CN119409837B - Chimeric conversion receptor for macrophages and application thereof - Google Patents
Chimeric conversion receptor for macrophages and application thereofInfo
- Publication number
- CN119409837B CN119409837B CN202411543869.0A CN202411543869A CN119409837B CN 119409837 B CN119409837 B CN 119409837B CN 202411543869 A CN202411543869 A CN 202411543869A CN 119409837 B CN119409837 B CN 119409837B
- Authority
- CN
- China
- Prior art keywords
- delivery system
- receptor
- chimeric
- lipid nanoparticle
- nucleic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
- A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
-
- 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
-
- 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
- C07K14/70521—CD28, CD152
-
- 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
- C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Toxicology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Inorganic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Nanotechnology (AREA)
- Dermatology (AREA)
- Neurosurgery (AREA)
- Dispersion Chemistry (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Optics & Photonics (AREA)
Abstract
The invention discloses a chimeric conversion receptor for macrophages and application thereof, belonging to the technical field of biological medicines. The chimeric transition type receptor for macrophages provided by the invention comprises an extracellular region, a transmembrane region and an intracellular region, wherein the extracellular region comprises an extracellular domain of IL-2 alpha, IL-2 beta or IL-2 gamma receptor, the transmembrane region comprises a CD8 or CD28 molecular transmembrane domain, and the intracellular region comprises a CD3 zeta, TLR4, CD40 or Dectin1 molecular intracellular domain. The chimeric transition type receptor of the invention is expressed on the surface of tumor-associated macrophages, so that after the exogenous IL-2 is combined with the chimeric transition type receptor on the cell surface, the M2 type macrophages are induced to transform towards the M1 phenotype, thereby fully playing the phagocytic killing effect on tumor cells.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a chimeric conversion type receptor for macrophages and application thereof.
Background
The information disclosed in the background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an admission or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
Renal cancer, also known as renal cell carcinoma (RENAL CELL carpinoma, RCC), is one of the common malignant tumors of the urinary system. Radical resection is an effective treatment for early stage renal cancer, but 20-30% of patients with renal cancer have undergone distant metastasis at the time of diagnosis, and about 40% of patients have suffered from recurrent problems after tumor resection. Patients with renal cancer metastasis are insensitive to conventional chemotherapy, and the effects of immunotherapy and molecular targeted therapy are also not ideal, so that the drug response rate is low. The existing clinic treatment of the advanced renal carcinoma mainly comprises anti-vascular factors, cytokines, monoclonal antibodies and kinase inhibitors, and has the problems of high cost, large side effect, easy generation of drug resistance and the like. Thus, there is a great need to develop new therapeutic strategies and drugs for the treatment of renal cancer.
Tumor immunotherapy has been rapidly developed in recent years, and has become a new weapon in clinical tumor therapy. Immune cell therapy, represented by CAR-T therapies (CHIMERIC ANTIGEN receptor T cells, chimeric antigen receptor modified T cell therapies), has shown good therapeutic effects in a variety of hematological tumors, and has become a leading-edge hotspot for current tumor immunotherapy. However, with the continuous popularization of clinical application, the side effects and limitations of CAR-T therapy are increasingly prominent. Macrophages (Macrophage, mΦ) are used as key cells in natural immunity and are involved in the progress of various diseases of the body including tumors and infections. Macrophages have strong tissue penetration capacity and good plasticity, and are largely infiltrated in a variety of solid tumors, which makes them more suitable than T cells as the basis cells for CAR immune cell therapies in solid tumors.
Interleukin-2 (IL-2) is an important cytokine in humans, which stimulates T cell proliferation and NK cell activation, and has immunoregulatory and antitumor effects. In 1992, the U.S. Food and Drug Administration (FDA) approved recombinant human IL-2 for the treatment of adult metastatic renal cancer, which was the first immunotherapy approved historically by humans for the treatment of cancer patients. In 1998, the FDA approved IL-2 for treating metastatic melanoma patients, and the IL-2 is currently used as an important clinical treatment means after kidney cancer operation, has a certain clinical effect on the comprehensive management of kidney cancer, and further widens the new application of the IL-2 cytokine therapy in kidney cancer based on the existing IL-2 cytokine therapy, thereby having important significance on the comprehensive treatment of kidney cancer.
Disclosure of Invention
In view of the above, the invention provides a chimeric conversion type receptor for macrophages and application thereof, and the chimeric conversion type receptor provided by the invention can induce M2 type immunosuppressive macrophages into M1 type immunocompetent macrophages, so that the chimeric conversion type receptor fully plays a role in phagocytosis and killing of tumor cells, can be entrapped by liposome and then is compounded with hydrogel, thereby reducing toxic and side effects and realizing the anti-tumor purpose.
