diseases. Therefore, they have good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The accompanying drawings in the specification, 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.

[0044] Figure 1 This is a flowchart of mouse immunization in an embodiment of the present invention.

[0045] Figure 2 This is the mouse CAR-T cell construction and killing experiment in the examples of the present invention.

[0046] Among them: A is the structure and original picture of PDGFRβCAR; B is the lentiviral vector used for construction; C is the flow cytometry identification of mouse T cells after sorting; D is the detection of PDGFRβCAR-T cell positive rate; E is the killing ability detection picture of PDGFRβCAR-T cells;

[0047] Figure 3 This is the example of the present invention where PDGFRβCAR-T cells prevent renal fibrosis caused by the UUO model.

[0048] Among them: A is Western blot detection of changes in fibrosis-related proteins Vimentin, Fibronectin, E-cadherin, and α-sma in the renal cortex of UUO model mice; B is immunohistochemistry detection of changes in fibrosis-related proteins Fibronectin and α-sma in the renal cortex of UUO model mice; C is Sirius Red and Masson staining to detect the degree of renal fibrosis in mice.

[0049] Figure 4 PDGFRβCAR-T cells improve renal fibrosis caused by UUO model in the embodiment of the present invention.

[0050] Among them: A is Western blot detection of changes in fibrosis-related proteins Vimentin, Fibronectin, Collagen I, and α-sma in the renal cortex of UUO model mice; B is immunohistochemistry detection of changes in fibrosis-related proteins Fibronectin and α-sma in the renal cortex of UUO model mice; C is Sirius Red and Masson staining to detect the degree of renal fibrosis in mice.

[0051] Figure 5 In the example of the present invention, PDGFRβCAR-T cells can improve fibrosis caused by the DOCA / salt hypertension model.

[0052] Among them: A is Western blot detection of the changes of fibrosis-related proteins Vimentin, Fibronectin, Collagen I, and α-sma in the renal cortex of DOCA / salt hypertension model mice; B is echocardiography detection of cardiac function of DOCA / salt hypertension model mice; C is HE staining detection of the changes in myocardial thickness of DOCA / salt hypertension model mice and Sirius Red detection of the degree of fibrosis of small blood vessels in the heart of DOCA / salt hypertension model mice; D is HE staining detection of the changes in aortic wall thickness of DOCA / salt hypertension model mice; E is the change in the ratio of kidney weight to body weight of each group of DOCA / salt hypertension model mice; F is the change in urinary microalbumin in each group of DOCA / salt hypertension model mice; G is immunohistochemistry detection of the changes in fibrosis-related protein α-sma in the renal cortex of DOCA / salt hypertension model mice; Sirius Red and Masson staining were used to detect the degree of renal fibrosis in DOCA / salt hypertension model mice.

[0053] Figure 6 In the embodiment of the present invention, PDGFRβCAR-T cells can improve fibrosis caused by AngII hypertension model.

[0054] Among them: A is Western blot detection of changes in fibrosis-related proteins α-sma, Vimentin, and E-cadherin in the renal cortex of AngII hypertension model mice; B is echocardiography detection of cardiac function of AngII hypertension model mice; C is HE staining detection of changes in myocardial thickness of AngII hypertension model mice and Sirius Red detection of the degree of fibrosis of small blood vessels in the heart of AngII hypertension model mice; D is HE staining detection of changes in aortic wall thickness of AngII hypertension model mice; E is the change in kidney weight to body weight ratio of each group of AngII hypertension mice; F is the change in urinary microalbumin in each group of AngII hypertension mice; G is immunohistochemistry detection of changes in fibrosis-related protein α-sma in the renal cortex of AngII hypertension model mice; Sirius Red and Masson staining detect the degree of renal fibrosis in AngII hypertension model mice. DETAILED DESCRIPTION

[0055] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0056] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0057] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out according to conventional conditions or the conditions recommended by the reagent company; the reagents and consumables used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0058] In the present invention, the term "antibody" is used as a general term to include full-length antibodies, their individual chains, and all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments, such as VHH domains or VH / VL domains, respectively) whether it refers to a heavy chain antibody or a conventional four-chain antibody. In addition, the term "sequence" used herein (such as in the terms "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence", etc.) is generally understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless a more limited explanation is required herein.

