WO2025209170A1 - PDGFRβ-TARGETING CHIMERIC ANTIGEN RECEPTOR, CAR-T CELL AND USE THEREOF - Google Patents

Abstract

A PDGFRβ-targeting chimeric antigen receptor, a CAR-T cell and the use thereof. For a PDGFRβ molecule, a PDGFRβ-targeting CAR-T cell is constructed by means of designing a monoclonal antibody and immunizing mice to obtain a murine single-chain antibody sequence, and it is proven that the CAR-T cell kills activated fibroblasts in vitro and has a therapeutic effect on renal and cardiac fibrosis associated with hypertension or diabetes, thus providing a basis for scavenging PDGFRβ+ muscle fibroblasts, fibroblasts, etc. in the clinical treatment of CKDs such as renal fibrosis, and provides a new concept for the clinical treatment of hepatic fibrosis, cardiovascular diseases and various tumor diseases, and therefore has good practical application value.

Description

A chimeric antigen receptor targeting PDGFRβ, CAR-T cells and their applications

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims priority to Chinese patent application No. 202410394636.2 filed with the State Intellectual Property Office of China on April 2, 2024, entitled “A Chimeric Antigen Receptor Targeting PDGFRβ, CAR-T Cells and Applications thereof”, and Chinese patent application No. 202410664607.3 filed with the State Intellectual Property Office of China on May 27, 2024, entitled “A Chimeric Antigen Receptor Targeting PDGFRβ, CAR-T Cells and Applications thereof”, the entire contents of which are incorporated by reference into the present invention and constitute a part of the present invention for all purposes.

Technical Field

The present invention belongs to the technical field of biomedicine and molecular biology, and specifically relates to a chimeric antigen receptor targeting PDGFRβ, CAR-T cells and applications.

Background Art

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

Fibrosis is a common pathological endpoint of chronic, progressive, or severe acute diseases in nearly all organs and a major bottleneck in the treatment of various chronic diseases. Chronic kidney disease (CKD) is a global public health issue that poses a serious threat to human health. With the aging population and the increasing prevalence of hypertension and diabetes, the global adult CKD prevalence has reached over 10%. It is projected that by 2040, CKD will become the fifth most common cause of death worldwide, and by the end of this century, it will be the second leading cause of death in countries with longer life expectancies. my country has the highest number of CKD patients globally, with over 130 million people undergoing hemodialysis and over 100,000 undergoing peritoneal dialysis, the highest total globally. This places a heavy burden on the national healthcare system. Renal fibrosis is a necessary stage in the progression of chronic kidney disease (CKD) from various causes to end-stage renal disease. The main clinical treatments for CKD, angiotensin-converting enzyme inhibitors and angiotensin receptor blockers, can slow the decline of renal function but cannot cure CKD and may cause serious side effects. Studies of renal protective drugs such as TGF-β inhibitors, endothelin inhibitors, and renin inhibitors have all failed. Although the antidiabetic SGLT2 inhibitor canagliflozin has recently been used to treat CKD, current treatment options are still very limited, and no method can effectively prevent renal fibrosis or reverse the loss of renal function in CKD.

In recent years, chimeric antigen receptor (CAR)-modified T cells, or CAR-T cells, have demonstrated promising anti-tumor effects in the treatment of malignancies such as leukemia, lymphoma, melanoma, and glioma. CAR-T immunotherapy involves genetically engineering T cells to enable them to specifically recognize and kill target cells. Numerous studies have demonstrated that CAR-T cells using 4-1BB as a co-stimulatory molecule exhibit greater persistence in vivo, with survival periods exceeding three months and even up to a year. In contrast, CAR-T cells using CD28 as a co-stimulatory molecule exhibit robust short-term proliferation but less persistence, generally surviving no more than one month. Recently, CAR-T cell therapy has been explored in the treatment of autoimmune diseases, metabolic disorders, and other non-tumor conditions, such as systemic lupus erythematosus, rheumatoid arthritis, and vascular diseases. Laboratory animal studies have demonstrated that CAR-T cells significantly improve the treatment of liver and myocardial fibrosis. However, the application of CAR-T cells in the treatment of chronic kidney disease and its related renal fibrosis remains blank.

