Lipid nanoparticles and uses thereof

Abstract

The application relates to a kind of lipid nanoparticles and its application.The lipid nanoparticles include: cationic lipid, anionic lipid DOPS (1,2-dioleoyl-sn-glycerol-3-phosphatidyl-L-serine), other lipids and targeting antibody.The application finds that doping anionic lipid DOPS and indirectly coupling targeting antibody can significantly improve the transfection efficiency of nanoparticles on spleen T cells.The application further loads nucleic acid encoding CAR protein targeting tumor vascular endothelial cells and tumor cells in the above lipid nanoparticles and is used for tumor treatment, and the results show that the above treatment significantly promotes intratumoral infiltration of immune cells, significantly inhibits the progression of various solid tumors, and provides a new idea and new means for the effective application of CAR-T therapy in solid tumors.

Description

Technical Field

[0001] This invention pertains to CAR-T cell therapy, specifically a novel combined treatment technique that utilizes lipid nanoparticles to efficiently edit spleen CAR-T cells in vivo, targeting solid tumor blood vessels and tumor cells. Background Technology

[0002] CAR-T cell therapy has become an important area of ​​anti-tumor immunotherapy. Currently, CAR-T therapy has achieved promising results in the treatment of hematologic malignancies and autoimmune diseases, including leukemia, lymphoma, multiple myeloma, and lupus erythematosus, and six CAR-T therapies have received FDA approval for B-cell-derived hematologic malignancies. However, CAR-T cell therapy has consistently failed to achieve significant progress in solid tumors. Tumor heterogeneity and the difficulty of T cells infiltrating the tumor stroma are the main reasons for its poor efficacy; therefore, there are currently no effective CAR-T therapies for solid tumors on the market. Tumor angiogenesis is crucial for maintaining tumor growth and metastasis. Targeting and killing tumor angiogenesis can block the tumor's acquisition of nutrients and the excretion of metabolites, inhibiting tumor progression, while simultaneously promoting immune cell infiltration. Furthermore, compared to tumor cells, tumor vascular endothelial cells have more stable antigen expression and are less prone to immune mutations. Therefore, combining CAR-T therapies that target both tumor angiogenesis and tumor cells can increase the intratumoral infiltration of CAR-T cells targeting tumor cells by disrupting vascular structure / barriers, thus providing new ideas and methods for improving the effective application of CAR-T therapy in solid tumors.

[0003] Currently, CAR-T cell therapy is a personalized treatment involving the collection, gene modification, and expansion of the patient's T cells, the removal of lymphocytes from the patient's body, and the infusion of CAR-T cells back into the patient. This personalized and multi-step nature makes it expensive, time-consuming, complex, and prone to side effects, hindering large-scale adoption. Therefore, developing in vivo in situ CAR-T editing technology holds promise for overcoming the shortcomings of traditional CAR-T therapy. While maintaining high anti-tumor efficacy, it could reduce production costs, shorten preparation time, and eliminate the lymphocyte removal steps required in traditional CAR-T therapy, thereby improving the safety of CAR-T treatment. Currently, several different delivery systems are used for in vivo CAR gene delivery. Compared to viral vectors with limited drug loading capacity and high immunogenicity and cytotoxicity, lipid nanoparticles offer advantages such as ease of preparation, high stability, efficient cellular uptake, and endosomal escape, making them one of the most clinically valuable non-viral gene delivery vectors. In particular, they have been successfully used to deliver mRNA COVID-19 vaccines and the CRISPR-Cas9 system, achieving effective gene editing, ideal therapeutic effects, and safety in clinical trials.

