Chimeric antigen receptors targeting ca9 and uses thereof

Abstract

The application discloses a chimeric antigen receptor targeting CA9 and application thereof, and belongs to the technical field of biological medicine. The chimeric antigen receptor targeting CA9 comprises a CD8 alpha leader signal peptide, an anti-CA9 single-chain variable fragment, a CD8 alpha hinge, a CD8 transmembrane domain and a CD3 zeta intracellular costimulatory signaling domain. The application adopts the anti-CA9 single-chain variable fragment as an antigen binding domain to construct a chimeric antigen receptor (CAR) molecule, and the macrophage expressing the anti-CA9 CAR can realize specific targeting and phagocytosis and killing of CA9 positive renal cancer cells, the constructed lipid nanoparticle delivery system can effectively carry a gene expression vector, so that the gene expression vector can realize effective target site delivery, and the constructed hydrogel-liposome combined drug delivery system has the performances of relieving toxic and side effects, controllable release and hemostasis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a chimeric antigen receptor targeting CA9 and applications thereof. Background Art

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

[0003] Malignant tumors have become a major social issue posing a serious threat to public health. They are characterized by rapid progression, high mortality, and low five-year survival rates. Kidney cancer, also known as renal cell carcinoma (RCC), is a common malignant tumor of the urinary system. Radical resection is an effective treatment for early-stage RCC, but 20-30% of patients have distant metastases at the time of diagnosis, and approximately 40% of patients experience recurrence after tumor resection. Patients with metastatic RCC are insensitive to conventional chemotherapy, while immunotherapy and molecularly targeted therapies are also ineffective, with low response rates. Current clinical treatments for advanced RCC primarily rely on anti-angiogenic agents, cytokines, monoclonal antibodies, and kinase inhibitors, which are associated with high costs, significant side effects, and the development of drug resistance. Therefore, the development of new therapeutic strategies and drugs for RCC is urgently needed.

[0004] Carbonic anhydrase 9 (CA9), a member of the carbonic anhydrase family of isoforms, is a transmembrane glycoprotein composed of acidic amino acids. It catalyzes the conversion of carbon dioxide to bicarbonate and participates in the regulation of intracellular and extracellular pH. By regulating pH, CA9 reduces cancer cell adhesion, promotes migration and invasion, and thus enhances malignant behavior. The CA9 antigen is expressed in approximately 90% of renal cell carcinomas, but not in normal renal tissue. It is known as a renal cancer-associated antigen and a potential target for renal cancer treatment. However, few reports exist on renal cancer immunotherapies targeting CA9. Summary of the Invention

[0005] In view of this, the present invention provides a chimeric antigen receptor targeting CA9 and its application. The chimeric antigen receptor targeting CA9 can edit local tumor macrophages in vivo, enabling them to specifically phagocytize CA9+ renal cancer cells.

[0006] In the first aspect, the present invention provides a chimeric antigen receptor targeting CA9, comprising a CD8α leader signal peptide, a single-chain antibody scFv that recognizes human CA9 antigen, a CD8α hinge region, a CD8 transmembrane region, and a CD3ζ intracellular co-stimulatory signaling domain.

[0007] Preferably, the amino acid sequence of the CD8α leader signal peptide is shown as SEQ ID NO. 1; the amino acid sequence of the single-chain antibody scFv that recognizes the human CA9 antigen is shown as SEQ ID NO. 2; the amino acid sequence of the CD8α hinge region is shown as SEQ ID NO. 3; the amino acid sequence of the CD8 transmembrane region is shown as SEQ ID NO. 4; and the amino acid sequence of the CD3ζ intracellular co-stimulatory signal transduction domain is shown as SEQ ID NO. 5.

[0008] In a second aspect, the present invention provides a nucleic acid molecule comprising a gene encoding the above-mentioned chimeric antigen receptor targeting CA9.

[0009] Preferably, the nucleic acid molecule is a circular RNA.

[0010] In a third aspect, the present invention provides a chimeric antigen receptor-macrophage, wherein the chimeric antigen receptor macrophage expresses the chimeric antigen receptor as described in the first aspect.

[0011] In a fourth 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.

[0012] 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):

[0013] Formula (I).

[0014] Preferably, the molar ratio of 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).