CAR-T immune cells rely on genetically engineered recognition sequences, such as single-chain antibodies, ligands, and receptors, that specifically recognize antibodies expressed by target cells, thereby effectively eliminating them. Currently, no suitable and effective CAR-T therapy targets renal fibroblasts and myofibroblasts have been reported. Platelet-derived growth factor receptor β (PDGFRβ) is a membrane molecule belonging to the receptor tyrosine kinase family and is primarily 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. Upon activation, PDGFRβ induces downstream signaling, triggering cell proliferation, migration, and differentiation. Increased PDGFRβ signaling, caused by activating mutations, gene translocations, or increased ligand abundance, has been implicated in 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 damage in various kidney disease models. Neutralization of PDGFRβ ligands PDGF-B or PDGF-D also has a protective effect in kidney 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 fibroblasts around blood vessels. Compared with these source cells, myofibroblasts have higher expression of PDGFRβ on the surface.

CAR-T cells targeting PDGFRβ can also potentially inhibit renal fibrosis, halting or even reversing the progression of CKD by eliminating myofibroblasts and reducing their source cells. CKD and cardiovascular disease (CVD) share common causes, such as diabetes and hypertension. Myofibroblasts in CVD hearts are derived from the expansion and differentiation of fibroblasts within damaged tissue. Therefore, CAR-T cells targeting PDGFRβ can simultaneously protect against fibrosis in the kidneys, heart, and other affected organs caused by the same underlying disease.

Summary of the Invention

In response to the above-mentioned prior art, the present invention aims to provide a chimeric antigen receptor, CAR-T cell, and application targeting PDGFRβ. Specifically, the present invention targets the PDGFRβ molecule, through monoclonal antibody design, immunization of mice to obtain mouse single-chain antibody sequences, and construction of CAR-T cells targeting PDGFRβ. The cells have been shown to kill activated fibroblasts in vitro and to have therapeutic effects 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 was completed.

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

In a first aspect of the present invention, there is provided a chimeric antigen receptor targeting PDGFRβ, comprising at least an antigen binding domain;

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

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

a1) the amino acid sequence shown in SEQ ID NO. 1; or

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

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

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

b2) A polypeptide having the same function as the amino acid sequence shown in SEQ ID NO.2, with one or several amino acid residues substituted and/or deleted and/or added.

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.

Furthermore, the antibody heavy chain and the antibody light chain can be connected directly or via 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.

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;

Furthermore, the signal peptide is CD8 SP signal peptide;

Furthermore, the hinge region of the chimeric receptor is CD8 Hinge;

Furthermore, the costimulatory signaling domain is a 4-1BB costimulatory signaling domain;

Furthermore, the signaling domain is a CD3ζ signaling domain;

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 costimulatory signaling domain, and a CD3ζ signaling domain in series.

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

It should be noted that those skilled in the art can readily mutate the nucleotide sequence encoding the chimeric antigen receptor targeting PDGFRβ of the present invention using known methods, such as directed evolution and point mutagenesis. Artificially modified nucleotide sequences that share 75% or greater identity with the nucleotide sequence of the present invention are derived from and are equivalent to the nucleotide sequence of the present invention, as long as they encode the chimeric antigen receptor and have the same function.

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

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

According to the present invention, the recombinant expression vector is a viral vector, which may be a retroviral vector or a lentiviral vector; more 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.

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

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 recombinant lentiviral vectors into lentiviral packaging cells and then culturing the cells; the lentiviral vector is obtained by inserting the coding gene of the above-mentioned chimeric antigen receptor into the lentiviral vector.

