CA9-targeted chimeric antigen receptor and application thereof

Abstract

The invention discloses a CA9-targeted chimeric antigen receptor and application thereof, and belongs to the technical field of biological medicines. The CA9 targeting chimeric antigen receptor provided by the invention comprises a CD8 alpha leading signal peptide, an anti-CA9 single-chain variable fragment, a CD8 alpha hinge, a CD8 transmembrane structural domain and a CD3 zeta intracellular costimulatory signal transduction structural domain. According to the invention, an anti-CA9 single-chain variable fragment is used as an antigen binding structural domain to construct a chimeric antigen receptor (CAR) molecule, the macrophage expressing the anti-CA9CAR can realize specific targeting and phagocytosis killing of CA9 positive kidney cancer cells, and the constructed lipid nanoparticle delivery system can effectively transfer a gene expression vector, so that the gene expression vector can realize effective target site delivery, and the CA9 positive kidney cancer cells can be effectively targeted and killed. The constructed hydrogel-liposome combined drug delivery system has the performance 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 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] Malignant tumors have become one of the important social problems that seriously threaten people's health. They progress rapidly, have high mortality rates, and low five-year survival rates. 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 renal cancer, but 20-30% of renal cancer patients have already developed distant metastasis when diagnosed, and about 40% of patients will face the problem of 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 treatment of advanced renal cancer in clinical practice 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, new treatment strategies and drugs are urgently needed for the treatment of renal cancer.

[0004] Carbonic anhydrase 9 (CA9) is one of the isomers of the carbonic anhydrase family. It is a transmembrane glycoprotein composed of acidic amino acids. It catalyzes the conversion of carbon dioxide into bicarbonate and participates in the regulation of intracellular and extracellular pH. Through pH regulation, CA9 reduces the adhesion of cancer cells, promotes their migration and invasion, and thus enhances their malignant behavior. CA9 antigen is expressed in about 90% of renal cell carcinomas, but not in normal renal tissue. It is called renal cancer-associated antigen and can be used as a target for renal cancer treatment. However, there are few reports on renal cancer immunotherapy targeting CA9. Summary of the invention

[0005] In view of this, the present invention provides a chimeric antigen receptor targeting CA9 and its application, wherein the chimeric antigen receptor targeting CA9 can edit local tumor macrophages in vivo, so that they can play the function of specifically phagocytizing CA9+ renal cancer cells.

[0006] In a first aspect, 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.

[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]

[0014] 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).

[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 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.

[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 an antigen binding domain to construct a chimeric antigen receptor (CAR) molecule, and 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 transfer gene expression vectors to achieve 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, so that they can 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, the controlled sustained release of loaded drugs can be achieved 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, and has good clinical promotion potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[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 2 is 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 4This 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 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 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 contour map showing the proportion of F4 / 80+GFP+ double-positive cells, and B is a statistical histogram showing the proportion of macrophages that have engulfed CA9-GFP+ renal cancer cells; G1 is a PBS control group, G2 is an IL-2 treatment group, and G3 is a CAR-mLNP / IL-2 treatment 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 in vivo imaging of small animals, 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 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.

[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" in English, refers to a fusion protein comprising at least one variable region antibody fragment comprising a light chain and at least one variable region antibody fragment comprising a heavy chain, wherein the light chain and heavy chain variable regions are adjacent via a short flexible polypeptide linker and can be expressed in the form of a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, as used herein, 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 scFv may include VL-linker-VH or may include 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] In this article, the term "macrophage" is a type of white blood cell located in tissues, derived from monocytes, and mainly performs phagocytosis, digestion, and antigen presentation. In the tumor microenvironment, macrophages polarize to the tumor-promoting M2 phenotype, which promotes tumor growth, invasion, metastasis, promotes the formation of new blood vessels, and participates in the formation of an immunosuppressive microenvironment. The transformation of the M2 phenotype to the M1 phenotype is conducive to reshaping the tumor microenvironment, allowing macrophages to resume their phagocytosis, digestion, 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 co-stimulatory signal transduction domain is shown in SEQ ID NO.5:

[0044] RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGK QQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATK DTYDALHMQTLAPR.