[0015] In a fifth aspect, the present invention provides a hydrogel-liposome combined drug delivery system, comprising the lipid nanoparticle delivery system described in the third aspect above, carbohydrazide-modified gelatin, aldehyde-modified hyaluronic acid and exogenous recombinant IL-2.

[0016] Preferably, the mass ratio of the aldehyde-modified 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 aldehyde-modified hyaluronic acid and the carbohydrazide-modified gelatin is (0.6~1.2) μg:(6~12) μg:(12~22) mg.

[0017] In the sixth aspect, the present invention provides the use of the chimeric antigen receptor targeting CA9 described in the first aspect, the nucleic acid molecule described in the second aspect, the lipid nanoparticle delivery system described in the fourth aspect, or the hydrogel-liposome combined drug delivery system described in the fifth aspect in the preparation of drugs for treating renal cancer.

[0018] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0019] (1) The present invention uses an anti-CA9 single-chain variable fragment as the antigen-binding domain to construct a chimeric antigen receptor (CAR) molecule. Macrophages expressing the anti-CA9 CAR can achieve specific targeting and phagocytic killing of CA9-positive renal cancer cells;

[0020] (2) The present invention constructs a lipid nanoparticle delivery system that can effectively carry gene expression vectors, enabling effective target site delivery.

[0021] (3) The hydrogel-liposome combined drug delivery system provided by the present invention can achieve CAR editing of local macrophages in postoperative tumor wounds, enabling them to specifically target CA9-positive tumor cells, reshape the immunosuppressive tumor microenvironment, and specifically kill tumor cells. In addition, the hydrogel-liposome combined drug delivery system avoids the systemic side effects caused by systemic administration; at the same time, it can achieve controlled sustained release of loaded drugs by controlling the degradation rate of the hydrogel skeleton; in addition, it also exhibits good hemostatic properties and can effectively prevent bleeding by forming a gel locally in the wound after renal cancer surgery, which has good clinical promotion potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings, which constitute part of the present invention, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute undue limitations thereon. It is obvious that one of ordinary skill in the art could derive other drawings based on these drawings without inventive effort.

[0023] Figure 1 Schematic diagram of the circRNA structure of Example 1 of the present invention (A) and gel electrophoresis analysis of the stability of circRNA to RNase digestion (B);

[0024] Figure 2is a transmission electron microscopy image of CAR-mLNP of Example 2 of the present invention;

[0025] Figure 3 This is a picture showing the hydrogel injection effect of Example 3 of the present invention;

[0026] Figure 4 This is a verification of the effect of CAR-mLNP of Example 2 of the present invention on the phagocytic function of macrophages in vitro, wherein A is the laser confocal microscope results of G1, G2 and G3 groups, and B is the statistical histogram of the number of magnetic beads phagocytosed by each macrophage; G1 is the PBS control group, G2 is the IL-2 treatment group, and G3 is the CAR-mLNP / IL-2 treatment group;

[0027] Figure 5 This is a verification of the effect of CAR-mLNP of Example 2 of the present invention on the phagocytic function of macrophages in vitro, wherein A is a flow cytometry contour map showing the proportion of F4 / 80+GFP+ double-positive cells, and B is a statistical histogram showing the proportion of macrophages that phagocytized CA9-GFP+ renal cancer cells; G1 is a PBS control group, G2 is an IL-2-treated group, and G3 is a CAR-mLNP / IL-2-treated group;

[0028] Figure 6 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 graph of mouse tumor fluorescence intensity, and C is a statistical graph of mouse survival; G1 is a PBS control group, G2 is an IL-2@gel treatment group, and G3 is a CAR-mLNP / IL-2@gel treatment group. DETAILED DESCRIPTION

[0029] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0030] In this article, the term "chimeric antigen receptor", also known as "Chimeric Antigen Receptor" in English, abbreviated as CAR, is an artificial fusion protein. It refers to the fusion of two or more different antigen-specific receptors or their fragments through genetic engineering technology to form a new receptor that can simultaneously or sequentially recognize and bind to multiple tumor-associated antigens and trigger an immune response.

[0031] As used herein, the term "scFv," or "single chain antibody fragment," refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are contiguous via a short, flexible polypeptide linker and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, an scFv may have the VL and VH variable regions in either order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and may comprise VL-linker-VH or VH-linker-VL.