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 a drug for preventing and/or treating diseases related to high PDGFRβ expression.

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.

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.

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.

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.

The drug may further 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 natural polymers and synthetic polymers), a lipoprotein, a polypeptide, a polysaccharide, a lipopolysaccharide, an artificial viral envelope, an inorganic (including metal) particle, and a bacterial, bacteriophage, clay or plasmid vector.

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.

The medicine of the present invention can be administered to the body in a known manner. For example, it can be delivered to the tissue of interest by systemic intravenous delivery or local injection. Alternatively, it can be administered intravenously, percutaneously, intranasally, through the mucosa, or other delivery methods. Such administration can be carried out via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be administered in the present invention can vary depending on various factors to a great extent, such as the target cell, biological type or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

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

Beneficial technical effects of one or more of the above technical solutions:

The above technical solution targets PDGFRβ, 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 the CAR, a T cell expressing a chimeric antigen receptor (CAR) targeting PDGFRβ was obtained. Experiments have shown that PDGFRβCAR-T cells can effectively eliminate PDGFRβ ⁺ target cells in vitro. In vivo experiments were carried out by constructing various mouse CKD models, including unilateral ureteral obstruction (UUO) models and chronic kidney disease models associated with diabetes and hypertension. After tail vein reinfusion of CAR-T cells, 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 were decreased. In vivo experiments have shown that PDGFRβCAR-T cells can effectively prevent and treat CKD in mice. This invention provides an antibody that recognizes and binds to mouse PDGFRβ antigen with high affinity. The CAR-T cells constructed based on this antibody have shown good therapeutic effects in mice. This study provides a basis for the clinical treatment of CKD diseases such as renal fibrosis by eliminating PDGFRβ ⁺ myofibroblasts and fibroblasts, and provides new ideas for the clinical treatment of liver fibrosis, cardiovascular disease and various tumor diseases. Therefore, it has good practical application value.

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG1 is a flowchart of mouse immunization in an embodiment of the present invention.

Figures 2A to 2E show the construction of mouse CAR-T cells and killing experiments in an embodiment of the present invention.

Among them: Figure 2A is the structure and original diagram of PDGFRβCAR; Figure 2B is the lentiviral vector used for construction; Figure 2C is the flow cytometry identification of mouse T cells after sorting; Figure 2D is the detection of the positive rate of PDGFRβCAR-T cells; Figure 2E is the detection of the killing ability of PDGFRβCAR-T cells;

3A to 3C show that PDGFRβ CAR-T cells prevent renal fibrosis caused by the UUO model in an embodiment of the present invention.

Among them: Figure 3A is a Western blot detection of the changes in fibrosis-related proteins Vimentin, Fibronectin, E-cadherin, and α-sma in the renal cortex of UUO model mice; Figure 3B is an immunohistochemistry detection of the changes in fibrosis-related proteins Fibronectin and α-sma in the renal cortex of UUO model mice; Figure 3C is Sirius Red and Masson staining to detect the degree of renal fibrosis in mice.

FIG4A to FIG4C show that PDGFRβCAR-T cells improve renal fibrosis caused by the UUO model in an embodiment of the present invention.

Among them: Figure 4A is a Western blot detection of the changes in fibrosis-related proteins Vimentin, Fibronectin, Collagen I, and α-sma in the renal cortex of UUO model mice; Figure 4B is an immunohistochemistry detection of the changes in fibrosis-related proteins Fibronectin and α-sma in the renal cortex of UUO model mice; Figure 4C is Sirius Red and Masson staining to detect the degree of renal fibrosis in mice.

Figures 5A to 5G show that PDGFRβ CAR-T cells can improve fibrosis caused by the DOCA/salt hypertension model in an embodiment of the present invention.