In a first aspect, the present invention provides a chimeric transition-type receptor for macrophages comprising:
An extracellular region comprising an extracellular domain of an IL-2α, IL-2β or IL-2γ receptor;
a transmembrane region comprising a CD8 or CD28 molecular transmembrane domain;
An intracellular region comprising a cd3ζ, TLR4, CD40, or Dectin1 molecular intracellular domain.
Preferably, the extracellular region is selected from the extracellular domain of the IL-2. Beta. Receptor, the transmembrane region is selected from the CD28 molecular transmembrane domain, and the intracellular region is selected from the intracellular domain of the TLR4 molecule.
Further, the amino acid sequence of the extracellular domain of the IL-2 beta receptor is shown as SEQ ID NO.1, the amino acid sequence of the CD28 molecular transmembrane domain is shown as SEQ ID NO.2, and the amino acid sequence of the intracellular domain of the TLR4 molecule is shown as SEQ ID NO. 3.
In a second aspect, the present invention provides a nucleic acid molecule encoding the chimeric transducer receptor described above.
In a third aspect, the present invention provides a lipid nanoparticle delivery system carrying a nucleic acid molecule as described in the second aspect above.
Preferably, the lipid nanoparticle delivery system further comprises NMS-C9H19, cholesterol, DMG-PEG, DSPE-PEG-mannose and DOPE, wherein the structural formula of the NMS-C9H19 is shown as the formula (I):
Preferably, the molar ratio of NMS-C9H19, cholesterol, DMG-PEG, DSPE-PEG-mannose to DOPE is (10-45): (20-35): (0.4-1.5): (10-40), and the mass ratio of the nucleic acid molecule to NMS-C9H19 is 1 (8-12).
In a fourth aspect, the present invention provides a hydrogel-liposome combination delivery system comprising the lipid nanoparticle delivery system of the third aspect, a carbohydrazide-modified gelatin, an aldehyde-modified hyaluronic acid, and exogenous recombinant IL-2.
Preferably, the mass ratio of the aldehyde group hyaluronic acid to the carbohydrazide modified gelatin is 1 (1-4), and the mass ratio of the exogenous recombinant IL-2 to the total mass of the aldehyde group hyaluronic acid and the carbohydrazide modified gelatin in the lipid nanoparticle delivery system is (0.6-1.2) mug (6-12) mug (12-22) mg.
In a fifth aspect, the present invention provides the use of a chimeric switch receptor according to the first aspect or a nucleic acid molecule according to the second aspect or a lipid nanoparticle delivery system according to the third aspect or a hydrogel-liposome combination delivery system according to the fourth aspect in the manufacture of a medicament for the treatment of renal cancer.
Compared with the prior art, the invention has the following beneficial effects:
(1) The chimeric conversion type receptor for the macrophage constructed by the invention is expressed on the surface of the tumor-associated macrophage, so that after the exogenous IL-2 is combined with the chimeric conversion type receptor on the cell surface, the M2 type macrophage is induced to be transformed towards the M1 phenotype, thereby fully playing the phagocytic killing effect on the tumor cells.
(2) The lipid nanoparticle delivery system constructed by the invention is used for locally delivering nucleic acid molecules encoding specific chimeric transition receptors to kidney tumors, so as to realize in-vivo reprogramming of tumor local macrophages. In addition, mannose molecules are connected to the surface of the lipid nanoparticle delivery system for modification, and the lipid nanoparticle delivery system can be specifically combined with macrophage surface receptors, so that active targeting and drug delivery to macrophages are realized, and the lipid nanoparticle delivery system has good biological characteristics and cell targeting.
(3) The hydrogel-liposome combined drug delivery system constructed by the invention uses the hydrogel system which takes the aldehyde hyaluronic acid (HA-CHO) and the carbohydrazide modified gelatin (Gel-CDH) as raw materials, HAs good bioadhesion and injectability, and can be used as a drug reservoir for carrying out local kidney injection, so that the carried drug is slowly released at local kidney tumor at a stable and controllable rate and with proper concentration, the accurate drug delivery is realized, the drug effect is fully exerted, the toxic and side effects caused by systemic drug administration are avoided, and the anti-tumor purpose is realized.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. It will be obvious to those skilled in the art that other figures may be obtained from these figures without the inventive effort.
