Chimeric transformant receptor for macrophages and application thereof

Abstract

The invention discloses a chimeric transformational receptor for macrophages and application of the chimeric transformational receptor, and belongs to the technical field of biological medicines. The chimeric transformational receptor for macrophages, provided by the invention, comprises an extracellular domain and an extracellular domain, wherein the extracellular domain comprises an extracellular structural domain of an IL-2alpha, IL-2beta or IL-2gamma receptor; a transmembrane region, wherein the transmembrane region comprises a CD8 or CD28 molecular transmembrane structural domain; and an intracellular region, wherein the intracellular region comprises a molecular intracellular structural domain of CD3 [zeta], TLR4, CD40 or Dectin1. The chimeric conversion type receptor is expressed on the surface of a tumor-related macrophage, so that after exogenous IL-2 is combined with the chimeric conversion type receptor on the surface of the cell, M2 type macrophage is induced to be converted towards M1 phenotype, and the phagocytosis and killing effects on tumor cells are fully exerted.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a chimeric switch receptor for macrophages and applications thereof. Background Art

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

[0003] Kidney cancer, also known as renal cell carcinoma (RCC), is one of the common malignant tumors of the urinary system. Radical resection is an effective treatment for early kidney cancer, but 20-30% of kidney cancer patients have distant metastasis when diagnosed, and about 40% of patients will face recurrence after tumor resection. Patients with renal cancer metastasis are insensitive to conventional chemotherapy, and immunotherapy and molecular targeted therapy are not ideal, with low drug response rates. At present, the clinical treatment of advanced kidney cancer is mainly based on anti-vascular factors, cytokines, monoclonal antibodies and kinase inhibitors, which have problems such as high cost, large side effects, and easy to develop drug resistance. Therefore, there is an urgent need to develop new treatment strategies and drugs for the treatment of kidney cancer.

[0004] Tumor immunotherapy has developed rapidly in recent years and has become a new weapon in clinical tumor treatment. Immune cell therapy represented by CAR-T therapy (Chimeric antigen receptor T cells, chimeric antigen receptor modified T cell therapy) has shown good therapeutic effects in a variety of hematological tumors and has become a frontier hotspot in tumor immunotherapy. However, with the continuous promotion of clinical applications, the side effects and limitations of CAR-T therapy have gradually become prominent. Macrophages (Macrophage, MΦ), as key cells in natural immunity, are involved in the process of various diseases in the body, including tumors and infections. Macrophages have strong tissue penetration ability and good plasticity, and infiltrate a large number of solid tumors, which makes them more suitable than T cells as the basic cells for CAR immune cell therapy in solid tumors.

[0005] Interleukin-2 (IL-2) is an important cytokine in the human body that can stimulate T cell proliferation and activate NK cells, and has immunomodulatory and anti-tumor 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 for the treatment of cancer patients in human history. In 1998, the FDA approved IL-2 for the treatment of patients with metastatic melanoma. Currently, IL-2 is an important clinical treatment for renal cancer after surgery, and it has a certain clinical effect on the comprehensive management of renal cancer. Based on the existing IL-2 cytokine therapy, further expanding its new application in renal cancer is of great significance to the comprehensive treatment of renal cancer. Summary of the invention

[0006] In view of this, the present invention provides a chimeric switch receptor for macrophages and its application. The chimeric switch receptor provided by the present invention can induce M2 immunosuppressive macrophages into M1 immune-activated macrophages, so that they can fully exert the phagocytic and killing effects on tumor cells, and can be encapsulated by liposomes and then compounded with hydrogels, thereby reducing toxic side effects and achieving the anti-tumor purpose.

[0007] In a first aspect, the present invention provides a chimeric switch receptor for macrophages, comprising:

[0008] An extracellular region, the extracellular region comprising the extracellular domain of an IL-2α, IL-2β or IL-2γ receptor;

[0009] A transmembrane region, wherein the transmembrane region includes a CD8 or CD28 molecule transmembrane domain;

[0010] The intracellular region includes the intracellular domain of CD3ζ, TLR4, CD40 or Dectin1 molecule.