Among them: Figure 5A is a Western blot detection of changes in fibrosis-related proteins Vimentin, Fibronectin, Collagen I, and α-sma in the renal cortex of DOCA/salt hypertension model mice; Figure 5B is an echocardiogram detection of cardiac function in DOCA/salt hypertension model mice; Figure 5C is HE staining detection of changes in myocardial thickness in DOCA/salt hypertension model mice and Sirius Red detection of the degree of fibrosis in small and medium blood vessels in the heart of DOCA/salt hypertension model mice; Figure 5D is HE staining detection of changes in aortic wall thickness in DOCA/salt hypertension model mice; Figure 5E is the change in kidney weight to body weight ratio in each group of DOCA/salt hypertension model mice; Figure 5F is the change in urinary microalbumin in each group of DOCA/salt hypertension model mice; Figure 5G is immunohistochemistry detection of changes in fibrosis-related protein α-sma in the renal cortex of DOCA/salt hypertension model mice; Sirius Red and Masson staining are used to detect the degree of renal fibrosis in DOCA/salt hypertension model mice.

6A to 6G show that PDGFRβ CAR-T cells can improve fibrosis caused by Ang II hypertension model in an embodiment of the present invention.

Among them: Figure 6A is a Western blot detection of changes in fibrosis-related proteins α-sma, Vimentin, and E-cadherin in the renal cortex of AngII hypertension model mice; Figure 6B is an echocardiogram detection of cardiac function in AngII hypertension model mice; Figure 6C is HE staining detection of changes in myocardial thickness in AngII hypertension model mice and Sirius Red detection of the degree of fibrosis in small blood vessels in the heart of AngII hypertension model mice; Figure 6D is HE staining detection of changes in aortic wall thickness in AngII hypertension model mice; Figure 6E is the change in kidney weight to body weight ratio in each group of AngII hypertension model mice; Figure 6F is the change in urinary microalbumin in each group of AngII hypertension model mice; Figure 6G 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 are used to detect the degree of renal fibrosis in AngII hypertension model mice.

DETAILED DESCRIPTION

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

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

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

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). In addition, the term "sequence" used herein (such as in terms such as "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence", or "protein sequence") should generally be 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.

The term "domain" (of a polypeptide or protein) refers to a folded protein structure that is capable of maintaining 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 losing the function of the rest of the protein and/or the domain.

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.

The term "affinity" is theoretically defined by the equilibrium association between intact antibodies and antigens. Affinity herein can be assessed or measured by _KD values (dissociation constants) (or other assays), such as bio-layer interferometry (BLI), using a FortebioRed96 instrument.

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.

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.

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.

The term "treatment/prevention" refers to an attempt to alter the natural course of a disease in a treated individual and can be a clinical intervention performed for prevention or during the course of clinical pathology. Desirable 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 development of the disease or slow the progression of the condition.

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

Among them, murine T cells were derived from the spleen of C57BL/6 mice. In the in vivo experiment, CAR-T cells were reinfused by tail vein injection, with a reinfusion dose of 1×10 ⁵ and a reinfusion system of 100-200 μL.

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

The lentiviral vector was named pLVX-IRES-ZsGreen1, and the lentivirus was provided by GeneCare Gene, NewHe Bio and other companies. The infection MOI was 100.

The present invention's mouse PDGFRβ-targeted CAR-T cells are capable of effectively killing mouse fibroblast cell lines in vitro and effectively preventing and improving kidney disease in vivo. In experiments conducted in the present invention, various kidney disease models were created, including unilateral ureteral obstruction (UUO), deoxycorticosterone acetate/salt (DOCA/salt), and angiotensin II (Ang II)-induced hypertension models. It was found that mouse PDGFRβ CAR-T cells were able to alleviate and improve these various kidney diseases. This study provides a novel treatment approach for CAR-T immunotherapy in the treatment of kidney diseases including renal fibrosis, liver diseases, cardiovascular diseases, and various tumor diseases with high PDGFRβ expression. More importantly, high-dose reinfusion of mouse PDGFRβ CAR-T cells to treat kidney disease did not cause significant side effects, providing a theoretical basis for the clinical application of PDGFRβ CAR-T cells.