FIG. 1 shows the construction of IL-2R/TLR4 CSR CIRCRNA and the verification of a downstream signal path in the embodiment 1 of the invention, wherein A is a schematic diagram of a circRNA structure, B is gel electrophoresis detection and analysis of stability of the circRNA to RNase digestion, and C is Western blot experiment detection of protein expression of a related signal path of BMDMs after different stimulations of a nano drug and PBS/IL-2;
FIG. 2 is a transmission electron microscope image of CSR-mLNP of example 2 of the present invention;
FIG. 3 is a hydrogel injection effect display picture of example 3 of the present invention;
FIG. 4 is a graph showing the effect of CSR-mLNP of example 2 on phagocytosis in vitro of macrophages, wherein A is the laser confocal result of groups G1, G2 and G3, B is the statistical histogram of the number of phagocytized magnetic beads per macrophage, G1 is PBS control group, G2 is IL-2 treated group, and G3 is CSR-mLNP/IL-2 treated group;
FIG. 5 shows the verification of the anti-tumor effect of the injectable liposome composite hydrogel of example 3 in a Renca kidney cancer mouse model, wherein A is a living animal imaging schematic diagram, B is a mouse tumor fluorescence intensity statistical diagram, C is a mouse kidney tumor general image, D is a mouse kidney weight statistical histogram, G1 is a PBS control group, G2 is a hydrogel-loaded IL-2 treatment group, and G3 is a hydrogel-loaded CSR-mLNP/IL-2 treatment group.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In the present invention, the term "chimeric transition-type Receptor", equivalent to "CSR", i.e., "CHIMERIC SWITCH Receptor", is an artificially constructed recombinant Receptor comprising an extracellular antigen-recognition domain, a transmembrane region and a signal transduction domain of an intracellular region. Unlike the extracellular antigen recognition domain of a chimeric antigen receptor which is composed of a single chain antibody, the extracellular antigen recognition domain of a chimeric switch receptor is mainly composed of an inhibitory surface receptor of an immune cell.
In the present invention, the term "extracellular domain" refers to a section of a membrane protein located outside a cell. These segments are often broken off during signal transduction by endopeptidases on the cell surface, which have the effect of modulating the signal pathway.
In the present invention, the term "transmembrane domain" (transmembrane domain, TM) is a structural feature of a protein that spans the cell membrane, typically consisting of an alpha helix or beta barrel structure. This structure connects the inside and outside of the cell and serves a variety of functions, such as modulating signal transduction across the cell membrane, substance transport, etc. The transmembrane domain is typically composed of hydrophobic amino acids, which are capable of intercalating into the hydrophobic interior of the cell membrane.
In the present invention, the term "intracellular domain" refers to a specific region of a protein molecule that is present in a cell, and is generally associated with functions such as intracellular signaling and regulation. These domains typically interact with other components within the cell through protein transport or translocation processes on the cell membrane or organelle membrane.
In the present invention, the term "treatment" is intended to mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects caused by the disease. "treating" as used herein encompasses diseases in mammals, particularly humans, including (a) preventing the occurrence of a disease or disorder in an individual susceptible to the disease but not yet diagnosed with the disease, (b) inhibiting the disease, e.g., arresting the development of the disease, or (c) alleviating the disease, e.g., alleviating symptoms associated with the disease. As used herein, "treating" or "treatment" encompasses any administration of a drug or transgenic immune cell to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administration of a drug comprising a chimeric transition-type receptor-containing cell of the invention to an individual in need thereof.
In the present invention, the term "IRES element" is a segment of non-coding RNA, also known as an internal ribosome entry site sequence (Internal ribosome ENTRY SITE), which recruits ribosomes for translation of mRNA.
The present invention provides a chimeric transition-type receptor for macrophages, comprising:
An extracellular region comprising an extracellular domain of an IL-2α, IL-2β or IL-2γ receptor;
a transmembrane region comprising a CD8 or CD28 molecular transmembrane domain;
An intracellular region comprising a cd3ζ, TLR4, CD40, or Dectin1 molecular intracellular domain.
In the present invention, the extracellular region is preferably the extracellular domain of IL-2. Beta. Receptor, the transmembrane region is preferably the CD28 molecular transmembrane domain, and the intracellular region is preferably the intracellular domain of TLR4 molecule. Further, the amino acid sequence of the extracellular domain of the IL-2 beta receptor is shown as SEQ ID NO.1, the amino acid sequence of the CD28 molecular transmembrane domain is shown as SEQ ID NO.2, and the amino acid sequence of the intracellular domain of the TLR4 molecule is shown as SEQ ID NO. 3.