[0011] Preferably, the extracellular region is selected from the extracellular domain of the IL-2β receptor; the transmembrane region is selected from the transmembrane domain of the CD28 molecule; and the intracellular region is selected from the intracellular domain of the TLR4 molecule.

[0012] Furthermore, the amino acid sequence of the extracellular domain of the IL-2β receptor is shown in SEQ ID NO.1; the amino acid sequence of the transmembrane domain of the CD28 molecule is shown in SEQ ID NO.2; and the amino acid sequence of the intracellular domain of the TLR4 molecule is shown in SEQ ID NO.3.

[0013] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned chimeric switch receptor.

[0014] In a third aspect, the present invention provides a lipid nanoparticle delivery system, wherein the lipid nanoparticle delivery system carries the nucleic acid molecule described in the second aspect.

[0015] Preferably, the lipid nanoparticle delivery system further comprises NMS-C9H19, cholesterol, DMG-PEG, DSPE-PEG-mannose and DOPE; wherein the structural formula of NMS-C9H19 is shown in formula (I):

[0016]

[0017] Preferably, the molar ratio of the NMS-C9H19, cholesterol, DMG-PEG, DSPE-PEG-mannose and DOPE is (10-45):(20-35):(0.4-1.5):(0.4-1.5):(10-40); the mass ratio of the nucleic acid molecule to the NMS-C9H19 is 1:(8-12).

[0018] In a fourth aspect, the present invention provides a hydrogel-liposome combined drug delivery system, comprising the lipid nanoparticle delivery system described in the third aspect, carbohydrazide-modified gelatin, aldehyde-modified hyaluronic acid and exogenous recombinant IL-2.

[0019] Preferably, the mass ratio of the aldehyded hyaluronic acid and the carbohydrazide-modified gelatin is 1:(1-4); the mass ratio of the exogenous recombinant IL-2, the nucleic acid molecules in the lipid nanoparticle delivery system and the total mass of the aldehyded hyaluronic acid and the carbohydrazide-modified gelatin is (0.6-1.2) μg:(6-12) μg:(12-22) mg.

[0020] In the fifth aspect, the present invention provides the use of the chimeric switch receptor described in the first aspect, the nucleic acid molecule described in the second aspect, the lipid nanoparticle delivery system described in the third aspect, or the hydrogel-liposome combined drug delivery system described in the fourth aspect in the preparation of drugs for treating renal cancer.

[0021] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0022] (1) The chimeric switch receptor for macrophages constructed by the present invention is expressed on the surface of tumor-associated macrophages, which can induce M2 macrophages to transform toward M1 phenotype after exogenous IL-2 binds to the chimeric switch receptor on the cell surface, thereby fully exerting the phagocytic killing effect on tumor cells.

[0023] (2) The lipid nanoparticle delivery system constructed by the present invention is used to locally deliver nucleic acid molecules encoding specific chimeric switch receptors to kidney tumors, thereby achieving in vivo reprogramming of local macrophages in tumors. In addition, the surface of the lipid nanoparticle delivery system is modified with mannose molecules, which can specifically bind to macrophage surface receptors to achieve active targeting and drug delivery to macrophages, and has good biological properties and cell targeting.

[0024] (3) The hydrogel-liposome combined drug delivery system constructed by the present invention uses a hydrogel system with aldehyde-modified hyaluronic acid (HA-CHO) and carbohydrazide-modified gelatin (Gel-CDH) as raw materials, which has good bioadhesion and injectability, and can be used as a drug reservoir for local injection into the kidney, so that the loaded drug is slowly released locally in the kidney tumor at a stable and controllable rate and appropriate concentration, thereby achieving precise drug delivery, thereby fully exerting the efficacy of the drug, avoiding the toxic and side effects caused by systemic medication, and achieving the purpose of anti-tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings constituting 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 description are used to explain the present invention and do not constitute an improper limitation of the present invention. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0026] Figure 1 The IL-2R / TLR4 CSR circRNA construction and downstream signaling pathway verification of Example 1 of the present invention; wherein A is a schematic diagram of the circRNA structure; B is a gel electrophoresis test to analyze the stability of circRNA to RNase digestion; C is a Western blot experiment to detect the expression of related signaling pathway proteins in BMDMs after different stimulations of nanomedicine and PBS / IL-2;

