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WO2025209170A1 - RÉCEPTEUR ANTIGÉNIQUE CHIMÉRIQUE CIBLANT PDGFRβ, CELLULE CAR-T ET UTILISATION ASSOCIÉE - Google Patents

RÉCEPTEUR ANTIGÉNIQUE CHIMÉRIQUE CIBLANT PDGFRβ, CELLULE CAR-T ET UTILISATION ASSOCIÉE

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Publication number
WO2025209170A1
WO2025209170A1 PCT/CN2025/083143 CN2025083143W WO2025209170A1 WO 2025209170 A1 WO2025209170 A1 WO 2025209170A1 CN 2025083143 W CN2025083143 W CN 2025083143W WO 2025209170 A1 WO2025209170 A1 WO 2025209170A1
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Prior art keywords
pdgfrβ
car
chimeric antigen
antigen receptor
cells
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Pending
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PCT/CN2025/083143
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English (en)
Chinese (zh)
Inventor
易凡
唐伟
赵松柏
李荣坤
张志岳
夏渊
王姿颖
张艳
刘志勇
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Shandong University
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Shandong University
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Publication of WO2025209170A1 publication Critical patent/WO2025209170A1/fr
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4207—Platelet-derived growth factor receptors [PDGFR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/13—Antibody-based
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    • C07K2317/515—Complete light chain, i.e. VL + CL
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    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
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    • C12N2510/00—Genetically modified cells
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present invention belongs to the technical field of biomedicine and molecular biology, and specifically relates to a chimeric antigen receptor targeting PDGFR ⁇ , CAR-T cells and applications.
  • 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
  • CKD chronic kidney disease
  • 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.
  • CKD chronic kidney disease
  • renal protective drugs such as TGF- ⁇ inhibitors, endothelin inhibitors, and renin inhibitors have all failed.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • recognition sequences such as single-chain antibodies, ligands, and receptors
  • Platelet-derived growth factor receptor ⁇ (PDGFR ⁇ ) 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.
  • PDGFR ⁇ is a tyrosine kinase receptor for PDGF-B and PDGF-D.
  • a chimeric antigen receptor targeting PDGFR ⁇ comprising at least an antigen binding domain
  • the amino acid sequence of the antibody light chain (VL) is selected from:
  • 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.
  • the chimeric antigen receptor targeting PDGFR ⁇ 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;
  • the signal peptide is CD8 SP signal peptide
  • the hinge region of the chimeric receptor is CD8 Hinge
  • the costimulatory signaling domain is a 4-1BB costimulatory signaling domain
  • the signaling domain is a CD3 ⁇ signaling domain
  • the chimeric antigen receptor is composed of a CD8 SP signal peptide, an antigen binding domain (VH-(GGGGS) 3 -VL) that binds to the PDGFR ⁇ antigen, a CD8 Hinge, a 4-1BB costimulatory signaling domain, and a CD3 ⁇ 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 PDGFR ⁇ .
  • nucleotide sequence encoding the chimeric antigen receptor targeting PDGFR ⁇ of the present invention can readily mutate the nucleotide sequence encoding the chimeric antigen receptor targeting PDGFR ⁇ 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.
  • 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 PDGFR ⁇ 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 CD3 ⁇ 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.
  • 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 PDGFR ⁇ .
  • the present invention provides a new therapeutic approach for CAR-T treatment of CKD, CVD, and other related diseases with high PDGFR ⁇ expression.
  • 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 PDGFR ⁇ expression.
  • the diseases associated with high PDGFR ⁇ 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 PDGFR ⁇ expression, wherein the active ingredient of the product may be the above-mentioned chimeric antigen receptor or the above-mentioned CAR-T cell.
  • the diseases associated with high PDGFR ⁇ 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 PDGFR ⁇ 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.
  • it can be delivered to the tissue of interest by systemic intravenous delivery or local injection.
  • 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.
  • 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 PDGFR ⁇ expression, which comprises administering an effective amount of the above-mentioned chimeric antigen receptor, CAR-T cell or product to a subject.
  • the above technical solution targets PDGFR ⁇ , successfully preparing an antibody targeting PDGFR ⁇ and constructing a second-generation CAR plasmid vector.
  • a T cell expressing a chimeric antigen receptor (CAR) targeting PDGFR ⁇ was obtained.
  • CAR chimeric antigen receptor
  • PDGFR ⁇ CAR-T cells can effectively eliminate PDGFR ⁇ + 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.
  • UUO unilateral ureteral obstruction
  • FIG1 is a flowchart of mouse immunization in an embodiment of the present invention.