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

Example

method:

The process of mouse immunization

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 immunized with PDGFRβ extracellular region protein via the tail vein. The mice were immunized once every 14 days for a total of four times, with an immunization dose of 100 μg/mouse/time.

The immunization process is shown in Figure 1.

Screening of high-affinity PDGFRβ scFv

Blood sampling and testing: This study followed established immunization protocols and completed titer determination and other testing. Due to the large amount of data collected, this example only summarizes the data from mice that effectively obtained the optimal cell line and antibody results. After the first, second, and third blood sampling tests, target mice with high titers that met the experimental requirements were selected for fusion. In this example, one mouse, mouse 3#, was selected from five mice for fusion. The blood sampling results are shown in Table 1 below.

Table 1 Blood test results

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.

3# Mouse strain identification and sequencing: After fusion, primary cloning by limiting dilution method and supernatant testing, 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.

Construction of PDGFRβCAR

The sequence of the CAR used in this study is CD8SP-VH-(G4S) ₃ -VL-CD8 Hinge-41BB-CD3ζ. This sequence was synthesized in one go by Qingke Biotech Co., Ltd. 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 Gene and NewHe Biotech using a second-generation viral packaging system for use in the present invention.

Obtaining mouse T cells

C57BL/6J mice were sacrificed by cervical dislocation and immersed in 75% alcohol for approximately 5 min. The spleens were removed in a biosafety cabinet and ground into a single cell suspension. Mouse T cells with a purity of 99% were isolated using the EasySep Mouse T Cell Isolation Kit (STEMCELL) (Figure 3B). CD3/CD28-activating magnetic beads (Miltenyi Biotec) were added for activation and cultured in RPMI1640 medium containing 10% fetal bovine serum, 50 IU ^mL⁻¹ IL-7 (Proteintech), and 100 IU ^mL⁻¹ IL-15 (Proteintech).

Construction of CAR-T cells

PDGFRβCAR virus was provided by GeneCare Gene Co., Ltd., and the virus was used to infect 1× ¹⁰⁷ T cells at an MOI of 100. The cells were infected by centrifugation in a 24-well plate at 600g for 60 min. After infection, the T cells were transferred to fresh culture medium for culture.

Flow cytometry

Take 2×10 ⁵ cells (Vec-T and PDGFRβCAR-T) in 500 μL pre-cooled PBS, add 1 μL of corresponding flow cytometry antibody (such as mouse CD3), incubate at 37°C in the dark for 15 minutes, wash three times with PBS, and then analyze with flow cytometer (Beckman) to measure the purity of isolated mouse T cells. The process of measuring GFP expression of CAR-T cells is as follows: take a certain amount of cells, centrifuge and wash once with pre-cooled PBS for detection.

LDH detection of CAR-T cell killing experiment

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

(1) 5 × 10 ⁵ target cells were placed in a 24-well plate. After adherence, the corresponding number of CAR-T (Vec-T) cells were added. The final volume was 1.5 ml. Target cell group and effector cell group were set up.

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

(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;

(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;

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

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

UUO modeling process

Before surgery, 6-8 week old male wild-type C57BL/6J mice (purchased from Weitonglihua) were selected and randomly divided into groups, weighing about 20-30 g. They were fasted for 12 hours before surgery, and the surgical instruments were sterilized in advance by high temperature and high pressure. 0.3% sodium pentobarbital was prepared to anesthetize the mice and injected intraperitoneally at 150 μL/10 g body weight. After anesthesia, the mice were fixed in a supine position on a sterilized operating board, the abdomen was opened along the midline of the abdomen, the left ureter was freed, the upper and lower ends were ligated, 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.