[0004] However, the accumulation of lipid nanoparticles in the liver severely limits the targeting and therapy of mRNA-lipid nanoparticle technology outside the liver; simply modifying the surface of clinically approved four-component lipid nanoparticles with targeting antibodies for in vivo editing to generate CAR-T cells generally results in low transfection efficiency, leading to limited efficacy. Therefore, it is essential to develop spleen-specific targeting lipid nanoparticles and improve antibody conjugation methods to enhance the efficiency of T cell editing in vivo. Summary of the Invention

[0005] This invention provides a lipid nanoparticle and its application to address the shortcomings of insufficient targeting and low transfection efficiency of in vivo CAR-T editing in the prior art, achieving spleen-targeted delivery and efficient transfection of mRNA, and further providing a targeted therapeutic drug for the treatment of solid tumors.

[0006] In a first aspect, the present invention provides a lipid nanoparticle comprising: cationic lipids, anionic lipids, other lipids, a linker, and an antibody targeting T cells;

[0007] The anionic lipid is DOPS; the cationic lipid is MC3.

[0008] This invention, through research and experimental comparison, found that doping DOPS (1,2-dioleoyl-sn-glycerol-3-phosphatidyl-L-serine) into MC3-LNP can improve the transfection efficiency of T cells in vitro and in vivo. Doping DOPS into lipid nanoparticles enables the lipid nanoparticles to achieve spleen-targeting properties, allowing for spleen-specific expression of mRNA. Furthermore, by indirectly coupling antibodies targeting T cell surface antigens to the surface of the lipid nanoparticles, the transfection efficiency in splenic T cells can be further improved.

[0009] Preferably, the other lipids in the above-mentioned lipid nanoparticles include one or more of cholesterol, phospholipids, PEG lipids, and reactive PEG lipids;

[0010] The anionic lipids account for 10% to 20% of the total lipid concentration, preferably 10%.

[0011] Preferably, the molar ratio of the cationic lipid MC3 to the other lipids in the above lipid nanoparticles is 1~2:1~2, more preferably 1:1;

[0012] The ratio of cationic lipids, cholesterol, phospholipids, PEG lipids and reactive PEG lipids in the lipid nanoparticles is 45~55:36~40:9~12:1.3~1.7:0.4~0.6, preferably 50:38:10:1.5:0.5.

[0013] Preferably, the Linker in the above lipid nanoparticles is an anti-rat IgG2α antibody, and the molar ratio of the Linker to the reactive PEG lipid is 1:4~6.5;

[0014] The targeting antibody is a CD3 or CD5 antibody, such as an anti-mouse CD5 antibody, and the mass ratio of the targeting antibody to the linker is 1:1.

[0015] The reactive PEG lipid in this invention can undergo a coupling reaction with the linker and further connect to the targeting antibody. This connection method can avoid the binding sites on the targeting antibody being occupied, which would reduce the targeting effect.

[0016] The above-mentioned ratio of linker, reactive PEG lipid, and target antibody can achieve the optimal antibody linking effect, ensuring the amount of target antibody linked while preserving its antibody binding site.

[0017] Doping DOPS into the quaternary component MC3-LNP enables spleen-targeting properties of lipid nanoparticles, allowing for spleen-specific mRNA expression. Furthermore, indirectly conjugating antibodies to the surface of the lipid nanoparticles significantly improves their transfection efficiency in T cells in vivo. Intravenous injection of lipid nanoparticles carrying CAR genes targeting tumor vascular endothelial cells (VEGFR2) and tumor cells generates CAR-T cells capable of specifically killing tumor vascular endothelial cells or tumor cells, thereby significantly inhibiting tumor progression.

[0018] Secondly, the present invention provides the application of the above-mentioned lipid nanoparticles in the preparation of drug delivery carriers or in the preparation of drugs, wherein the drugs are gene therapy drugs.

[0019] Thirdly, the present invention provides a drug delivery carrier containing the aforementioned lipid nanoparticles.

[0020] Fourthly, the present invention provides a targeted drug for in vivo CAR-T editing, the targeted drug comprising:

[0021] The lipid nanoparticles described above; or the drug delivery carriers described above;

[0022] Nucleic acid for encoding CAR protein, the nucleic acid being loaded into lipid nanoparticles.