[0059] The term "domain" (of a polypeptide or protein) refers to a folded protein structure that is able to maintain its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and in many cases can be added, removed or transferred to other proteins without loss of the function of the rest of the protein and / or the domain.

[0060] The term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

[0061] The term "affinity" is theoretically defined by the equilibrium association between intact antibody and antigen. Affinity herein can be measured by K D The dissociation constant (or other determination method) is evaluated or determined, for example, by bio-layer interferometry (BLI) using a FortebioRed96 instrument.

[0062] An "effective amount" of an agent is that amount necessary to effect a physiological change in the cell or tissue to which it is administered.

[0063] A "therapeutically effective amount" of an agent, such as a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or preventive result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes or prevents the adverse effects of a disease.

[0064] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the individual or subject is a human.

[0065] The term "treatment / prevention" refers to an attempt to alter the natural course of a disease in a treated individual, and may be a clinical intervention performed for prevention or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and eliminating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the formation of a disease or slow the progression of a condition.

[0066] Specifically, the cells designed in the present invention are mouse T cells.

[0067] Among them, mouse T cells were derived from the spleen of C57BL / 6 mice. In the in vivo experiment, the CAR-T cells were injected into the tail vein, and the infusion dose was 1×10 5 , the return system is 100-200μL.

[0068] The CAR-T cells used in the present invention are constructed by lentiviral infection.

[0069] Among them: the name of the lentiviral vector is pLVX-IRES-ZsGreen1, the lentivirus is provided by GeneCare Gene, NewHe Bio and other companies, and the infection MOI=100.

[0070] The CAR-T cells targeting mouse PDGFRβ of the present invention can kill mouse fibroblast cell lines well in vitro, and can effectively prevent and improve kidney disease in vivo. In the experiment of the present invention, by creating a variety of kidney disease models, including unilateral ureteral obstruction model (UUO), deoxycorticosterone acetate-induced hypertension model, etc., it was found that mouse PDGFRβCAR-T cells can alleviate and improve the above various kidney diseases. This study provides a novel treatment method for CAR-T immunotherapy for kidney diseases including renal fibrosis, liver diseases, cardiovascular diseases, and various tumor diseases with high expression of PDGFRβ. More importantly, high-dose reinfusion of mouse PDGFRβCAR-T cells to treat kidney disease did not cause obvious side effects, providing a theoretical basis for the clinical application of PDGFRβCAR-T cells.

[0071] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods for the specific conditions in the following examples are usually carried out under normal conditions.

[0072] Example

[0073] method:

[0074] The process of mouse immunization

[0075] Gene synthesis design was performed, and the optimized DNA was constructed into the PGEX-4T-1 vector using BamH1-XhoI as the restriction site. The amino acid sequence used for mouse PDGFRβ immunization was the extracellular region (PDGFRβECD, SEQ ID NO.8). Four-week-old Balb / c mice were taken and immunized with PDGFRβ extracellular region protein through the tail vein. The mice were immunized once every 14 days for 4 times, and the immunization dose was 100 μg / mouse / time.

[0076] Immune process such as Figure 1 shown.

[0077] Screening of high affinity PDGFRβ scFv

[0078] Blood collection test: This study performed immunization according to the mature immune process and completed the titer determination and other index tests. Due to the large amount of test data, this example only summarizes and summarizes the mice and related data corresponding to the effective optimal cell lines and antibody results. After the first, second, and third blood collection tests, target mice with higher titers that meet the experimental requirements are selected for fusion. In this example, one mouse 3# was selected from five mice for fusion, and the blood collection test results are shown in Table 1 below.

[0079] Table 1 Blood test results

[0080]

[0081] Fusion and cloning: SP2 / 0 mouse myeloma cells were used for cell fusion with spleen 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.

[0082] 3# Mouse strain identification and sequencing: After fusion, primary cloning by limiting dilution method and supernatant detection, 14 cell lines with 31 cells that met the project requirements were obtained. By comparison, a hybridoma with high titer and optimal affinity was sequenced to obtain the full-length PDGFRβ antibody sequence. The antibody heavy chain / light chain variable region genes were obtained by PCR to obtain PDGFRβscFv.