[0032] As used herein, the term "exogenous" refers to any substance that is introduced from or produced outside an organism, cell, tissue, or system.

[0033] As used herein, the term "macrophage" refers to a tissue-resident white blood cell derived from monocytes that primarily functions in phagocytosis, digestion, and antigen presentation. Within the tumor microenvironment, macrophages polarize to the pro-tumor M2 phenotype, promoting tumor growth, invasion, and metastasis, as well as neovascularization and participating in the formation of an immunosuppressive microenvironment. The transition from the M2 phenotype to the M1 phenotype facilitates the remodeling of the tumor microenvironment, enabling macrophages to resume their phagocytic, digestive, and antigen presentation functions.

[0034] The present invention provides a chimeric antigen receptor targeting CA9, comprising a CD8α leader signal peptide, a single-chain antibody scFv recognizing human CA9 antigen, a CD8α hinge region, a CD8 transmembrane region and a CD3ζ intracellular co-stimulatory signal transduction domain.

[0035] Preferably, the amino acid sequence of the CD8α leader signal peptide is shown in SEQ ID NO. 1:

[0036] MASPLTRFLSLNLLLLGESIILGSGEA;

[0037] The amino acid sequence of the single-chain antibody scFv that recognizes human CA9 antigen is shown in SEQ ID NO. 2:

[0038] QVQLKESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSKIYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRMAYYGYFDVWGAGTTVTVSSGGGG SGGGGSGGGGSDIVMTQAPSSLAMSVGQKVTMSCKSSQSLLNSRNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQYYSTPLTFGAGTKLELK;

[0039] The amino acid sequence of the CD8α hinge region is shown in SEQ ID NO. 3:

[0040] LQKVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIY;

[0041] The amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO. 4:

[0042] IWAPLAGICVALLLSLIITLI;

[0043] The amino acid sequence of the CD3ζ intracellular costimulatory signaling domain is shown in SEQ ID NO. 5:

[0044] RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR.

[0045] The present invention provides a nucleic acid molecule comprising a gene encoding the aforementioned chimeric antigen receptor targeting CA9. It should be noted that, when nucleic acid molecules are referred to herein, those skilled in the art will understand that they encompass either or both complementary double strands. Furthermore, the molecular sequences herein include DNA or RNA forms, and disclosure of one implies disclosure of the other. The RNA may be mRNA, siRNA, miRNA, saRNA, etc. In the present invention, the nucleic acid molecule is preferably a circular RNA, which exhibits excellent stability. The present invention has designed and constructed a circRNA encoding an anti-CA9 CAR, which can achieve stable expression of the anti-CA9 CAR in macrophages, thereby inducing phenotypic remodeling and functional changes in macrophages, and achieving specific targeting and phagocytic killing of CA9-positive renal cancer cells.

[0046] The present invention also provides a chimeric antigen receptor-macrophage, wherein the chimeric antigen receptor macrophage expresses the chimeric antigen receptor.

[0047] The present invention also provides a lipid nanoparticle delivery system, wherein the lipid nanoparticle delivery system carries the nucleic acid molecule described in the second aspect.

[0048] In the present invention, the lipid membrane preferably includes NMS-C9H19, cholesterol, DMG-PEG (phospholipid-polyethylene glycol), DSPE-PEG-mannose (phospholipid-polyethylene glycol-mannose) and DOPE (dioleoylphosphatidylethanolamine).

[0049] DMG-PEG is preferably DMG-PEG2000, i.e. dimyristoylglycerol-polyethylene glycol 2000. The lipid nanoparticle delivery system is externally connected to a mannose molecule (Mannose), which can serve as a molecular target for targeting macrophages; wherein, the structural formula of NMS-C9H19 is shown in formula (I):

[0050] Formula (I).

[0051] In the present invention, the molar ratio of 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); more preferably 15:25:0.5:0.5:20; the mass ratio of the nucleic acid molecule to the NMS-C9H19 is 1:(8~12), more preferably 1:10.

[0052] The lipid nanoparticle delivery system constructed in the application can effectively carry a gene expression vector, so that the gene expression vector is effectively delivered to a target site.

[0053] The application does not make special restrictions on the preparation method of the lipid nanoparticle delivery system, and a preparation method commonly used in the art can be used.