Establishment of DOCA/salt hypertension model

At least one week after recovery from single nephrectomy, half of the mice were randomly selected and anesthetized after fasting for 12 hours. They were then fixed to the operating table and the scapular area of the mice was depilated with depilatory cream. After disinfection, a small incision approximately 1 cm was made and a pre-prepared 21-day sustained-release deoxycorticosterone acetate tablet (Innovative Research of America, Sarasota, FL, Cat# M-121) was implanted subcutaneously under the scapula. The wound was sutured and disinfected, and the mice were kept warm until they recovered. After recovery, their drinking water was replaced with 1% saline and maintained for 3 weeks. The sham group underwent the same procedure, except that the DOCA sustained-release tablet was replaced with a sedative, while the drinking water remained unchanged.

Establishment of AngII hypertension model

Male mice of appropriate age and weight were selected. Neither kidney was removed. After fasting and anesthesia, they were fixed on an operating table. The hair at the back of the neck was shaved. After disinfection, a 1 cm incision was made. A pre-fabricated Ang II sustained-release pump (1000 ng/kg/min, Alzet, model 2004) was implanted subcutaneously in the back of the mouse. The wound was sutured, and the mouse was kept warm until it recovered and housed for 28 days. The sham group was treated with the same procedure, except that the Ang II was replaced with saline.

Immunohistochemical staining

(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).

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

(3) After repair, remove the slides and cool them to room temperature. Wash them with PBS three times, 5 minutes each time.

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

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

(6) The next day, bring the wet box to room temperature for 1 hour, wash it with PBS three times, 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 with PBS three times, 5 minutes each time.

(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.

(8) Prepare DAB colorimetric solution according to the kit instructions, 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 colored at the same time.

(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.

(10) Dehydrate, seal, and air dry in a fume hood before observing and photographing using an optical microscope.

Sirius red staining

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

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

(2) Wash the sections with triple-distilled water three times, 2 minutes each time.

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

(4) Add hematoxylin staining solution and stain the nucleus for 8 minutes. Rinse with running water to remove the staining solution on the surface of the slice.

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

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

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

CAR-T cell infusion process

In the UUO prevention model, 1× ^10⁶ Vec-T or CAR-T cells were injected into each C57 mouse via tail vein infusion. Five days later, the UUO model was established. One week later, the mice were treated, and renal fibrosis markers were measured. In the UUO treatment model, 1× ^10⁶ Vec-T or CAR-T cells were injected into each C57 mouse via tail vein infusion one week after model establishment. One week later, the mice were treated, and renal fibrosis markers were measured. In the DOCA/salt hypertension model, 1× ^10⁶ Vec-T or CAR-T cells were injected into each C57 mouse via tail vein infusion one week after model establishment. Two weeks later, the mice were treated, and cardiac, renal, and vascular fibrosis markers were measured. In the AngII hypertension model, 1×10 ⁶ Vec-T or CAR-T cells were injected into each C57 mouse via tail vein infusion one week after model establishment. Three weeks later, the mice were treated and samples were collected to detect fibrosis indicators such as heart, kidney, and blood vessels.

result:

CAR structure and CAR-T cell construction:

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

Mouse T cell acquisition and CAR-T cell construction: C57BL/6J mice were sacrificed by cervical dislocation and soaked in 75% alcohol for approximately 5 minutes. The spleens were removed in a biosafety cabinet and ground into a single cell suspension. Mouse T cells with a purity greater than 99% were isolated using the EasySep Mouse T Cell Isolation Kit (STEMCELL) (Figure 2C). CD3/CD28-activating magnetic beads (Miltenyi Biotec) were added for activation and cultured in RPMI1640 medium containing 10% fetal bovine serum, 50 IU mL ^-1 IL-7 (Proteintech), and 100 IU mL ^-1 IL-15 (Proteintech). After 48 hours, the cells were infected with lentivirus at an MOI of 50 for 6 hours, resulting in a positive rate of more than 70% mouse PDGFRβ CAR-T cells (Figure 2D).