[0027] Figure 2 is a transmission electron microscopy image of CSR-mLNP of Example 2 of the present invention;

[0028] Figure 3 This is a picture showing the hydrogel injection effect of Example 3 of the present invention;

[0029] Figure 4 This is a verification of the effect of CSR-mLNP of Example 2 of the present invention on the phagocytic function of macrophages in vitro, wherein A is the laser confocal results of G1, G2 and G3 groups, and B is a statistical histogram of the number of magnetic beads phagocytosed by each macrophage; G1 is a PBS control group, G2 is an IL-2 treatment group, and G3 is a CSR-mLNP / IL-2 treatment group;

[0030] Figure 5 This is a verification of the anti-tumor effect of the injectable liposome composite hydrogel of Example 3 of the present invention in the Renca renal cancer mouse model; wherein A is a schematic diagram of small animal in vivo imaging, B is a statistical diagram of mouse tumor fluorescence intensity, C is a gross image of mouse kidney tumor, and D is a statistical histogram of mouse kidney weight; 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

[0031] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0032] In the present invention, the term "chimeric switch receptor" is equivalent to "CSR", i.e. "Chimeric Switch Receptor", which is an artificially constructed recombinant receptor, comprising an extracellular antigen recognition domain, a transmembrane region and an intracellular signal transduction domain. Unlike the extracellular antigen recognition domain of the chimeric antigen receptor, which is composed of a single-chain antibody, the extracellular antigen recognition domain of the chimeric switch receptor is mainly composed of inhibitory surface receptors of immune cells.

[0033] In the present invention, the term "extracellular domain" refers to the segment of a membrane protein located outside the cell. These segments are often hydrolyzed and shed by endopeptidases on the cell surface during signal transduction and have the function of regulating signal pathways.

[0034] In the present invention, the term "transmembrane domain" (TM) refers to a structural feature of a protein that spans the cell membrane, usually composed of an α-helix or β-barrel structure. This structure connects the inside and outside of the cell and plays a variety of functions, such as regulating signal transduction and substance transport across the cell membrane. The transmembrane domain is usually composed of hydrophobic amino acids and can be embedded in the hydrophobic interior of the cell membrane.

[0035] In the present invention, the term "intracellular domain" refers to a specific region of a protein molecule that exists in a cell and is usually associated with intracellular signal transduction, regulation, and other functions. These domains usually interact with other components in the cell through protein transport or translocation processes on the cell membrane or organelle membrane.

[0036] In the present invention, the term "treatment" refers to obtaining the desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or adverse effects caused by the disease. "Treatment" as used in the present invention covers diseases in mammals, especially humans, including: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or transgenic immune cell to an individual to treat, cure, alleviate, improve, reduce or inhibit an individual's disease, including but not limited to administering a drug containing cells containing a chimeric switch receptor as described in the present invention to an individual in need.

[0037] In the present invention, the term "IRES element" refers to a non-coding RNA, also known as an internal ribosome entry site sequence (Internal ribosome entry site), which can recruit ribosomes to translate mRNA.

[0038] The present invention provides a chimeric switch receptor for macrophages, comprising:

[0039] An extracellular region, the extracellular region comprising the extracellular domain of an IL-2α, IL-2β or IL-2γ receptor;

[0040] A transmembrane region, wherein the transmembrane region includes a CD8 or CD28 molecule transmembrane domain;

[0041] The intracellular region includes the intracellular domain of CD3ζ, TLR4, CD40 or Dectin1 molecule.