[0023] Preferably, the CAR protein in the above-mentioned targeted drug includes proteins that target vascular endothelial cells and those that target tumors;

[0024] The proteins that target tumor vascular endothelial cells include VEGFR2, TAM1, CLEC14A, etc., with VEGFR2 being preferred.

[0025] The tumor-targeting proteins include one or more of TRP1, CLDN18.2, HER2, GPC3, and MSLN;

[0026] The ratio of VEGFR2 to the tumor-targeting protein is 3 to 5:1, preferably 4:1.

[0027] By intravenously injecting lipid nanoparticles carrying the CAR gene targeting tumor vascular endothelial cells VEGFR2 and tumor cells, CAR-T cells capable of specifically killing tumor vascular endothelial cells or tumor cells can be generated in vivo, thereby significantly inhibiting tumor progression.

[0028] Preferably, the amino acid sequence of the CAR molecule encoding the VEGFR2 target in the above-mentioned targeted drug is shown in SEQ ID NO.1, the amino acid sequence of the CAR molecule encoding the TRP1 target is shown in SEQ ID NO.2, and the amino acid sequence of the CAR molecule encoding the CLDN18.2 target is shown in SEQ ID NO.3.

[0029] Preferably, the nucleic acid used to encode the target CAR protein in the above-mentioned targeted drug is mRNA or a CRISPR-Cas system, preferably mRNA;

[0030] The mass ratio of the lipid nanoparticles to the mRNA is 4:1.

[0031] Fifthly, the present invention provides the use of the above-mentioned lipid nanoparticles and / or the above-mentioned drug delivery carriers and / or the above-mentioned targeted drugs in the preparation of drugs for treating tumors, preferably in the preparation of drugs for treating solid tumors.

[0032] This invention reveals that by doping DOPS anionic lipids into MC3-lipid nanoparticles and indirectly coupling them with an anti-mouse CD5 antibody via an anti-rat IgGα antibody as a linker, the lipid nanoparticles can be enriched in the spleen and efficiently transfected into T cells in vivo, generating CAR-T cells with specific cytotoxic functions. Furthermore, a combined CAR-T therapy strategy targeting tumor vascular endothelial cells and tumor cells can effectively inhibit tumor angiogenesis, promote cytotoxic T cell infiltration into the tumor, and thus enhance anti-tumor efficacy.

[0033] The nanocarrier provided by this invention can efficiently transfect T cells in vivo to generate CAR-T cells that can specifically kill target cells; the treatment strategy provided by this invention can effectively inhibit tumor growth and improve survival rate, and has important value in the field of CAR-T cell therapy for solid tumors. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0035] Figure 1 This is a schematic diagram of the in vivo edited CAR-T tumor-targeting drug preparation process provided by the present invention.

[0036] Figure 2 These are the test results of the targeting of the nanoparticles prepared in Examples 1, 1, and 2 of this invention to various organs of mice.

[0037] Figure 3 This is a cryo-electron microscopy image of the in vivo edited CAR-T tumor-targeting drug prepared in Example 2 of the present invention. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0039] Example 1

[0040] This invention provides nanoparticles for targeted delivery of mRNA to the spleen in vivo, which are prepared by the following method:

[0041] (1) First, prepare an organic phase containing lipid components: mix MC3, cholesterol, DSPC, DMG-PEG2000 and DOPS into the organic phase in a molar ratio of 45:34.2:9:1.8:10, and the total molar concentration of all components is 2.57mM; among them, DOPS needs to be dissolved in tetrahydrofuran before being mixed with other lipids.

[0042] (2) Luciferase mRNA was then dissolved in sodium citrate buffer (10 mM, pH=4) at a concentration of 0.025 mg / mL. The aqueous and organic phases were passed through a microfluidic chip at a rate ratio of 3:1 to obtain lipid nanoparticles loaded with Luciferase mRNA. The nanoparticles were then centrifuged at 4000 g for 40 min and washed twice with PBS to remove excess anhydrous ethanol, yielding lipid nanoparticles loaded with specific mRNA.