The results showed that the present invention successfully constructed a second-generation CAR targeting mouse PDGFRβ antigen (Figure 2A). By using lentivirus to infect high-purity mouse T cells (Figure 2C), CAR-T cells with a high positive rate were obtained (Figure 2D).

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

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.

Mouse PDGFRβCAR-T cells can effectively kill PDGFRβ ⁺ cells in vitro:

To investigate the killing ability of mouse PDGFRβCAR-T cells on target cells, we co-cultured PDGFRβCAR-T cells with PDGFRβ ⁺ NIH3T3 and L929 cells, respectively, with effector-target ratios of 5:1 and 1:1, and the killing time was 16 h. LDH kit (Promega) was used for detection.

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 2E).

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

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.

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

To investigate 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. One week after ureteral ligation, we perfused the kidneys and detected changes in fibrosis-related proteins in the renal cortex of UUO model mice at the molecular level by Western blot and IHC. We also used picrosirius red staining and Masson's staining to detect changes in mouse kidney morphology and the degree of fibrosis.

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 3A). Immunohistochemistry further verified this trend. 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 3B), and was able to significantly improve the tubular interstitial fibrosis and massive deposition of collagen fibers induced by the UUO model (Figure 3C).

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

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

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

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 injected back into the tail vein. One week after the reinfusion of CAR-T cells, the 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's staining were also used to detect changes in mouse kidney morphology and the degree of fibrosis.

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 4A). Immunohistochemistry further verified this trend. 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 4B), and was able to significantly improve the tubular interstitial fibrosis and massive deposition of collagen fibers induced by the UUO model (Figure 4C).

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

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

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

To further investigate the role of mouse PDGFRβ CAR-T in the treatment of hypertensive fibrosis, we established a DOCA/salt hypertension model. Blood pressure was monitored weekly in mice. One week after modeling, PDGFRβ CAR-T cells were reinfused via tail vein injection. Two weeks later, the heart, kidneys, and blood vessels were harvested by perfusion. At the molecular level, Western blot and IHC were used to examine changes in fibrosis-related proteins in the renal cortex of DOCA/salt mice. Picrosirius red staining, Masson's staining, and hematoxylin-eosin staining were also used to examine changes in heart, kidney, and vascular morphology, as well as changes in the degree of fibrosis. Echocardiography was also used to assess the effects of PDGFRβ CAR-T cells on cardiac function.

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 the DOCA/salt hypertension model (Figure 5A). Echocardiography results showed that PDGFRβCAR-T could further improve the increase in left ventricular posterior wall thickness, ejection fraction, and left ventricular short-axis shortening caused by DOCA/salt in mice (Figure 5B). Morphological staining results showed that PDGFRβCAR-T could improve the damage and fibrosis of the heart, kidney, and blood vessels in DOCA/salt hypertensive mice (Figure 5C-G). Kidney weight to body weight ratio and urine microalbumin test results showed that PDGFRβCAR-T could improve renal function damage (Figure 5E-F).

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

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

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

To comprehensively investigate the role of mouse PDGFRβ CAR-T in the treatment of hypertensive fibrosis-related diseases, we established an Ang II-induced hypertension model. Blood pressure was monitored weekly in mice. One week after modeling, PDGFRβ CAR-T cells were reinfused via tail vein injection. Three weeks later, the heart, kidneys, and blood vessels were perfused and harvested. At the molecular level, Western blot and IHC were used to examine changes in fibrosis-related proteins in the renal cortex of Ang II hypertension mice. Picrosirius red staining, Masson's staining, and hematoxylin-eosin staining were also used to examine changes in heart, kidney, and vascular morphology, as well as changes in the degree of fibrosis. Echocardiography was also used to examine the effects of PDGFRβ CAR-T cells on mouse cardiac function.