[0042] In the present invention, the extracellular region is preferably the extracellular domain of the IL-2β receptor; the transmembrane region is preferably the transmembrane domain of the CD28 molecule; and the intracellular region is preferably the intracellular domain of the TLR4 molecule. Further, the amino acid sequence of the extracellular domain of the IL-2β receptor is shown in SEQ ID NO.1; the amino acid sequence of the transmembrane domain of the CD28 molecule is shown in SEQ ID NO.2; and the amino acid sequence of the intracellular domain of the TLR4 molecule is shown in SEQ ID NO.3.

[0043] SEQ ID NO.1:

[0044] MATIALPWSLSLYVFLLLLATPWASAAVKNCSHLECFYNSRANVSCMWSHEEALNVTTCHVHAKSNLRHWNKTCELTLVRQASWACNLILGSFPESQSLTSVDLLDINVVCWEEKGWRRV KTCDFHPFDNLRLVAPHSLQVLHIDTQRCNISWKVSQVSHYIEPYLEFEARRRLLGHSWEDASVLSLKQRQQWLFLEMLIPSTSYEVQVRVKAQRNNTGTWSPWSQPLTFRTRPADPMKE;

[0045] SEQ ID NO.2:

[0046] FWALVVVAGVLFCYGLLVTVALCVIWT;

[0047] SEQ ID NO.3:

[0048] AGCKKYSRGESIYDAFVIYSSQNEDWVRNELVKNLEEGVPRFHLCLHYRDFIPGVAIAANIIQEGFHKSRKVIVVVSRHFIQSRWCIFEYEIAQTWQFLSSRSGIIFIVLEKVEKSLLRQQVELYRLLSRNTYLEWEDNPLGRHIFWRRLKNALLDGKASNPEQTAEEEQETATWT.

[0049] The present invention innovatively constructs an IL-2R / TLR4 chimeric switch receptor (CSR) and expresses it on the surface of tumor-associated macrophages. It can enable exogenous IL-2 to bind to the cell surface IL-2R / TLR4 CSR, convert the upstream IL-2 stimulation signal into the downstream TLR4 activation signal, achieve phosphorylation activation of the TLR4 downstream signaling pathway, and ultimately induce macrophages to transform toward the M1 phenotype, so that they can fully exert their phagocytic and killing effects on tumor cells.

[0050] The present invention also provides a nucleic acid molecule encoding the above-mentioned chimeric conversion receptor. Immune cells carrying the above-mentioned nucleic acid molecules can express the aforementioned chimeric conversion receptor. It should be noted that for the nucleic acid molecules mentioned herein, those skilled in the art should understand that they actually include any one or two of the complementary double strands. In addition, the molecular sequences in the present invention include DNA forms or RNA forms, and disclosing one of them means that the other is also disclosed; the RNA can be mRNA, siRNA, miRNA, saRNA, etc. The present invention preferably constructs circular RNA (circRNA).

[0051] The present invention also provides a lipid nanoparticle delivery system, which is loaded with the above-mentioned nucleic acid molecule. The present invention uses lipid nanoparticles loaded with the nucleic acid molecule to locally deliver circRNA encoding a specific chimeric switch receptor to a renal tumor, thereby achieving in vivo reprogramming of local macrophages in the tumor.

[0052] In the present invention, the lipid nanoparticle delivery system further comprises NMS-C9H19, cholesterol, DMG-PEG (phospholipid-polyethylene glycol), DSPE-PEG-mannose (phospholipid-polyethylene glycol-mannose) and DOPE (dioleoylphosphatidylethanolamine); wherein the structural formula of NMS-C9H19 is shown in formula (I):

[0053]

[0054] DMG-PEG is preferably DMG-PEG 2000 , that is, dimyristoylglycerol-polyethylene glycol 2000.

[0055] In the present invention, the molar ratio of the NMS-C9H19, cholesterol, DMG-PEG, DSPE-PEG-mannose and DOPE is (10-45):(20-35):(0.4-1.5):(0.4-1.5):(10-40); the mass ratio of the nucleic acid molecule to the NMS-C9H19 is 1:(8-12); more preferably 1:10.

[0056] The present invention does not impose any particular restrictions on the preparation method of the lipid nanoparticle delivery system, and any common preparation method in the art can be used. The present invention preferably uses microfluidic technology to prepare the lipid nanoparticle delivery system. The present invention does not impose any particular restrictions on the specific steps of using microfluidic technology to prepare the lipid nanoparticle delivery system.