  • Figure 4A is a Western blot detection of the changes in fibrosis-related proteins Vimentin, Fibronectin, Collagen I, and ⁇ -sma in the renal cortex of UUO model mice
  • Figure 4B is an immunohistochemistry detection of the changes in fibrosis-related proteins Fibronectin and ⁇ -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.
  • Figure 6A is a Western blot detection of changes in fibrosis-related proteins ⁇ -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 ⁇ -sma in the renal cortex of
  • 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).
  • 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.
  • domain refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein.
  • 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.
  • 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.
  • 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.
  • KD values dissociation constants
  • BLI bio-layer interferometry
  • 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 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.
  • 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).
  • domesticated animals e.g., cows, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats
  • 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.
  • the antibodies of the invention are used to delay the development of the disease or slow the progression of the condition.
  • the cells designed in the present invention are mouse T cells.
  • murine T cells were derived from the spleen of C57BL/6 mice.
  • CAR-T cells were reinfused by tail vein injection, with a reinfusion dose of 1 ⁇ 10 5 and a reinfusion system of 100-200 ⁇ L.
  • 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 PDGFR ⁇ -targeted CAR-T cells are capable of effectively killing mouse fibroblast cell lines in vitro and effectively preventing and improving kidney disease in vivo.
  • various kidney disease models were created, including unilateral ureteral obstruction (UUO), deoxycorticosterone acetate/salt (DOCA/salt), and angiotensin II (Ang II)-induced hypertension models.
  • UUO unilateral ureteral obstruction
  • DOCA/salt deoxycorticosterone acetate/salt
  • Ang II angiotensin II
  • mice 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 ⁇ L/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.
  • mice 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.
  • mice of appropriate age and weight 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.
  • Ang II sustained-release pump 1000 ng/kg/min, Alzet, model 2004
  • Paraffin tissue sections were stained using the Regen Sirius Red staining kit.
  • Paraffin sections were baked at 65°C for 2 h and dewaxed to water while still hot, following the same procedure as for IHC.
  • 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.
  • mice Two weeks later, the mice were treated, and cardiac, renal, and vascular fibrosis markers were measured.
  • 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.
  • the mice were treated and samples were collected to detect fibrosis indicators such as heart, kidney, and blood vessels.
  • 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-CD3 ⁇ ( 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 PDGFR ⁇ CAR-T cells ( Figure 2D).
  • mouse PDGFR ⁇ CAR 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 PDGFR ⁇ CAR-T cells can effectively kill PDGFR ⁇ + cells in vitro:
  • PDGFR ⁇ CAR-T cells can prevent renal fibrosis induced by unilateral ureteral ligation (UUO):
  • PDGFR ⁇ CAR-T cells can significantly prevent tubular interstitial fibrosis in UUO mice, significantly reduce the changes in Fibronectin, ⁇ -SMA, E-cadherin and Vimentin in the renal cortex of mice, and slow down the progression of renal fibrosis.
  • PDGFR ⁇ CAR-T cells can alleviate the progression of renal fibrosis induced by UUO:
  • PDGFR ⁇ CAR-T cells can significantly improve the degree of renal damage and fibrosis in UUO mice, clarifying the role of PDGFR ⁇ CAR-T cells in preventing the progression of renal fibrosis.
  • PDGFR ⁇ CAR-T cells have the effect of alleviating CKD (deoxycorticosterone acetate-induced hypertension model):
  • Picrosirius red staining was 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 PDGFR ⁇ CAR-T cells on cardiac function.
  • PDGFR ⁇ CAR-T cells can improve DOCA/salt-induced hypertension, cardiovascular remodeling, and renal damage.
  • PDGFR ⁇ CAR-T cells have the effect of alleviating cardiovascular disease (angiotensin II-induced hypertension model):
  • Picrosirius red staining was 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 PDGFR ⁇ CAR-T cells on mouse cardiac function.
  • PDGFR ⁇ CAR-T cells can improve AngII-induced hypertension, cardiovascular remodeling, and renal damage.
  • the present invention obtained a high-affinity single-chain antibody targeting mouse PDGFR ⁇ antigen by immunizing mice, and applied it to the second-generation CAR.
  • the constructed CAR-T cells can effectively kill PDGFR ⁇ + 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 PDGFR ⁇ CAR-T cells, and provides a new idea for the treatment of kidney disease, cardiovascular disease and various tumor diseases by clearing PDGFR ⁇ + cells. It has extremely attractive value for further development and application prospects.
  • 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.
  • the light chain sequence is:
  • the CD8 SP sequence is:
  • the CD8 Hinge region sequence is:

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Abstract

L'invention concerne un récepteur antigénique chimérique ciblant PDGFRβ, une cellule CAR-T et l'utilisation associée. Pour une molécule PDGFRβ, une cellule CAR-T ciblant PDGFRβ est construite au moyen de la conception d'un anticorps monoclonal et de l'immunisation de souris pour obtenir une séquence d'anticorps monocaténaire murine, et il est prouvé que la cellule CAR-T tue les fibroblastes activés in vitro et a un effet thérapeutique sur la fibrose rénale et cardiaque associée à l'hypertension ou au diabète, fournissant ainsi une base pour piéger les fibroblastes musculaires PDGFRβ+, les fibroblastes, etc. dans le traitement clinique des CKD tels que la fibrose rénale, et fournit un nouveau concept pour le traitement clinique de la fibrose hépatique, de maladies cardiovasculaires et de diverses maladies tumorales, et a donc une bonne valeur d'application pratique.
PCT/CN2025/083143 2024-04-02 2025-03-18 RÉCEPTEUR ANTIGÉNIQUE CHIMÉRIQUE CIBLANT PDGFRβ, CELLULE CAR-T ET UTILISATION ASSOCIÉE Pending WO2025209170A1 (fr)

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CN101707882A (zh) * 2007-04-17 2010-05-12 伊姆克罗尼责任有限公司 PDGFRβ特异性抑制剂
US20140193402A1 (en) * 2013-01-09 2014-07-10 Regeneron Pharmaceuticals, Inc. ANTI-PDGFR-beta ANTIBODIES AND USES THEREOF
CN117247462A (zh) * 2023-08-18 2023-12-19 济南泰和医药科技有限公司 一种靶向ror1的嵌合抗原受体、car-t细胞及其用途
CN118440214A (zh) * 2024-04-02 2024-08-06 山东大学 一种靶向PDGFRβ的嵌合抗原受体、CAR-T细胞及应用

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WO2021158110A1 (fr) * 2020-02-07 2021-08-12 Biorion Technologies B.V. Anticorps anti-récepteur du facteur de croissance dérivé des plaquettes (pdgfr), conjugués, compositions et leurs utilisations
CN117695278A (zh) * 2023-12-19 2024-03-15 南京市儿童医院 Ptprj激动剂在制备预防和/或治疗肾脏纤维化的药物中的应用

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US20070037224A1 (en) * 2005-08-11 2007-02-15 Hamer Peter J Quantitative assays for PDGFR-beta in body fluids
CN101707882A (zh) * 2007-04-17 2010-05-12 伊姆克罗尼责任有限公司 PDGFRβ特异性抑制剂
US20140193402A1 (en) * 2013-01-09 2014-07-10 Regeneron Pharmaceuticals, Inc. ANTI-PDGFR-beta ANTIBODIES AND USES THEREOF
CN104936614A (zh) * 2013-01-09 2015-09-23 瑞泽恩制药公司 抗-PDGFR-β抗体及其使用
CN117247462A (zh) * 2023-08-18 2023-12-19 济南泰和医药科技有限公司 一种靶向ror1的嵌合抗原受体、car-t细胞及其用途
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