SEQ ID NO.1:
MATIALPWSLSLYVFLLLLATPWASAAVKNCSHLECFYNSRANVSCMWSHEEALNVTTCHVHAKSNLRHWNKTCELTLVRQASWACNLILGSFPESQSLTSVDLLDINVVCWEEKGWRRVKTCDFHPFDNLRLVAPHSLQVLHIDTQRCNISWKVSQVSHYIEPYLEFEARRRLLGHSWEDASVLSLKQRQQWLFLEMLIPSTSYEVQVRVKAQRNNTGTWSPWSQPLTFRTRPADPMKE;
SEQ ID NO.2:
FWALVVVAGVLFCYGLLVTVALCVIWT;
SEQ ID NO.3:
AGCKKYSRGESIYDAFVIYSSQNEDWVRNELVKNLEEGVPRFHLCLHYRDFIPGVAIAANIIQEGFHKSRKVIVVVSRHFIQSRWCIFEYEIAQTWQFLSSRSGIIFIVLEKVEKSLLRQQVELYRLLSRNTYLEWEDNPLGRHIFWRRLKNALLDGKASNPEQTAEEEQETATWT.
The invention innovatively constructs an IL-2R/TLR4 chimeric conversion receptor (CSR) so as to enable the IL-2 chimeric conversion receptor to be expressed on the surface of tumor-related macrophages, and can convert an upstream IL-2 stimulation signal into a downstream TLR4 activation signal after the exogenous IL-2 is combined with the cell surface IL-2R/TLR4 CSR, thereby realizing the phosphorylation activation of a TLR4 downstream signal channel, and finally inducing the macrophages to transform towards an M1 phenotype so as to fully play the phagocytic killing effect on tumor cells.
The invention also provides a nucleic acid molecule which encodes the chimeric transition receptor. Immune cells carrying the nucleic acid molecules described above may express chimeric transducer receptors as described above. It is noted that, for the nucleic acid molecules mentioned herein, one skilled in the art will understand that either one or both of the complementary double strands are actually included. In addition, the molecular sequence in the present invention includes a DNA form or an RNA form, one of which is disclosed, meaning the other is also disclosed, and the RNA may be mRNA, siRNA, miRNA, saRNA or the like, and the present invention preferably constructs a circular RNA (circRNA).
The invention also provides a lipid nanoparticle delivery system that supports the nucleic acid molecules described above. The present invention uses lipid nanoparticles to load the nucleic acid molecules for local delivery of circRNA encoding specific chimeric turnover receptors to renal tumors, enabling reprogramming of tumor local macrophages in vivo.
In the invention, the lipid nanoparticle delivery system also comprises NMS-C9H19, cholesterol, DMG-PEG (phospholipid-polyethylene glycol), DSPE-PEG-mannose (phospholipid-polyethylene glycol-mannose) and DOPE (dioleoyl phosphatidylethanolamine), wherein the structural formula of the NMS-C9H19 is shown as the formula (I):
the DMG-PEG is preferably DMG-PEG 2000, namely dimyristoylglycerol-polyethylene glycol 2000.
In the invention, the mol ratio of NMS-C9H19, cholesterol, DMG-PEG, DSPE-PEG-mannose to DOPE is (10-45): (20-35): (0.4-1.5): (10-40), and the mass ratio of the nucleic acid molecule to NMS-C9H19 is 1 (8-12), more preferably 1:10.
The preparation method of the lipid nanoparticle delivery system is not particularly limited, and the lipid nanoparticle delivery system is prepared by adopting a preparation method commonly used in the field, and preferably adopts a microfluidic technology. The specific steps for preparing the lipid nanoparticle delivery system using microfluidic technology are not particularly limited in the present invention.
The invention also provides a hydrogel-liposome combined administration system, which comprises the lipid nanoparticle delivery system, carbohydrazide modified gelatin, aldehyde hyaluronic acid and exogenous recombinant IL-2.
The aldehyde hyaluronic acid and the carbohydrazide modified gelatin are mixed to generate Schiff base reaction to quickly form gel, so that the gel has good bioadhesion and injectability, and can be used as a drug reservoir for local injection of kidneys, so that the carried drugs are slowly released at local parts of kidney tumors at a stable and controllable rate and with proper concentration, accurate drug administration is realized, and the drug effect is fully exerted.