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 6A). Echocardiography results showed that PDGFRβCAR-T could further improve the increase in left ventricular posterior wall thickness, ejection fraction, and left ventricular short-axis shortening rate caused by AngII in mice (Figure 6B). Morphological staining results showed that PDGFRβCAR-T could improve heart, kidney, and vascular damage and fibrosis in DOCA/salt hypertensive mice (Figure 6C-G). Kidney weight to body weight ratio and urine microalbumin test results showed that PDGFRβCAR-T could improve renal function damage (Figure 6E-F).

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

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

In summary, the present invention obtained a high-affinity single-chain antibody targeting mouse PDGFRβ antigen by immunizing mice, and applied it to the second-generation CAR. The constructed CAR-T cells can effectively kill PDGFRβ ⁺ cells and have the function of preventing and alleviating mouse CKD. It provides a new strategy for the clinical treatment of CKD and other diseases including renal fibrosis with PDGFRβCAR-T cells, and provides a new idea for the treatment of kidney disease, cardiovascular disease and various tumor diseases by clearing PDGFRβ ⁺ cells. It has extremely attractive value for further development and application prospects.

Amino acid sequence information used in the examples:

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

in:

The heavy chain sequence is:

The light chain sequence is:

The G4S linker sequence is:

The CD8 SP sequence is:

The CD8 Hinge region sequence is:

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

The CD3ζ chain sequence is:

The PDGFRβ ECD sequence is:

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

Claims (17)

A chimeric antigen receptor targeting PDGFRβ, characterized in that it comprises at least an antigen binding domain; the antigen binding domain is an anti-PDGFRβ antibody, and the anti-PDGFRβ antibody is a single-chain antibody 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. The chimeric antigen receptor targeting PDGFRβ according to claim 1, wherein the antibody heavy chain is directly connected to the antibody light chain or is connected through a connecting fragment. The chimeric antigen receptor targeting PDGFRβ according to claim 2, wherein the connecting fragment is selected from a polypeptide chain consisting of alanine and/or serine and/or glycine. The chimeric antigen receptor targeting PDGFRβ according to claim 2, wherein the length of the connecting fragment is 3 to 30 amino acids. The chimeric antigen receptor targeting PDGFRβ according to claim 2, wherein the connecting fragment is (GGGGS)n, where n is 1-6. The chimeric antigen receptor targeting PDGFRβ according to claim 1, characterized in that it comprises a signal peptide, an antigen binding domain, a chimeric receptor hinge region, a co-stimulatory signaling domain and a signaling domain connected in series. The chimeric antigen receptor targeting PDGFRβ as described in claim 6 is characterized in that the signal peptide is CD8 SP signal peptide. The chimeric antigen receptor targeting PDGFRβ according to claim 6, wherein the chimeric receptor hinge region is CD8Hinge. The chimeric antigen receptor targeting PDGFRβ according to claim 6, wherein the costimulatory signaling domain is a 4-1BB costimulatory signaling domain. The chimeric antigen receptor targeting PDGFRβ according to claim 6, wherein the signaling domain is a CD3ζ signaling domain. A nucleic acid molecule, characterized in that it comprises nucleotides encoding the chimeric antigen receptor targeting PDGFRβ according to any one of claims 1 to 10. A recombinant expression vector, characterized in that the recombinant expression vector comprises the nucleic acid molecule according to claim 11. A CAR-T cell, characterized in that the CAR-T cell is a T lymphocyte modified by the chimeric antigen receptor targeting PDGFRβ according to any one of claims 1 to 10. 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 a medicament for preventing or treating renal fibrosis; c2) preparing a medicament for preventing or treating DOCA/salt-induced hypertension, cardiovascular remodeling, and renal damage; c3) preparing drugs for preventing or treating Ang II-induced hypertension, cardiovascular remodeling, and renal damage; The renal fibrosis is UOO-induced renal fibrosis. 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 according to any one of claims 1 to 10 or the CAR-T cell according to claim 13. The product according to claim 15, wherein the product is a medicine. The product according to claim 16, wherein the drug further comprises a pharmaceutically acceptable carrier.