[0083] Construction of PDGFRβCAR

[0084] The sequence of CAR in this example is CD8SP-VH-(G4S)3-VL-CD8Hinge-41BB-CD3ζ. This sequence was synthesized by Qingke Biotechnology Co., Ltd. at one time. The CAR was constructed into the lentiviral vector pLVX-IRES-ZsGreen1 ( Figure 2B), the vector is Amp-resistant and was packaged into lentivirus by GeneCare Genetics and NewHerb using the second-generation virus packaging system for use in the research of the present invention.

[0085] Obtaining mouse T cells

[0086] C57BL / 6J mice were killed by cervical dislocation and soaked in 75% alcohol for about 5 minutes. The spleen of the mice was removed in a biosafety cabinet and ground into a single cell suspension. Mouse T cells with a purity of 99% were obtained after sorting using a kit (EasySep Mouse T Cell Isolation Kit, STEMCELL) Figure 3 B), add CD3 / CD28 activation beads (Miltenyi Biotec) for activation, using a medium containing 10% fetal bovine serum, 50 IU mL -1 IL-7 (Proteintech) and 100 IU mL - 1 IL-15 (Proteintech) was cultured in RPMI1640 medium

[0087] Construction of CAR-T cells

[0088] PDGFRβCAR virus was provided by GeneCare Gene Corporation, and the virus volume with MOI=100 was used to infect 1×10 7 T cells were infected by centrifugation in a 24-well plate at 600 g for 60 min. After infection, the T cells were transferred to fresh culture medium for culture.

[0089] Flow cytometry

[0090] Take 2×10 5 Each cell (Vec-T and PDGFRβCAR-T) was placed in 500 μL pre-cooled PBS, 1 μL of the corresponding flow cytometry antibody (such as mouse CD3) was added, and the cells were incubated at 37°C in the dark for 15 min, washed three times with PBS, and then analyzed by flow cytometry (Beckman). The purity of the isolated mouse T cells was measured. The process of measuring the GFP expression of CAR-T cells is as follows: a certain amount of cells were taken, centrifuged, and washed once with pre-cooled PBS for detection.

[0091] LDH detection of CAR-T cell killing experiment

[0092] The killing experiment of PDGFRβCAR-T cells on NIH3T3 and L929 was mainly carried out by analyzing LDH ( The test was performed by Non-Radioactive Cytotoxicity Assay, with two groups of effector-target ratios of 1:1 and 5:1 respectively. The details are as follows:

[0093] (1) Take 5×10 5 The target cells were placed in a 24-well plate. After the cells adhered to the wall, the corresponding number of CAR-T (Vec-T) cells were added. The final system was 1.5 ml. There were target cell group and effector cell group.

[0094] (2) Effector cells and target cells were co-cultured for 16 h, centrifuged, and the supernatant was collected;

[0095] (3) The supernatant of the target cell group was removed, leaving only the cells, and lysis buffer was added to react for 45 min to completely release LDH;

[0096] (4) Take 50 μL of the supernatant from each group and place it in a 96-well plate, add 50 μL of detection reagent, and react in the dark for 30 min;

[0097] (5) After 30 minutes, add 50 μL of stop solution, react for 1 hour, and read the results using an ELISA reader. The killing value of each group is calculated as follows:

[0098] Killing efficiency of each group (%) = (experimental group value - effector cell natural death value - target cell natural death value) / (target cell complete death value - target cell natural death value) × 100%

[0099] UUO modeling process

[0100] Preoperatively, 6-8 week old male wild-type C57BL / 6J mice (purchased from Vital River) were selected and randomly divided into groups, weighing about 20-30 g, fasting for 12 hours before surgery, and surgical instruments were sterilized in advance by high temperature and high pressure. 0.3% sodium pentobarbital was prepared to anesthetize the mice, and 150 μL / 10g body weight was injected intraperitoneally. After anesthetizing the mice, they were fixed in a supine position on a sterilized surgical board, the abdomen was opened along the midline of the abdomen, the left ureter was freed and ligated at the upper and lower ends, and the middle was cut and the abdomen was closed and sutured. After opening the abdomen of the control group (Sham) mice, the ureters were freed and the abdomen was closed directly. Keep warm until the mice are fully awake. Tissue samples were collected on the 7th day after ureteral obstruction.