In the invention, the mass ratio of the aldehyde hyaluronic acid to the carbohydrazide modified gelatin is 1 (1-4), and the mass ratio of the exogenous recombinant IL-2 to the total mass of the aldehyde hyaluronic acid and the carbohydrazide modified gelatin in the lipid nanoparticle delivery system is (0.6-1.2) mug (6-12) mug (12-22) mg.
The preparation methods of the carbohydrazide modified gelatin and the aldehyde hyaluronic acid are not particularly limited, and the carbohydrazide modified gelatin and the aldehyde hyaluronic acid are prepared by adopting the preparation methods disclosed in the art.
The hydrogel-liposome combined drug delivery system provided by the invention has good bioadhesion and injectability, and can be used as a drug reservoir for local injection of the kidney, so that the carried drug can be slowly released at local part of kidney tumor at a stable and controllable rate and with proper concentration, and accurate drug delivery is realized, thereby fully playing the drug effect and avoiding toxic and side effects caused by systemic drug delivery.
The invention also provides application of the chimeric conversion type receptor, the nucleic acid molecule, the lipid nanoparticle delivery system or the hydrogel-liposome combined administration system in preparation of medicines for treating renal cancer.
The technical scheme of the invention is further described below by combining specific embodiments.
In the following examples, NMS-C9H19 was prepared as follows:
(1) In a 250mL three-necked flask, precisely weighed N-Boc-ethylenediamine (1.05 g,6.58 mmol), 1-bromononane (3.0 g,14.5 mmol), potassium carbonate (1.82 g,13.16 mol) and anhydrous acetonitrile (30 mL) were mixed. The reaction temperature was maintained at 80℃with a heated magnetic stirrer, stirred at constant speed for 72 hours, and the reaction of the mixture was monitored by Thin layer chromatography (Thin-layer chromatography, TLC) until the reaction was complete. The reaction solution was collected and concentrated under reduced pressure using a rotary evaporator, and the product was separated and purified by silica gel column chromatography using an eluent (PE/EA) to give intermediate (tert-butyl 2- (dinonylamino) ethyl) carbamate.
(2) The resulting tert-butyl (2- (dinonylamino) ethyl) carbamate (5 g,12.1 mmol) was dissolved in 30mL of 1, 4-dioxane solution followed by the addition of 30mL of HCl-1, 4-dioxane (1M) solution. The resulting solution was mixed at room temperature for 3 hours with a magnetic stirrer. The reaction of the mixture was monitored by TLC until the reaction was complete. The reaction was washed with saturated NaHCO 3 and saturated NaCl solution, respectively, the organic layer was collected and anhydrous MgSO 4 was added to dry the sample, which was left to stand overnight for treatment. After filtration the reaction was concentrated by rotary evaporator. The product was used directly in the subsequent reaction without purification.
(3) To a 250mL round bottom flask equipped with citric acid (0.8 g,4.2 mmol), HOBt (2.0 g,14.8 mmol) and DIC (3.2 mL,20 mmol) was added 80mL of anhydrous DMF and activated by stirring with a magnetic stirrer for 15 minutes, followed by addition of N1, N1-dinonylethane-1, 2-diamine (4.4 g,14.1 mmol) prepared in step (2) and stirring at room temperature for reaction for 10 hours. The reaction was washed with saturated NaHCO 3 and saturated NaCl solution, respectively, the organic layer was collected and anhydrous MgSO 4 was added to dry the sample, which was left to stand overnight for treatment. After filtration the reaction was concentrated by rotary evaporator. The product was separated and purified by silica gel column chromatography using eluent (CH 2Cl2/MeOH) to obtain NMS-C9H19, structural formula shown below:
Example 1
This example provides the construction of IL-2R/TLR4 CSR CIRCRNA.
IL-2R/TLR4 CSR is the sequence of the extracellular domain of IL-2 receptor (IL-2R), the sequence of CD28 transmembrane domain and the sequence of TLR4 intracellular domain in sequence, the amino acid sequence of the extracellular domain of IL-2 beta receptor is shown as SEQ ID NO.1, the amino acid sequence of CD28 transmembrane domain is shown as SEQ ID NO.2, and the amino acid sequence of TLR4 intracellular domain is shown as SEQ ID NO. 3.