[0054] The application also provides a hydrogel-liposome combined drug delivery system, comprising the lipid nanoparticle delivery system of the third aspect, carbonyl hydrazine modified gelatin, aldehyde-based hyaluronic acid and exogenous recombinant IL-2.

[0055] The aldehyde-based hyaluronic acid and the carbonyl hydrazine modified gelatin are mixed to rapidly form a gel through a Schiff base reaction, have good biological adhesion and injectability, and can be used as a drug reservoir for local injection of the kidney, so that the loaded drug is slowly released at a stable and controllable rate and a suitable concentration in the local kidney tumor, precise drug delivery is achieved, and the drug efficacy is fully exerted.

[0056] In the application, the mass ratio of the aldehyde-based hyaluronic acid and the carbonyl hydrazine modified gelatin is 1:(1-4); the mass ratio of the exogenous recombinant IL-2, the nucleic acid molecule in the lipid nanoparticle delivery system and the total mass of the aldehyde-based hyaluronic acid and the carbonyl hydrazine modified gelatin is (0.6-1.2) μg:(6-12) μg:(12-22) mg.

[0057] The application does not make special restrictions on the preparation method of the carbonyl hydrazine modified gelatin and the aldehyde-based hyaluronic acid, and a preparation method disclosed in the art can be used.

[0058] The application also provides application of the targeting CA9 chimeric antigen receptor of the first aspect, the nucleic acid molecule of the second aspect, the lipid nanoparticle delivery system of the fourth aspect or the hydrogel-liposome combined drug delivery system of the fifth aspect in preparation of a drug for treating kidney cancer.

[0059] The technical solutions of the application will be further described below with reference to specific embodiments.

[0060] In the following embodiments, the preparation method of NMS-C9H19 is as follows:

[0061] (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 heated magnetic stirrer and stirred at a constant speed for 72 hours. The reaction mixture was monitored by thin-layer chromatography (TLC) until the reaction was complete. The reaction solution was collected and concentrated under reduced pressure using a rotary evaporator. The product was separated and purified by silica gel column chromatography using an eluent (PE / EA) to obtain the intermediate tert-butyl (2-(dinonylamino)ethyl)carbamate.

[0062] (2) Dissolve the prepared tert-butyl (2-(dinonylamino)ethyl)carbamate (5 g, 12.1 mmol) in 30 mL of 1,4-dioxane solution, and then add 30 mL of HCl-1,4-dioxane (1 M) solution. Mix the resulting solution with a magnetic stirrer at room temperature for 3 hours. Monitor the reaction of the mixture by TLC until the reaction is complete. Wash the reactants with saturated NaHCO3 and saturated NaCl solutions, respectively. Collect the organic layer and add anhydrous MgSO4 to dry the sample, and leave it overnight. After filtration, concentrate the reactants by rotary evaporation. The product can be used directly in subsequent reactions without purification.

[0063] (3) Add 80 mL of anhydrous DMF 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 stir with a magnetic stirrer for 15 minutes to activate. Then add N1,N1-dinonylethane-1,2-diamine (4.4 g, 14.1 mmol) prepared in step (2) and stir 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 left overnight. After filtration, the reactants were concentrated by rotary evaporation. 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:

[0064] .

[0065] Example 1

[0066] This example provides the construction of anti-CA9 CAR circRNA.

[0067] The anti-CA9 CAR is composed of a CD8α leader signal peptide, a single-chain antibody scFv that recognizes the human CA9 antigen, a CD8α hinge region, a CD8 transmembrane region, and a CD3ζ intracellular co-stimulatory signaling domain, connected in series. The amino acid sequence of the CD8α leader signal peptide is shown in SEQ ID NO. 1; the amino acid sequence of the single-chain antibody scFv that recognizes the human CA9 antigen is shown in SEQ ID NO. 2; the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO. 3; the amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO. 4; and the amino acid sequence of the CD3ζ intracellular co-stimulatory signaling domain is shown in SEQ ID NO. 5.