[0101] Establishment of DOCA / salt hypertension model

[0102] After at least 1 week of recovery from unilateral kidney removal, half of the mice were randomly selected, anesthetized after fasting for 12 hours, fixed on the operating table, and the hair on the shoulder blade of the mouse back was removed with a depilatory cream. After disinfection, a small incision of about 1 cm was made, and the 21-day sustained-release deoxycorticosterone acetate tablets (Innovative Research of America, Sarasota FL, Cat#M-121) prepared in advance were implanted under the skin of the mouse shoulder blade. After suturing the wound, disinfection was performed, and the mice were kept warm and waited for awakening. After the mice recovered, their drinking water was replaced with 1% saline and raised for 3 weeks. The same operation was performed in the sham group, and the DOCA sustained-release tablets were replaced with an induction agent, and the drinking water remained unchanged.

[0103] Establishment of AngII hypertension model

[0104] Male mice of appropriate age and weight were selected without removing a single kidney. After fasting and anesthesia, they were fixed on the operating table, the hair on the back of the neck was removed, and a 1 cm small incision was made after disinfection. The AngII sustained-release pump (1000 ng / kg / min, Alzet, model 2004) prepared in advance according to the weight was implanted subcutaneously on the back of the mouse. The wound was sutured, and the mouse was kept warm until it recovered and raised for 28 days. The sham group was operated in the same way, but AngII was replaced with normal saline.

[0105] Immunohistochemical staining

[0106] (1) Place the slices in a 65°C oven for more than 1 hour. Dewax and hydrate the slices while they are still hot according to the following procedure: xylene I (20 min), xylene II (20 min), anhydrous ethanol (10 min), 90% ethanol (10 min), 80% ethanol (5 min), 70% ethanol (3 min), and triple-distilled water (10 min).

[0107] (2) Place the dewaxed slides in freshly prepared antigen retrieval solution and heat them in a microwave oven at high heat for 5 minutes, then keep them warm at low heat for 15 minutes.

[0108] (3) After repair, remove the slides and cool them to room temperature, then wash them with PBS three times, 5 min each time.

[0109] (4) Add appropriate amount of 5% BSA and seal the plate at 37°C for 1 h.

[0110] (5) Add the primary antibody prepared in an appropriate ratio and incubate overnight at 4°C in a light-proof humid box.

[0111] (6) The next day, bring the wet box to room temperature for 1 hour, wash it three times with PBS, 5 minutes each time, add appropriate amount of primary and secondary antibody enhancement solution, incubate at 37 degrees for half an hour, and wash it three times with PBS, 5 minutes each time.

[0112] (7) Add appropriate amount of secondary antibody, incubate at 37°C for 2 h, and wash three times with PBS, each time for 5 min.

[0113] (8) Prepare DAB colorimetric solution according to the instructions of the kit, add an appropriate amount of DAB colorimetric solution to start staining and observe under a microscope. When the color development reaches the appropriate effect, stop the color development with tap water and record the time. All subsequent slides should be developed at the same time.

[0114] (9) Then start staining the cell nucleus, add an appropriate amount of hematoxylin, stain the nucleus for 2 minutes, stop with tap water, quickly differentiate with 1% hydrochloric acid alcohol, stop with tap water, turn blue with ammonia water, and rinse with tap water.

[0115] (10) Dehydrate, seal the slides, and air dry them in a fume hood before observing and photographing them under an optical microscope.

[0116] Sirius red staining

[0117] Paraffin tissue sections were stained using the Regen Sirius Red staining kit.

[0118] (1) Paraffin sections were baked at 65°C for 2 h and dewaxed while hot, following the same procedure as for IHC.

[0119] (2) Wash the sections three times with triple-distilled water, 2 min each time.

[0120] (3) Add Sirius red dye solution and stain at room temperature for 1-2 hours (adjustable according to the staining situation), rinse with running water to remove the dye solution on the surface of the slice.

[0121] (4) Add hematoxylin staining solution to stain the nucleus for 8 minutes, rinse with running water to remove the staining solution on the surface of the slice.

[0122] (5) Dehydration and transparency: rapid dehydration with 95% ethanol for 3 seconds; dehydration with anhydrous ethanol three times, 8 seconds each time; and standing in xylene for 10-20 minutes.

[0123] (6) Sealing: Seal the slides with neutral gum and dry them in a fume hood.