IL-2R/TLR4CSR CIRCRNA was synthetically provided by Gicemetery Biotechnology Inc., guangzhou, which comprises a nucleotide sequence encoding IL-2R/TLR4 CSR. As shown in FIG. 1A, circular RNA (IL-2R/TLR 4 CSRCIRCRNA) was prepared by in vitro transcriptional cyclization of a validated IRES active element and a validated aptamer T4RNA ligase 2 (T4 Rnl-2) added upstream and downstream of the linear RNA encoding anti-CA9 CAR. The preparation process comprises the following steps:
(1) In vitro cyclization, a cyclization reaction system for RNA was formulated as in table 1:
TABLE 1 cyclization reaction system
| Component (A) | Dosage of |
| DEPC-H2O | Up to 400μL |
| RNA inhibitor(40U/μL) | 20μL |
| Linear RNA | 100μg |
| 10×T4 Rnl2 Buffer | 40μL |
| T4 Rnl-2(10U/μL) | 60μL |
After gentle pipetting and mixing, the reaction was carried out at 25℃for 3h and maintained at 4℃to give the cyclized product.
(2) And (3) cyclizing product purification:
① To 400. Mu.L of the above cyclized product, 200. Mu.L of LiCI (8M, RNase-free) was added.
② After mixing well, placing at-20 ℃ for at least 30min, centrifuging at maximum rotation speed and 4 ℃ for 15min, and collecting precipitate.
③ RNA pellet was washed by adding 500. Mu.L of ice-chilled 70% ethanol.
④ RNA pellet was dissolved with 20. Mu.L RNase-freeH 2 O.
⑤ RNA concentrations were detected using a micro nucleic acid meter.
⑥ The purified RNA solution was stored at-80 ℃.
(3) RNase R digestion removes linear RNA:
linear RNA in the circularized product was digested with RNaseR (Geneseed, cat. No.: R0301) under the reaction conditions shown in Table 2, and the digested product was recovered by lithium chloride precipitation.
TABLE 2 reaction conditions for RNase R digestion to remove Linear RNA
| Component (A) | Dosage of |
| 10×Buffer | 20μL |
| Cyclized product | 100μg |
| RNA inhibitor(40U/μL) | 10μL |
| RNase R(20U/μL) | 20μL |
| DEPC-H2O | Up to 200μL |
After mixing by pipetting, the mixture was gently blown and aspirated, and the reaction was carried out at 37℃for 15min, maintaining the temperature at 4 ℃. After digestion, purification was performed using LiCI precipitate, and finally, circular RNA (circRNA) used in this example was obtained.
The stability of the constructed circRNA against RNase degradation was tested, and IL-2R/TLR4 CSR in the form of linear RNA (Linear RNA) and circular RNA (circRNA) was incubated with RNase R for 30min and then analyzed using gel electrophoresis, as shown in FIG. 1B, it was seen that the linear RNA was completely degraded after incubation with RNase, whereas the circular RNA was hardly degraded, indicating that the IL-2R/TLR4 CSR in the form of circular RNA had good stability.
As shown in C in fig. 1, using flow cytometry to detect the phosphorylation level of TLR 4-related signaling pathway molecules in cells expressing IL-2R/TLR4 CSR under exogenous recombinant IL-2 stimulation, it can be seen that exogenous recombinant IL-2 is capable of significantly activating TLR4 downstream signaling pathways through IL-2R/TLR4 CSR, thereby providing the basis for downstream function.
Example 2
The embodiment provides a preparation method of lipid nanoparticles for encapsulating circRNA.
NMS-C9H19, cholesterol, DMG-PEG2000, DSPE-PEG-mannose and DOPE were co-dissolved in ethanol as organic phases in a molar ratio of 15:25:0.5:0.5:20.
The circRNA in example 1 was dissolved in citrate buffer at ph=4 as aqueous phase.
The aqueous phase and the organic phase were mixed in a 3:1 volume ratio in a microfluidic chip device, wherein the mass ratio of NMS-C9H19 to circRNA was 10:1, and ethanol was removed by ultrafiltration to obtain lipid nanoparticles (CSR-mLNP) encapsulating the circRNA, the transmission electron microscopy image of which is shown in FIG. 2, and the average particle size was about 120 nm.
Example 3
The embodiment provides a preparation method of an injectable liposome composite hydrogel.
3.00G of gelatin and 2.20g of carbohydrazide were dissolved in 300mL of ultrapure deionized water with stirring at 55 ℃. Then, 0.50g of EDC (1- [ 3-dimethylaminopropyl ] -3-ethylcarbodiimide hydrochloride) and 0.50g of HOBt (1-hydroxybenzotriazole) were added to the solution. Finally, the pH of the above solution was adjusted to 5 using 0.1M HCl solution, and the mixed solution was stirred overnight to give carbohydrazide-modified gelatin (Gel-CDH).