[0068] The anti-CA9 CAR circRNA was synthesized and provided by Guangzhou Gisai Biotechnology Co., Ltd., and contains the nucleotide sequence encoding the anti-CA9 CAR. Figure 1 As shown in Figure A, a validated IRES activity element and a validated adapter, T4 RNA ligase 2 (T4 Rnl-2), were added upstream and downstream of the linear RNA encoding anti-CA9 CAR for in vitro transcription and circularization to prepare circular RNA (anti-CA9 CAR circRNA). The specific preparation process is as follows:

[0069] (1) In vitro cyclization: Prepare the RNA cyclization reaction system according to Table 1:

[0070] Table 1 Cyclization reaction system

[0071]

[0072] After gently pipetting to mix, the mixture was reacted at 25°C for 3 h and maintained at 4°C to obtain a cyclized product.

[0073] (2) Purification of cyclization product:

[0074] To the above 400 μL of cyclized product, 200 μL of LiCI (8 M, RNase-free) was added.

[0075] After mixing evenly, place at -20℃ for at least 30min, centrifuge at maximum speed and 4℃ for 15min, and collect the precipitate.

[0076] Add 500 μL of ice-cold 70% ethanol to wash the RNA pellet.

[0077] Dissolve the RNA pellet in 20 μL RNase-free H2O.

[0078] The RNA concentration was measured using a micro-nucleic acid analyzer.

[0079] The purified RNA solution was stored at -80°C.

[0080] (3) RNase R digestion to remove linear RNA:

[0081] The linear RNA in the circularized product was digested with RNase R (Geneseed, Cat. No: R0301). The reaction conditions are shown in Table 2. The digested product was recovered by lithium chloride precipitation.

[0082] Table 2 Reaction conditions for RNase R digestion to remove linear RNA

[0083]

[0084] After gently pipetting to mix, the mixture was reacted at 37°C for 15 min and maintained at 4°C. After digestion, the mixture was purified using LiCI precipitation to obtain the circular RNA (circRNA) used in this example.

[0085] To test the stability of the constructed circRNA against RNase degradation, linear RNA (Linear RNA) and circular RNA (circRNA) forms of CA9-CAR were incubated with RNase R for 30 min and then analyzed by gel electrophoresis. Figure 1 As shown in Figure 3B, linear RNA was completely degraded after incubation with RNase, while circular RNA was hardly degraded, indicating that CA9-CAR in the form of circular RNA has good stability.

[0086] Example 2

[0087] This example provides a method for preparing circRNA-loaded lipid nanoparticles (CAR-mLNPs).

[0088] 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.

[0089] The circRNA in Example 1 was dissolved in a citrate buffer at pH 4 as the aqueous phase.

[0090] 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. Subsequently, ethanol was removed by ultrafiltration to obtain lipid nanoparticles (CAR-mLNPs) encapsulating circRNA. The transmission electron microscopy image is shown below. Figure 2 As shown, it can be seen that the average particle size is about 120 nm.

[0091] Example 3

[0092] This example provides a method for preparing an injectable liposome composite hydrogel (CAR-mLNP / IL-2@gel).

[0093] 3.00 g of gelatin and 2.20 g of carbohydrazide were dissolved in 300 mL of ultrapure deionized water at 55°C with stirring. 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 of the solution was adjusted to 5 using 0.1 M HCl solution, and the mixture was stirred overnight to obtain carbohydrazide-modified gelatin (Gel-CDH).

[0094] Dissolve 2g of hyaluronic acid (HA) in 200mL of pure water and add 10mL of 0.5M NaIO4 dropwise. Stir and oxidize for 2 hours in the dark. Then, add 4mL of ethylene glycol to terminate the reaction, yielding aldehyde-modified hyaluronic acid (HA-CHO).

[0095] HA-CHO and Gel-CDH were mixed (mass ratio of 3:5) to obtain injectable hydrogel.

[0096] 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 (CAR-mLNP / IL-2@gel).

[0097] The hydrogel is prepared into a gel and sucked into a syringe. It can be molded into various shapes through a 26-gauge syringe needle, such as Figure 3 As shown, it can be seen that the hydrogel of the present invention has good injectable properties.

[0098] Test example

[0099] 1. Verification of the phagocytic effect of circRNA-loaded lipid nanoparticles (CAR-mLNPs) on macrophages in vitro:

[0100] Mouse bone marrow-derived macrophages (BMDMs) cultured in vitro were treated with 25 ng / mL M-CSF (macrophage colony-stimulating factor) for 7 days. The cells were then cultured in M2 macrophage-conditioned medium containing 20 ng / mL IL-4 for a further 2 days to polarize them into M2 macrophages. After induction, the macrophages were treated with CAR-mLNPs and cultured for an additional 24 hours. Subsequently, 20 ng / mL IL-2 was added to the cell culture medium and stimulated for 24 hours before phagocytosis experiments were performed.