[0124] (7) Take photos under an optical microscope and analyze the results.

[0125] CAR-T cell infusion process

[0126] In the UUO model prevention experiment, 1×10 6 Vec-T or CAR-T cells were used to establish the UUO model 5 days later. The mice were treated one week later and samples were collected to detect renal fibrosis indicators. In the UUO model treatment experiment, 1×10 6 In the DOCA / salt hypertension model, 1×10 Vec-T or CAR-T cells were injected into each C57 mouse through the tail vein one week after modeling. 6 Vec-T or CAR-T cells were injected into each C57 mouse through the tail vein one week after modeling. 6 The mice were treated with Vec-T or CAR-T cells three weeks later, and samples were taken to detect fibrosis indicators such as heart, kidney and blood vessels. Results:

[0127] The structure of CAR and the construction of CAR-T cells:

[0128] The CAR-T used in this example is a traditional second-generation structure. The specific molecular structure sequence of the CAR is: CD8 SP-VL-(G4S)3-VH-CD8 Hinge-41BB-CD3ζ( Figure 2 A). The CAR was constructed into the lentiviral vector pLVX-IRES-ZsGreen1 ( Figure 2 B), the vector is Amp-resistant and was packaged into lentivirus by GeneCare Genetics and NewHerb using the second-generation virus packaging system for use in the research of this invention.

[0129] Obtaining mouse T cells and constructing CAR-T cells: C57BL / 6J mice were killed by cervical dislocation and soaked 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) Figure 2 C), add CD3 / CD28 activation magnetic beads (Miltenyi Biotec) for activation, use a medium containing 10% fetal bovine serum, 50 IU mL -1 IL-7 (Proteintech) and 100 IU mL -1 IL-15 (Proteintech) was cultured in RPMI1640 medium. After 48 hours, the cells were infected with lentivirus at an MOI of 50 for 6 hours, and the positive rate was 70% ( Figure 2 D) The above mouse PDGFRβCAR-T cells.

[0130] The results showed that the present invention successfully constructed a second-generation CAR targeting mouse PDGFRβ antigen ( Figure 2 A), by using lentivirus to encode highly purified mouse T cells ( Figure 2 C) were infected and CAR-T cells with a high positive rate were obtained ( Figure 2 D).

[0131] From the above experiments and their results, we can draw the following conclusions:

[0132] Through molecular experiments, the mouse PDGFRβCAR was successfully constructed and verified by sequencing. After obtaining mouse spleen-derived T cells, mouse CAR-T cells were successfully constructed, laying the foundation for subsequent functional exploration of CAR-T cells and the smooth progress of in vivo and in vitro experiments.

[0133] Mouse PDGFRβCAR-T cells can effectively kill PDGFRβ in vitro + cell:

[0134] In order to explore the killing ability of mouse PDGFRβCAR-T on target cells, PDGFRβCAR-T cells were isolated and cultured separately with PDGFRβ + NIH3T3 and L929 cells were co-cultured, the effector-target ratio was set to 5:1 and 1:1, the killing time was 16 h, and the LDH kit (Promega) was used for detection.

[0135] The results showed that the target cell death rate in the Vec-T (Vector transduced T cell) cell group was very low, while the target cell death rate in the PDGFRβCAR-T cell group was relatively high ( Figure 2 E).

[0136] From the above experiments and their results, we can draw the following conclusions:

[0137] PDGFRβCAR-T cells have a strong killing ability against PDGFRβ+ cells in vitro, and the killing efficiency gradually increases with the increase of the effector-target ratio.

[0138] PDGFRβCAR-T cells can prevent renal fibrosis induced by unilateral ureteral ligation (UUO):

[0139] To explore whether mouse PDGFRβCAR-T can prevent the occurrence of renal fibrosis, we injected PDGFRβCAR-T cells into the tail vein before establishing the mouse fibrosis UUO model, and perfused the kidneys one week after ureteral ligation. At the molecular level, Western blot and IHC were used to detect changes in fibrosis-related proteins in the renal cortex of UUO model mice. Sirius red staining and Masson staining were also used to detect changes in mouse kidney morphology and changes in the degree of fibrosis.