[0043] Example 2

[0044] This embodiment further provides nanoparticles transfected with T cells in vivo and CAR-T tumor-targeting drugs edited in vivo.

[0045] The amino acid sequence of the CAR molecule targeting VEGFR2 is shown in SEQ ID NO.1, and the amino acid sequence of the CAR molecule targeting TRP1 is shown in SEQ ID NO.2. The mRNA was obtained by in vitro transcription as follows:

[0046] The target sequence was cloned into the pVAX1 vector; subsequently, the supercoiled plasmid was linearized using BspQⅠ, and the linearized plasmid was used as a template for in vitro transcription using the T7 high-yield RNA co-transcription kit to synthesize the target mRNA sequence containing a poly A tail of approximately 109 adenosine residues; then, nucleoside modification was performed using N1-methylpseudouracil triphosphate (N1-Me-pUTP), and capping was performed using the cap analogue GAG; finally, the mRNA was purified using lithium chloride precipitation.

[0047] Its flowchart is as follows Figure 1 As shown, it is prepared by the following method:

[0048] (1) First, prepare an organic phase containing lipid components: Mix MC3, cholesterol, DSPC, DMG-PEG2000, DSPE-PEG-Mal and DOPS into the organic phase in a molar ratio of 45:34.2:9:1.35:0.45:10. The total molar concentration of all components is 10.3 mM. Among them, DOPS needs to be dissolved in tetrahydrofuran before being mixed with other lipids.

[0049] (2) Subsequently, the mRNA encoding the target sequence was dissolved in sodium citrate buffer (10 mM, pH=4) at a concentration of 0.1 mg / mL. The aqueous phase and organic phase were then passed through a microfluidic chip at a rate ratio of 3:1 to obtain lipid nanoparticles encapsulating the target sequence, such as... Figure 1 As shown in the figure. Subsequently, the mixture was centrifuged at 4000g for 40 min and washed twice with PBS to remove excess anhydrous ethanol, yielding lipid nanoparticles encapsulating specific mRNA.

[0050] (3) The anti-rat IgGα antibody and Traut's Reagent were mixed at a molar ratio of 1:20 and reacted at room temperature for 1 hour. Unreacted Traut's Reagent was removed by a Zeba desalting column. The activated anti-rat IgGα antibody was then mixed with lipid nanoparticles and incubated at 4°C for 2 hours. Anti-mouse CD5 antibody was then added and incubated at 4°C for 0.5 hours. The nanoparticles were then concentrated by passing the mixture through a 4 ml 10 KD ultrafiltration tube.

[0051] (4) The lipid nanoparticles loaded with tumor vascular endothelial cell mRNA (VEGFR2 CAR mRNA) and lipid nanoparticles loaded with tumor cell mRNA (TRP1 CAR mRNA) were mixed at a mass ratio of 4:1 to obtain in vivo edited CAR-T tumor-targeting drugs.

[0052] Example 3

[0053] The amino acid sequence of the CAR molecule targeting VEGFR2 is shown in SEQ ID NO.1, and the amino acid sequence of the CAR molecule targeting CLDN18.2 is shown in SEQ ID NO.3. The mRNA was obtained via in vitro transcription as follows:

[0054] The target sequence was cloned into the pVAX1 vector; subsequently, the supercoiled plasmid was linearized using BspQⅠ, and the linearized plasmid was used as a template for in vitro transcription using the T7 high-yield RNA co-transcription kit to synthesize the target mRNA sequence containing a poly A tail of approximately 109 adenosine residues; then, nucleoside modification was performed using N1-methylpseudouracil triphosphate (N1-Me-pUTP), and capping was performed using the cap analogue GAG; finally, the mRNA was purified using lithium chloride precipitation.