[0057] The present invention also provides a hydrogel-liposome combined drug delivery system, comprising the lipid nanoparticle delivery system described in the third aspect, carbohydrazide-modified gelatin, aldehyde-modified hyaluronic acid and exogenous recombinant IL-2.

[0058] After the aldehyde-modified hyaluronic acid and carbohydrazide-modified gelatin are mixed, a Schiff base reaction occurs and a gel is quickly formed. The gel has good bioadhesion and injectability, and can be used as a drug reservoir for local injection into the kidney, so that the loaded drug can be slowly released locally in the kidney tumor at a stable and controllable rate and appropriate concentration, achieving precise drug delivery and fully exerting the drug efficacy.

[0059] In the present invention, the mass ratio of the aldehyded hyaluronic acid and the carbohydrazide-modified gelatin is 1:(1-4); the mass ratio of the exogenous recombinant IL-2, the nucleic acid molecules in the lipid nanoparticle delivery system and the total mass of the aldehyded hyaluronic acid and the carbohydrazide-modified gelatin is (0.6-1.2) μg:(6-12) μg:(12-22) mg.

[0060] The present invention does not impose any special restrictions on the preparation methods of carbohydrazide-modified gelatin and aldehyde-modified hyaluronic acid, and they can be prepared by the preparation methods disclosed in the art.

[0061] The hydrogel-liposome combined drug delivery system provided by the present invention has good bioadhesion and injectability, and can be used as a drug reservoir for local kidney injection, so that the loaded drug is slowly released locally in the kidney tumor at a stable and controllable rate and appropriate concentration, achieving precise drug delivery, thereby fully exerting the drug efficacy and avoiding toxic side effects caused by systemic medication.

[0062] The present invention also provides the use of the chimeric switch receptor, the nucleic acid molecule, the lipid nanoparticle delivery system or the hydrogel-liposome combined drug delivery system in the preparation of drugs for treating renal cancer.

[0063] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0064] In the following examples, the preparation method of NMS-C9H19 is as follows:

[0065] (1) In a 250 mL 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° C. using a heating magnetic stirrer, and the mixture was stirred at a constant speed for 72 hours. The reaction was monitored by thin-layer chromatography (TLC) until the reaction was completed. 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 obtain the intermediate (2-(dinonylamino)ethyl)carbamic acid tert-butyl ester.

[0066] (2) The prepared tert-butyl (2-(dinonylamino)ethyl)carbamate (5 g, 12.1 mmol) was dissolved in 30 mL of 1,4-dioxane solution, and then 30 mL of HCl-1,4-dioxane (1 M) solution was added. The resulting solution was mixed with a magnetic stirrer at room temperature for 3 hours. The reaction of the mixture was monitored by TLC until the reaction was completed. The reactants were washed with saturated NaHCO3 and saturated NaCl solutions, respectively, and the organic layer was collected and anhydrous MgSO4 was added to dry the sample, which was left overnight. After filtering, the reactants were concentrated by rotary evaporation. The product can be directly used for subsequent reactions without purification.

[0067] (3) 80 mL of anhydrous DMF was added to a 250 mL round-bottom flask containing citric acid (0.8 g, 4.2 mmol), HOBt (2.0 g, 14.8 mmol) and DIC (3.2 mL, 20 mmol) and activated by stirring with a magnetic stirrer for 15 minutes, followed by the addition of N1, N1-dinonylethane-1,2-diamine (4.4 g, 14.1 mmol) obtained in step (2) and stirred at room temperature for 10 hours. The reactants were washed with saturated NaHCO3 and saturated NaCl solutions, respectively, the organic layer was collected and anhydrous MgSO4 was added to dry the sample, and the sample was placed overnight for treatment. After filtering, the reactants were concentrated by a rotary evaporator. The product was separated and purified by silica gel column chromatography using an eluent (CH2Cl2 / MeOH) to obtain NMS-C9H19, the structural formula of which is shown below:

[0068]

[0069] Example 1

[0070] This example provides the construction of IL-2R / TLR4 CSR circRNA.