[0140] The results showed that PDGFRβCAR-T significantly reduced the upregulation of fibrosis-related proteins Vimentin, Fibronectin, E-cadherin, and α-SMA in the kidneys of mice induced by the UUO model ( Figure 3 A), immunohistochemistry further verified this trend, and PDGFRβCAR-T significantly reduced the upregulation of fibrosis-related proteins Fibronectin and α-SMA in the kidneys of mice induced by the UUO model ( Figure 3 B), and can significantly improve the tubulointerstitial fibrosis and collagen fiber deposition induced by UUO model ( Figure 3 C).

[0141] From the above experiments and their results, we can draw the following conclusions:

[0142] PDGFRβCAR-T cells can significantly prevent tubulointerstitial fibrosis in UUO mice, significantly reduce the changes of Fibronectin, α-SMA, E-cadherin and Vimentin in the renal cortex of mice, and slow down the progression of renal fibrosis.

[0143] PDGFRβCAR-T cells can alleviate the progression of renal fibrosis induced by UUO:

[0144] To further explore the role of mouse PDGFRβCAR-T in renal interstitial fibrosis, a mouse fibrosis UUO model was constructed. One week after ureteral ligation, PDGFRβCAR-T cells were reinfused via tail vein injection. One week after reinfusion of CAR-T cells, kidneys were perfused and collected. At the molecular level, Western blot and IHC were used to detect changes in fibrosis-related proteins in the renal cortex of UUO model mice. Sirius red staining and Masson staining were also used to detect changes in mouse kidney morphology and changes in the degree of fibrosis.

[0145] The results showed that PDGFRβCAR-T significantly reduced the upregulation of fibrosis-related proteins Vimentin, Fibronectin, Collagen I, and α-SMA in the kidneys of mice induced by the UUO model ( Figure 4 A), immunohistochemistry further verified this trend, and PDGFRβCAR-T significantly reduced the upregulation of fibrosis-related proteins Fibronectin and α-SMA in the kidneys of mice induced by the UUO model ( Figure 4 B), and can significantly improve the tubulointerstitial fibrosis and collagen fiber deposition induced by UUO model ( Figure 4 C).

[0146] From the above experiments and their results, we can draw the following conclusions:

[0147] PDGFRβCAR-T cells can significantly improve the renal damage and fibrosis of UUO mice, clarifying the role of PDGFRβCAR-T cells in preventing the progression of renal fibrosis.

[0148] PDGFRβCAR-T cells have the effect of alleviating CKD (deoxycorticosterone acetate-induced hypertension model):

[0149] In order to further explore the role of mouse PDGFRβCAR-T in the treatment of hypertensive fibrosis-related diseases, we constructed a DOCA / salt hypertension model, tested the blood pressure of mice every week, and reinfused PDGFRβCAR-T cells through tail vein injection one week after modeling. After two weeks, the heart, kidneys, and blood vessels were perfused. At the molecular level, Western blot and IHC were used to detect changes in fibrosis-related proteins in the renal cortex of DOCA / salt model mice. Sirius red staining, Masson staining, and hematoxylin-eosin staining were also used to detect changes in the morphology of the mouse heart, kidney, and blood vessels, as well as changes in the degree of fibrosis. At the same time, echocardiography was used to detect the effect of PDGFRβCAR-T cells on mouse cardiac function.

[0150] The results showed that PDGFRβCAR-T significantly reduced the upregulation of fibrosis-related proteins Vimentin, Collagen I, α-SMA, and Fibronectin in the kidneys of mice induced by DOCA / salt hypertension model ( Figure 5 A); Echocardiography results showed that PDGFRβCAR-T could further improve the increase in left ventricular posterior wall thickness, the decrease in ejection fraction and left ventricular short axis shortening rate caused by DOCA / salt in mice ( Figure 5 B); Morphological staining results showed that PDGFRβCAR-T can improve the damage and fibrosis of the heart, kidney and blood vessels in DOCA / salt hypertensive mice ( Figure 5 CG); kidney weight to body weight ratio and urine microalbumin test results showed that PDGFRβCAR-T can improve renal function damage ( Figure 5 EF).

[0151] From the above experiments and their results, we can draw the following conclusions:

[0152] PDGFRβCAR-T cells can improve DOCA / salt-induced hypertension, cardiovascular remodeling, and renal damage.