[0101] For the tumor cell phagocytosis magnetic bead test, 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: CAR-mLNP / IL-2 treatment group. After 6 hours of co-culture, BMDMs were stained with a live cell dye, and macrophage phagocytosis was imaged using a laser confocal imaging system. Figure 4 As shown in the confocal microscopy, the number of BMDMs phagocytosed magnetic beads in the CAR-mLNP / IL-2 treatment group was significantly higher than that in other control groups, indicating that the nanomedicine of the present invention can induce enhanced phagocytic function of mouse macrophages in vitro.

[0102] For the tumor cell phagocytosis assay, BMDMs stimulated by the above culture were co-cultured with Renca cells expressing GFP and specifically expressing human CA9. After 6 hours of co-culture, BMDMs were collected, stained with Anti-F4 / 80 antibodies, and analyzed by flow cytometry. PBS control group (G1) and IL-2 treatment group (G2) were used as control groups. Figure 5 As shown, the flow cytometry results showed that the proportion of double-positive cells in the CAR-mLNP / IL-2 treatment group (G3) was significantly higher than that in other control groups, indicating that the CAR-mLNP / IL-2 of Example 2 of the present invention can induce enhanced phagocytic function of mouse macrophages in vitro.

[0103] 2. Investigation of the inhibitory effect of the injectable liposome composite hydrogel (CAR-mLNP / IL-2@gel) of Example 3 on renal cancer:

[0104] In this example, an orthotopic renal cancer tumor-bearing mouse model was constructed using the mouse Renca cell line. Visible renal tumors were surgically removed on the 7th day after tumor inoculation, and the injectable liposome composite hydrogel (CAR-mLNP / IL-2@gel, G3) developed in Example 3 of the present invention was locally injected into the incision. A PBS control group (G1) and a hydrogel-loaded IL-2 treatment group (IL-2@gel, G2) were used as comparisons. The mortality of mice in each group was recorded, and the growth of the tumor was tracked and monitored using a small animal imaging system. Figure 6As shown, it can be seen that the injectable liposome complex hydrogel developed in the embodiment 3 of the present application can significantly inhibit the recurrence and tumor growth after the kidney cancer surgery, and effectively prolong the survival period of the tumor-bearing mice.

[0105] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. The present application can be variously changed and modified by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A lipid nanoparticle delivery system, characterized in that The lipid nanoparticle delivery system is loaded with nucleic acid molecules; The nucleic acid molecule includes a gene encoding a chimeric antigen receptor targeting CA9; The chimeric antigen receptor targeting CA9 includes a CD8α leader signal peptide, a single-chain antibody scFv that recognizes human CA9 antigen, a CD8α hinge region, a CD8 transmembrane region, and a CD3ζ intracellular costimulatory signal transduction domain; The amino acid sequence of the CD8α leader signal peptide is shown in SEQ ID NO. 1; the amino acid sequence of the single-chain antibody scFv that recognizes the human CA9 antigen is shown in SEQ ID NO. 2; the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO. 3; the amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO. 4; and the amino acid sequence of the CD3ζ intracellular costimulatory signaling domain is shown in SEQ ID NO.

5. 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): Formula (I).

2. The lipid nanoparticle delivery system according to claim 1, wherein 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).

3. The lipid nanoparticle delivery system according to claim 1, wherein The nucleic acid molecule is a circular RNA.

4. A hydrogel-liposome combined drug delivery system, characterized in that: The invention comprises the lipid nanoparticle delivery system according to any one of claims 1 to 3, carbohydrazide-modified gelatin, aldehyde-modified hyaluronic acid and exogenous recombinant IL-2.

5. The hydrogel-liposome combined drug delivery system according to claim 4, wherein: The mass ratio of the aldehyde-modified 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 aldehyde-modified hyaluronic acid and the carbohydrazide-modified gelatin is (0.6-1.2) μg:(6-12) μg:(12-22) mg.

6. Use of the lipid nanoparticle delivery system according to any one of claims 1 to 3 or the hydrogel-liposome combined drug delivery system according to claim 4 or 5 in the preparation of a drug for treating renal cancer.

Images