[0071] The IL-2R / TLR4 CSR is, in sequence, the sequence of the extracellular domain of the IL-2 receptor (IL-2R), the sequence of the CD28 transmembrane domain and the sequence of the TLR4 intracellular domain. The amino acid sequence of the extracellular domain of the IL-2β receptor is shown in SEQ ID NO.1; the amino acid sequence of the transmembrane domain of the CD28 molecule is shown in SEQ ID NO.2; and the amino acid sequence of the intracellular domain of the TLR4 molecule is shown in SEQ ID NO.3.

[0072] The IL-2R / TLR4 CSR circRNA was synthesized and provided by Guangzhou Gisai Biotechnology Co., Ltd., which contains the nucleotide sequence encoding IL-2R / TLR4 CSR. Figure 1As shown in A, a verified IRES active element and a verified adapter T4 RNA ligase 2 (T4 Rnl-2) were added upstream and downstream of the linear RNA encoding anti-CA9CAR for in vitro transcription and circularization to prepare circular RNA (IL-2R / TLR4CSRcircRNA). The specific preparation process is as follows:

[0073] (1) In vitro cyclization: prepare the RNA cyclization reaction system according to Table 1:

[0074] Table 1 Cyclization reaction system

[0075] Components Dosage <![CDATA[DEPC-H2O]]> Up to 400μL RNA inhibitor (40U / μL) 20μL Linear RNA 100 μg 10×T4 Rnl2 Buffer 40μL T4 Rnl-2 (10U / μL) 60μL

[0076] After gently mixing by pipetting, the mixture was reacted at 25°C for 3 h and maintained at 4°C to obtain a cyclized product.

[0077] (2) Purification of cyclization product:

[0078] ① Add 200 μL of LiCI (8 M, RNase-free) to the above 400 μL cyclization product.

[0079] ② After mixing evenly, place at -20℃ for at least 30min, centrifuge at maximum speed and 4℃ for 15min, and collect the precipitate.

[0080] ③ Add 500 μL of ice-cold 70% ethanol to wash the RNA precipitate.

[0081] ④Dissolve the RNA precipitate with 20 μL RNase-free H2O.

[0082] ⑤ Use a micro-nucleic acid analyzer to detect RNA concentration.

[0083] ⑥The purified RNA solution was stored at -80℃.

[0084] (3) RNase R digestion to remove linear RNA:

[0085] The linear RNA in the cyclized product was digested with RNaseR (Geneseed, Cat. No: R0301). The reaction conditions are shown in Table 2. The digested product was recovered by lithium chloride precipitation.

[0086] Table 2 Reaction conditions for RNase R digestion to remove linear RNA

[0087] Components Dosage 10×Buffer 20μL Cyclization product 100 μg RNA inhibitor (40U / μL) 10μL RNase R (20U / μL) 20μL <![CDATA[DEPC-H2O]]> Up to 200μL

[0088] After gently pipetting and mixing, the mixture was reacted at 37° C. for 15 min and maintained at 4° C. After digestion, LiCI precipitation was used for purification to finally obtain the circular RNA (circRNA) used in this example.

[0089] To test the stability of the constructed circRNA against RNase degradation, the linear RNA (Linear RNA) and circular RNA (circRNA) forms of IL-2R / TLR4 CSR were incubated with RNase R for 30 min and then analyzed by gel electrophoresis. Figure 1 As shown in B, it can be seen that linear RNA is completely degraded after co-incubation with RNase, while circular RNA is hardly degraded, indicating that the circular RNA form of IL-2R / TLR4 CSR has good stability.

[0090] like Figure 1 As shown in C, flow cytometry was used to detect the phosphorylation levels of TLR4-related signaling pathway molecules in cells expressing IL-2R / TLR4 CSR under the condition of exogenous recombinant IL-2 stimulation. It can be seen that exogenous recombinant IL-2 can significantly activate the TLR4 downstream signaling pathway through IL-2R / TLR4 CSR, thus providing the basis for exerting downstream functions.