[0153] PDGFRβCAR-T cells have the effect of alleviating cardiovascular disease (angiotensin II-induced hypertension model):

[0154] In order to comprehensively study the role of mouse PDGFRβCAR-T in the treatment of hypertension-related diseases, we constructed an AngII-induced hypertension model, tested the blood pressure of mice weekly, and reinfused PDGFRβCAR-T cells through tail vein injection one week after modeling. After three weeks, the heart, kidneys, and blood vessels were perfused. At the molecular level, Western blot and IHC were used to detect changes in fibrosis-related proteins in the renal cortex of AngII hypertension model mice. Sirius red staining, Masson staining, and hematoxylin-eosin staining were also used to detect changes in the morphology of the mouse heart, kidney, and blood vessels, as well as changes in the degree of fibrosis. At the same time, echocardiography was used to detect the effect of PDGFRβCAR-T cells on mouse cardiac function.

[0155] The results showed that PDGFRβCAR-T significantly reduced the upregulation of fibrosis-related proteins α-SMA, Vimentin, and E-cadherin in the kidneys of mice induced by the DOCA / salt hypertension model ( Figure 6 A) Echocardiography results showed that PDGFRβCAR-T can further improve the increase in left ventricular posterior wall thickness, the decrease in ejection fraction and left ventricular short axis shortening rate caused by AngII in mice ( Figure 6 B); Morphological staining results showed that PDGFRβCAR-T can improve the damage and fibrosis of the heart, kidney and blood vessels in DOCA / salt hypertensive mice ( Figure 6 CG); kidney weight to body weight ratio and urine microalbumin test results showed that PDGFRβCAR-T can improve renal function damage ( Figure 6 EF).

[0156] From the above experiments and their results, we can draw the following conclusions:

[0157] PDGFRβCAR-T cells can improve AngII-induced hypertension, cardiovascular remodeling, and renal damage.

[0158] In summary, the present invention obtains a single-chain antibody with high affinity targeting mouse PDGFRβ antigen by immunizing mice, and applies it to the second-generation CAR. The constructed CAR-T cells can effectively kill PDGFRβ. + The cells have the function of preventing and alleviating CKD in mice, providing a new strategy for the clinical use of PDGFRβCAR-T cells in the treatment of diseases such as CKD including renal fibrosis, and for the treatment of CKD by clearing PDGFRβ + Cell therapy for kidney disease, cardiovascular disease and various tumor diseases provides a completely new approach with extremely attractive value for further development and application prospects.

[0159] Amino acid sequence information used in the examples:

[0160] The scFv of the present invention is a high-affinity single-chain antibody obtained by the team through screening of immunized mice, in which the heavy chain and the light chain are connected by a G4S linker.

[0161] in:

[0162] The heavy chain sequence is:

[0163] QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKLRPGQGFEWI GEINPSNGGTNYSEKFKRKATLTVDKSSSTAYMQLSSLTSEDSAVYYCTIWDY VIDYWGQGTILTVSS(SEQ ID NO.1)

[0164] The light chain sequence is:

[0165] DIMMTQTPLSLPVSLGDQASISCRSSQSLVHSYGNTYLHWYLQKPGQSPK LLIYKVSNRFSGVPDRFRGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFG GGTKLEIK(SEQ ID NO.2)

[0166] The G4S linker sequence is:

[0167] GGGGSGGGGSGGGGS (SEQ ID NO. 3)

[0168] The CD8 SP sequence is:

[0169] MASPLTRFLSLNLLLLGESIILGSGEA(SEQ ID NO.4)

[0170] The CD8 Hinge region sequence is:

[0171] TTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLA GICVALLLSLIITLICYHRSRKWIRKKFPHIFKQPFKKTTGAAQEEDACSCRCPQ EEEGGGGGYEL(SEQ ID NO.5)

[0172] The sequence of the 4-1BB intracellular region is:

[0173] KWIRKKFPHIFKQPFKKTTGAAQEEDACSCRCPQEEEGGGGGYEL(SEQ ID NO.6)

[0174] The CD3ζ chain sequence is:

[0175] FSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQR RRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTY DALHMQTLAPR(SEQ ID NO.7)

[0176] The PDGFRβ ECD sequence is:

[0177] LVVTPGPELVLNVSSTFVLTCSGSAPVVWERMSQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHNDSRGLETDERKRLYIFVPDPTVGFLPNDAEELFIFLTEITEITIPCRVT DPQLVVTLHEKKGDVALVPYDHQRGFSGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSINVSVNAVQTVVRQGENITLMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDMPYHI RSILHIPSAELEDSGTYTCNVTESVNDHQDEKAINITVVESGYVRLLGEVGTLQFAELHRSRTLQVVFEAYPPPTVLWFKDNRTLGDSSAGEIALSTRNVSETRYVSELTLVRVKVAEAG HYTMRAFHEDAEVQLSFQLQINVPVRVLELSESHPDSGEQTVRCRGRGMPQPNIIWSACRDLKRCPRELPPTLLGNSSEEESQLETNVTYWEEEQEFEVVSTLRLQHVDRPLSVRC(SEQ ID NO.8)

[0178] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they 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 included in the protection scope of the present invention.

Claims

1. A chimeric antigen receptor targeting PDGFRβ, characterized in that: It at least comprises an antigen binding domain; the antigen binding domain is an anti-PDGFRβ antibody, and the anti-PDGFRβ antibody is a single-chain antibody, which is composed of an antibody heavy chain and an antibody light chain; The amino acid sequence of the antibody heavy chain is selected from: a1) the amino acid sequence shown in SEQ ID NO.1; The amino acid sequence of the antibody light chain is selected from: b1) The amino acid sequence shown in SEQ ID NO.

2.

2. The chimeric antigen receptor targeting PDGFRβ according to claim 1, characterized in that The antibody heavy chain is connected to the antibody light chain directly or through a connecting fragment.

3. The chimeric antigen receptor targeting PDGFRβ according to claim 2, characterized in that The connecting fragment is selected from a polypeptide chain consisting of alanine and / or serine and / or glycine.

4. The chimeric antigen receptor targeting PDGFRβ according to claim 2, characterized in that The length of the connecting fragment is 3 to 30 amino acids.

5. The chimeric antigen receptor targeting PDGFRβ according to claim 2, characterized in that The connecting fragment is (GGGGS)n, where n is 1-6.

6. The chimeric antigen receptor targeting PDGFRβ according to claim 1, characterized in that It contains a signal peptide, an antigen binding domain, a chimeric receptor hinge region, a co-stimulatory signaling domain and a signaling domain connected in series.

7. The chimeric antigen receptor targeting PDGFRβ according to claim 6, characterized in that The signal peptide is a CD8 SP signal peptide.

8. The chimeric antigen receptor targeting PDGFRβ according to claim 6, characterized in that The chimeric receptor hinge region is CD8 Hinge.

9. The chimeric antigen receptor targeting PDGFRβ according to claim 6, characterized in that The co-stimulatory signaling domain is a 4-1BB co-stimulatory signaling domain.

10. The chimeric antigen receptor targeting PDGFRβ according to claim 6, characterized in that The signaling domain is the CD3 zeta signaling domain.

11. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises nucleotides encoding the chimeric antigen receptor targeting PDGFRβ according to any one of claims 1 to 10.

12. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid molecule of claim 11.

13. A CAR-T cell, characterized in that: The CAR-T cell is a T lymphocyte modified by the chimeric antigen receptor targeting PDGFRβ as described in any one of claims 1-10.

14. Use of the chimeric antigen receptor according to any one of claims 1 to 10, the nucleic acid molecule according to claim 11, the recombinant expression vector according to claim 12, or the CAR-T cell according to claim 13 in any one of the following: c1) preparing drugs for preventing or treating renal fibrosis; c2) preparing drugs for preventing or treating DOCA / salt-induced hypertension, cardiovascular remodeling, and kidney damage; c3) preparing drugs for preventing or treating hypertension, cardiovascular remodeling and kidney damage caused by Ang II; The renal fibrosis is UOO-induced renal fibrosis.

15. A product for preventing and / or treating UOO-induced renal fibrosis, DOCA / salt or AngII-induced hypertension, cardiovascular remodeling and renal damage, characterized in that: The active ingredient of the product is the chimeric antigen receptor described in any one of claims 1 to 10 or the CAR-T cell described in claim 13.

16. The product according to claim 15, characterized in that The product described is a drug.

17. The product according to claim 16, characterized in that The medicament further includes a pharmaceutically acceptable carrier.

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