2G of Hyaluronic Acid (HA) was dissolved in 200mL of pure water and 10mL of 0.5M NaIO 4 was added dropwise. The reaction was stirred under light-shielding conditions for 2 hours, and then 4mL of ethylene glycol was added to terminate the reaction, to obtain the hydroformylation hyaluronic acid (HA-CHO).
HA-CHO and Gel-CDH (mass ratio 3:5) were mixed to give an injectable hydrogel.
1. Mu.g of exogenous recombinant IL-2, 10. Mu.g of the circRNA-entrapped lipid nanoparticle of example 2, and 20mg of the injectable hydrogel described above were mixed to give an injectable liposome composite hydrogel (CSR-mLNP/IL-2@gel).
The hydrogel is prepared into gel and then inhaled into a syringe, and can be molded into various shapes through a syringe needle, as shown in fig. 3, so that the hydrogel has good injectability.
Test examples
1. Injectable liposomal composite hydrogel (CSR-mLNP/IL-2@gel) for macrophage in vitro phagocytosis validation:
In vitro cultured mouse bone marrow-derived macrophages (BMDMs) were treated with 25ng/mL M-CSF (macrophage colony stimulating factor) for 7 days, after which the cells were further cultured in M2 macrophage conditioned medium containing 20ng/mL IL-4 for 2 days to polarize them into M2 macrophages. After completion of the induction culture, macrophages were treated with CSR-mLNP and then cultured for a further 24 hours, after which 20ng/mL IL-2 was added to the cell culture medium and stimulated for 24 hours, the cell phagocytosis assay was performed.
For the tumor cell phagocytosis assay, BMDMs stimulated by the above culture was co-cultured with antibody-coated magnetic beads, wherein G1 was PBS-treated group, G2 was IL-2-treated group, and G3 was CSR-mLNP +IL-2-treated group. After 6 hours of co-culture, BMDMs was stained with a living cell dye and imaged for macrophage phagocytic function using a laser confocal imaging system. As shown in fig. 4, the confocal results showed that the number of phagocytic beads in CSR-mLNP/IL-2 treated group BMDMs was significantly higher than that in other control groups, demonstrating that CSR-mLNP of example 2 of the present invention was able to induce enhanced phagocytic function in vitro in mouse macrophages.
2. Investigation of the renal carcinoma inhibiting Effect of injectable Liposome composite hydrogel (CSR-mLNP/IL-2@gel):
The kidney cancer inhibition effect of the injectable liposome composite hydrogel (CSR-mLNP/IL-2@gel) developed in the embodiment 3 of the invention is verified by constructing an in-situ kidney cancer tumor-bearing mouse model by using a mouse Renca cell line, and each prepared group system (G1: PBS control group; G2: hydrogel load IL-2 treatment group; G3: hydrogel load CSR-mLNP/IL-2 treatment group) is subjected to kidney local injection by using an ultrasonic guidance method, and when the growth condition of tumors is monitored by using a small animal imaging system, the tumors are taken out, photographed and weighed when the experimental end point is reached, as shown in fig. 5, the hydrogel load CSR-mLNP/IL-2 treatment group shows the most remarkable tumor inhibition effect (tumor fluorescence intensity and tumor size and weight are the lowest), so that the injectable liposome composite hydrogel developed in the embodiment 3 of the invention can remarkably inhibit the growth of kidney cancer.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411543869.0A CN119409837B (en) | 2024-10-31 | 2024-10-31 | Chimeric conversion receptor for macrophages and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411543869.0A CN119409837B (en) | 2024-10-31 | 2024-10-31 | Chimeric conversion receptor for macrophages and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN119409837A CN119409837A (en) | 2025-02-11 |
| CN119409837B true CN119409837B (en) | 2025-09-19 |
Family
ID=94469057
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202411543869.0A Active CN119409837B (en) | 2024-10-31 | 2024-10-31 | Chimeric conversion receptor for macrophages and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN119409837B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN119371557A (en) * | 2024-10-31 | 2025-01-28 | 山东大学齐鲁医院 | A chimeric antigen receptor targeting CA9 and its application |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11654158B2 (en) * | 2016-11-17 | 2023-05-23 | 2Seventy Bio, Inc. | TGFBeta signal convertor |
| EP3624834A1 (en) * | 2017-05-17 | 2020-03-25 | Thunder Biotech Inc. | Transgenic macrophages, chimeric antigen receptors, and associated methods |