[0091] Example 2

[0092] This embodiment provides a method for preparing lipid nanoparticles encapsulating circRNA.

[0093] NMS-C9H19, cholesterol, DMG-PEG2000, DSPE-PEG-mannose and DOPE were co-dissolved in ethanol at a molar ratio of 15:25:0.5:0.5:20 as the organic phase.

[0094] The circRNA in Example 1 was dissolved in a citrate buffer at pH = 4 as the aqueous phase.

[0095] The aqueous phase and the organic phase were mixed in a microfluidic chip device at a volume ratio of 3:1, wherein the mass ratio of NMS-C9H19 to circRNA was 10:1, and then the ethanol was removed by ultrafiltration to obtain lipid nanoparticles (CSR-mLNP) encapsulating circRNA. The transmission electron microscopy image is shown in FIG. Figure 2 As shown, it can be seen that the average particle size is about 120 nm.

[0096] Example 3

[0097] This embodiment provides a method for preparing an injectable liposome composite hydrogel.

[0098] 3.00 g of gelatin and 2.20 g of carbohydrazide were stirred and dissolved in 300 mL of ultrapure deionized water at 55°C. Then, 0.50 g of EDC (1-[3-dimethylaminopropyl]-3-ethylcarbodiimide hydrochloride) and 0.50 g of HOBt (1-hydroxybenzotriazole) were added to the solution. Finally, the pH value of the above solution was adjusted to 5 using 0.1 M HCl solution, and the mixed solution was stirred overnight to obtain carbohydrazide-modified gelatin (Gel-CDH).

[0099] 2 g of hyaluronic acid (HA) was dissolved in 200 mL of pure water, and 10 mL of 0.5 M NaIO4 was added dropwise. The oxidation reaction was stirred for 2 hours under light-proof conditions, and then 4 mL of ethylene glycol was added to terminate the reaction to obtain aldehyde-modified hyaluronic acid (HA-CHO).

[0100] HA-CHO and Gel-CDH (mass ratio of 3:5) were mixed to obtain an injectable hydrogel.

[0101] 1 μg of exogenous recombinant IL-2, the lipid nanoparticles encapsulating 10 μg of circRNA of Example 2, and 20 mg of the above-mentioned injectable hydrogel were mixed to obtain an injectable liposome composite hydrogel (CSR-mLNP / IL-2@gel).

[0102] After the hydrogel is prepared into a gel, it is sucked into a syringe and can be molded into various shapes through the syringe needle, such as Figure 3 As shown, it can be seen that the hydrogel of the present invention has good injectable properties.

[0103] Test example

[0104] 1. Verification of the in vitro phagocytic effect of injectable liposome composite hydrogel (CSR-mLNP / IL-2@gel) on macrophages:

[0105] Mouse bone marrow-derived macrophages (BMDMs) cultured in vitro were treated with 25 ng / mL M-CSF (macrophage colony stimulating factor) for 7 days, and then the cells were further cultured in M2 macrophage conditioned medium containing 20 ng / mL IL-4 for 2 days to polarize them into M2 macrophages. After the induction culture was completed, the macrophages were treated with CSR-mLNPs and then cultured for another 24 hours. Subsequently, 20 ng / mL IL-2 was added to the cell culture medium and stimulated for 24 hours before the cell phagocytosis experiment was performed.

[0106] For the tumor cell phagocytosis test, the BMDMs stimulated by the above culture were co-cultured with antibody-coated magnetic beads, where G1: PBS treatment group; G2: IL-2 treatment group; G3: CSR-mLNP+IL-2 treatment group. After 6 hours of co-culture, BMDMs were stained with live cell dyes, and the macrophage phagocytosis function was imaged using a laser confocal imaging system. Figure 4 As shown, the confocal results showed that the number of BMDMs phagocytosed magnetic beads in the CSR-mLNPs / IL-2 treatment group was significantly higher than that in other control groups, indicating that the CSR-mLNPs in Example 2 of the present invention can induce enhanced phagocytic function of mouse macrophages in vitro.