[0045] The present invention provides a nucleic acid molecule, which includes the coding gene of the chimeric antigen receptor targeting CA9. 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 of the complementary double strands, or two. In addition, the molecular sequence in the present invention includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed; the RNA can be mRNA, siRNA, miRNA, saRNA, etc. In the present invention, the nucleic acid molecule is preferably a circular RNA, which has good stability. The present invention designs and constructs a circRNA encoding an anti-CA9 (anti-CA9) CAR, which can achieve stable expression of anti-CA9 CAR in macrophages, thereby inducing macrophage phenotypic remodeling and functional changes, 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 be used as a molecular target for targeting macrophages; wherein the structural formula of NMS-C9H19 is shown in formula (I):

[0050]

[0051] 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); 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 by the present invention can effectively transfer the gene expression vector, so that it can achieve effective target site delivery.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] The present invention also 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.

[0059] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0060] In the following examples, 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 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.

[0062] (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.

[0063] (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:

[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 human CA9 antigen, a CD8α hinge region, a CD8 transmembrane region, and a CD3ζ intracellular co-stimulatory signal transduction domain connected in series, wherein 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 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 signal transduction 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 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-CA9 CAR for in vitro transcription and circularization to prepare circular RNA (anti-CA9 CARcircRNA). 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] 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

[0072] 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.

[0073] (2) Purification of cyclization product:

[0074] ① Add 200 μL of LiCI (8 M, RNase-free) to the above 400 μL cyclization product.

[0075] ② After mixing evenly, place at -20℃ for at least 30min, centrifuge at maximum speed and 4℃ for 15min, and collect the precipitate.

[0076] ③ Add 500 μL of ice-cold 70% ethanol to wash the RNA precipitate.

[0077] ④Dissolve the RNA precipitate with 20 μL RNase-free H2O.

[0078] ⑤ Use a micro-nucleic acid analyzer to detect RNA concentration.

[0079] ⑥The purified RNA solution was stored at -80℃.

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

[0081] 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.

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

[0083] 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

[0084] 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.

[0085] To test the stability of the constructed circRNA against RNase degradation, 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 B, 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 embodiment provides a method for preparing lipid nanoparticles (CAR-mLNPs) encapsulating circRNA.

[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, and then the ethanol was removed by ultrafiltration to obtain lipid nanoparticles (CAR-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.

[0091] Example 3

[0092] This embodiment 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 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).

[0094] 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).

[0095] HA-CHO and Gel-CDH (mass ratio of 3:5) were mixed to obtain an 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] After the hydrogel is prepared into a gel, it is sucked into a syringe and 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 circRNA-loaded lipid nanoparticles (CAR-mLNP) of Example 2 on the phagocytic effect of 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, 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 CAR-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.

[0101] For the tumor cell phagocytosis magnetic bead 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: CAR-mLNPs+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 engulfing magnetic beads in the CAR-mLNPs / 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 antibody, and analyzed by flow cytometry. The PBS control group (G1) and the 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 mouse Renca cell line. The macroscopic renal tumor was 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 death of mice in each group was recorded, and the growth of the tumor was tracked and monitored using a small animal imaging system. Figure 6 As shown, it can be seen that the injectable liposome composite hydrogel developed in Example 3 of the present invention can significantly inhibit the recurrence and tumor growth of renal cancer after surgery and effectively prolong the survival of tumor-bearing mice.

[0105] 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 antigen receptor targeting CA9, characterized in that It 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 co-stimulatory 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 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 signal transduction domain is shown in SEQ ID NO.

5.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule includes the coding gene of the chimeric antigen receptor targeting CA9.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecule is a circular RNA.

4. A chimeric antigen receptor-macrophage, characterized in that The chimeric antigen receptor macrophage expresses the chimeric antigen receptor of claim 1.

5. A lipid nanoparticle delivery system, characterized in that: The lipid nanoparticle delivery system is loaded with the nucleic acid molecule described in the second aspect above.

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 5, 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 lipid nanoparticle delivery system according to any one of claims 5 to 7, carbohydrazide-modified gelatin, aldehyde-modified hyaluronic acid and exogenous recombinant IL-2.

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 antigen receptor targeting CA9 as claimed in claim 1, the nucleic acid molecule as claimed in claim 2 or 3, the lipid nanoparticle delivery system as claimed in any one of claims 5 to 7, or the hydrogel-liposome combined drug delivery system as claimed in claim 8 or 9 in the preparation of drugs for treating renal cancer.

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