| WO2024193687A1 (en) * | 2023-03-23 | 2024-09-26 | Nanjing Legend Biotech Co., Ltd. | Inverted cytokine receptors and methods of use thereof |
-
2024
- 2024-10-31 CN CN202411543869.0A patent/CN119409837B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN119371557A (en) * | 2024-10-31 | 2025-01-28 | 山东大学齐鲁医院 | A chimeric antigen receptor targeting CA9 and its application |
Non-Patent Citations (1)
| Title |
|---|
| An in situ engineered chimeric IL-2 receptor potentiates the tumoricidal activity of proinflammatory CAR macrophages in renal cell carcinoma;Weiqiang Jing等;nature cancer;20250429;838-853 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN119409837A (en) | 2025-02-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN119371557B (en) | A chimeric antigen receptor targeting CA9 and its application | |
| WO2012014945A1 (en) | Novel nucleic acid having adjuvant activity and use thereof | |
| WO2024131403A1 (en) | Aptamer-based mrna targeted delivery system for spleen and subcells thereof | |
| Liang et al. | Manganese immunotherapy for treating osteosarcoma: glycosylating 1V209 anchored MnO2 nanosheets prompt pro-inflammatory macrophage polarization | |
| CN116286637A (en) | Targeted peptide functionalized dendritic cell exosome, and preparation method and application thereof | |
| WO2008036421A2 (en) | Reishi-mediated enhancement of human tissue progenitor cell adhesion and differentiation | |
| CN114191539A (en) | Exosome nano particle for composite co-transport of small molecule nucleic acid and active protein, and preparation method and application thereof | |
| WO2025001335A1 (en) | Intervention target for advanced fibrosis of ischemic heart disease and use thereof | |
| CN117598980A (en) | An injectable nucleic acid hydrogel in situ tumor vaccine and its application | |
| US12221471B2 (en) | Nanocarriers for lung inflammation therapy | |
| CN119409837B (en) | Chimeric conversion receptor for macrophages and application thereof | |
| WO2022160374A1 (en) | Aptamer that targets cd206-positive cells, and application thereof | |
| CN1434054A (en) | Dowble-stranded RNA and use thereof | |
| WO2023155861A1 (en) | Aluminum nanocrystalline composite immune drug and preparation method therefor and use thereof | |
| CN111777667B (en) | Small peptides and their application in the preparation of immunomodulatory drugs | |
| CN119792502A (en) | mRNA vaccines and their application in the preparation of drugs for treating tumors | |
| KR20240124200A (en) | Lipid Nanoparticles for the treatment of Inflammatory Bowel Disease | |
| CN115261384B (en) | A deoxyribozyme targeting PD-L1 gene, a targeted delivery vector, a tumor-targeted nanocomplex and applications thereof | |
| CN104721835A (en) | Melanoma inhibiting DNA plasmid vaccine and preparation method thereof | |
| CN117143250A (en) | An IL-15/IL-15Rα fusion gene, vector, engineered adipocytes and preparation method thereof | |
| CN117414426A (en) | Use of K-ras gene in preparing reagents for regulating adenosine content in the body and anti-tumor applications | |
| CN116077676A (en) | Protein targeted degradation chimeric body, preparation method and application thereof | |
| CN115350282A (en) | Drug-loaded exosomes targeting liver cancer, rapid preparation methods and applications | |
| CN114381426A (en) | Natural killer cell and application | |
| CN112138018A (en) | Application of dendrobium officinale polysaccharide in preparation of medicine for treating tumors by targeting tumor-related macrophages |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20250228 Address after: 266000 1st and 2nd floors, 7th Building, Incubation Center of Qingdao Blue Biomedical Industrial Park, 368 Hedong Road, Qingdao Hi-tech Zone, Qingdao City, Shandong Province Applicant after: QINGDAO RESTORE BIOTECHNOLOGY Co.,Ltd. Country or region after: China Address before: 250012 No. 107 west Wenhua Road, Lixia District, Shandong, Ji'nan Applicant before: QILU HOSPITAL OF SHANDONG University Country or region before: China |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20260112 Address after: 266000 Shandong Province, Qingdao High-tech Industrial Development Zone, Zhu Yuan Road No. 18, Building 6, Room 505 Patentee after: Life Valley (Qingdao) Health Technology Co.,Ltd. Country or region after: China Address before: 266000, No. 1, 2, building 7, Qingdao blue bio Pharmaceutical Industrial Park, 368 Hedong Road, Hedong District, Qingdao, Shandong Patentee before: QINGDAO RESTORE BIOTECHNOLOGY Co.,Ltd. Country or region before: China |