[0107] 2. Investigation of the inhibitory effect of injectable liposome composite hydrogel (CSR-mLNP / IL-2@gel) on renal cancer:

[0108] An in situ renal cancer tumor-bearing mouse model was constructed using the mouse Renca cell line, and the renal cancer inhibitory effect of the injectable liposome composite hydrogel (CSR-mLNP / IL-2@gel) developed in Example 3 of the present invention was verified. The prepared groups of systems (G1: PBS control group; G2: hydrogel-loaded IL-2 treatment group; G3: hydrogel-loaded CSR-mLNP / IL-2 treatment group) were injected locally into the kidneys using an ultrasound-guided method, and the growth of the tumor was tracked and monitored using a small animal imaging system. When the experiment reached the end point, the tumor was removed, photographed and weighed. Figure 5 As shown in the figure, it can be seen that the hydrogel-loaded CSR-mLNP / IL-2 treatment group showed the most significant tumor inhibition effect (tumor fluorescence intensity, tumor size and weight were the lowest). This shows that the injectable liposome composite hydrogel developed in Example 3 of the present invention can significantly inhibit the growth of renal cancer.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A chimeric switch receptor for macrophages, characterized in that include: An extracellular region, the extracellular region comprising the extracellular domain of an IL-2α, IL-2β or IL-2γ receptor; A transmembrane region, wherein the transmembrane region includes a CD8 or CD28 molecule transmembrane domain; The intracellular region includes the intracellular domain of CD3ζ, TLR4, CD40 or Dectin1 molecule.

2. The chimeric switch receptor according to claim 1, wherein The extracellular region is selected from the extracellular domain of the IL-2β receptor, and its amino acid sequence is shown in SEQ ID NO.1; the transmembrane region is selected from the transmembrane domain of the CD28 molecule, and its amino acid sequence is shown in SEQ ID NO.2; the intracellular region is selected from the intracellular domain of the TLR4 molecule, and its amino acid sequence is shown in SEQ ID NO.

3.

3. A nucleic acid molecule, characterized in that It encodes the chimeric switch receptor according to any one of claims 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that The nucleic acid molecule is a circular RNA.

5. A lipid nanoparticle delivery system, characterized in that: The lipid nanoparticle delivery system carries the nucleic acid molecule of claim 3 or 4.

6. The lipid nanoparticle delivery system according to claim 5, characterized in that The lipid nanoparticle delivery system further comprises NMS-C9H19, cholesterol, DMG-PEG, DSPE-PEG-mannose and DOPE; wherein the structural formula of NMS-C9H19 is shown in formula (I):

7. The lipid nanoparticle delivery system according to claim 6, characterized in that The molar ratio of the NMS-C9H19, cholesterol, DMG-PEG, DSPE-PEG-mannose and DOPE is (10-45):(20-35):(0.4-1.5):(0.4-1.5):(10-40); the mass ratio of the nucleic acid molecule to the NMS-C9H19 is 1:(8-12).

8. A hydrogel-liposome combined drug delivery system, characterized in that: The invention comprises the liposome according to any one of claims 5 to 7, carbohydrazide-modified gelatin and aldehyde-modified hyaluronic acid.

9. The hydrogel-liposome combined drug delivery system according to claim 8, characterized in that: The mass ratio of the aldehyded hyaluronic acid and the carbohydrazide-modified gelatin is 1:(1-4); the mass ratio of the exogenous recombinant IL-2, the nucleic acid molecules in the lipid nanoparticle delivery system and the total mass of the aldehyded hyaluronic acid and the carbohydrazide-modified gelatin is (0.6-1.2) μg:(6-12) μg:(12-22) mg.

10. Use of the chimeric switch receptor according to claim 1 or 2, or the nucleic acid molecule according to claim 3 or 4, or the lipid nanoparticle delivery system according to any one of claims 5 to 7, or the hydrogel-liposome combined drug delivery system according to claim 8 or 9 in the preparation of drugs for treating renal cancer.

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