[0055] This embodiment further provides nanoparticles transfected with T cells in vivo and CAR-T tumor-targeting drugs edited in vivo, the flowchart of which is shown below. Figure 1 As shown, it is prepared by the following method:

[0056] (1) First, prepare an organic phase containing lipid components: Mix MC3, cholesterol, DSPC, DMG-PEG2000, DSPE-PEG-Mal and DOPS into the organic phase in a molar ratio of 45:34.2:9:1.35:0.45:10. The total molar concentration of all components is 10.3 mM. Among them, DOPS needs to be dissolved in tetrahydrofuran before being mixed with other lipids.

[0057] (2) Subsequently, the mRNA encoding the target sequence was dissolved in sodium citrate buffer (10 mM, pH=4) at a concentration of 0.1 mg / mL. The aqueous phase and organic phase were then passed through a microfluidic chip at a rate ratio of 3:1 to obtain lipid nanoparticles encapsulating the target sequence, such as... Figure 1 As shown in the figure. Subsequently, the mixture was centrifuged at 4000g for 40 min and washed twice with PBS to remove excess anhydrous ethanol, yielding lipid nanoparticles encapsulating specific mRNA.

[0058] (3) The anti-rat IgGα antibody and Traut's Reagent were mixed at a molar ratio of 1:20 and reacted at room temperature for 1 hour. Unreacted Traut's Reagent was removed by a Zeba desalting column. The activated anti-rat IgGα antibody was then mixed with lipid nanoparticles and incubated at 4°C for 2 hours. Anti-mouse CD5 antibody was then added and incubated at 4°C for 0.5 hours. The nanoparticles were then concentrated by passing the mixture through a 4 ml 10 KD ultrafiltration tube.

[0059] (4) The lipid nanoparticles loaded with tumor vascular endothelial cell mRNA (VEGFR2 CAR mRNA) and lipid nanoparticles loaded with tumor cell mRNA (CLDN18.2 CAR mRNA) were mixed at a mass ratio of 4:1 to obtain in vivo edited CAR-T tumor-targeting drugs.

[0060] The difference between Example 3 and Example 2 is that the CAR protein combination used is VEGFR2 and CLDN18.2.

[0061] Comparative Example 1

[0062] This invention provides nanoparticles for in vivo delivery of mRNA. The nanoparticles are the same as those in Example 1, except that the molar ratio of MC3, cholesterol, DSPC, DMG-PEG2000 and DOPS is 50:38:10:2:0.

[0063] Comparative Example 2

[0064] This invention provides nanoparticles for targeted delivery of mRNA to the spleen in vivo. The nanoparticles are the same as those in Example 1, except that the molar ratio of MC3, cholesterol, DSPC, DMG-PEG2000 and DOPS is 40:30.4:8:1.6:20.

[0065] Comparative Example 3

[0066] This invention provides nanoparticles for targeted delivery of mRNA to the spleen in vivo. The nanoparticles are the same as those in Example 1, except that DOPS is replaced with DOPG.

[0067] Comparative Example 4

[0068] This invention provides nanoparticles for targeted delivery of mRNA to the spleen in vivo. The nanoparticles are the same as those in Example 1, except that DOPS is replaced with DOPA.

[0069] Comparative Example 5

[0070] The present invention provides a nanoparticle for targeted delivery of CAR genes in a comparative example. The nanoparticle is the same as that in Example 2, except that the targeting antibody is not modified on the surface of the nanoparticle.

[0071] Comparative Example 6

[0072] The present invention provides a nanoparticle for targeted delivery of CAR genes in a comparative example. The nanoparticle is the same as that in Example 2, except that the linker is not modified, and the target antibody anti-mouse CD5 antibody is directly modified on the surface of the nanoparticle.

[0073] Comparative Example 7

[0074] This invention provides a nanoparticle for targeted delivery of the CAR gene, which is the same as that in Comparative Example 5, except that the molar ratio of MC3, cholesterol, DSPC, DMG-PEG2000, DSPE-PEG-Mal, and DOPS is 50:38:10:2:0.

[0075] Comparative Example 8

[0076] This invention provides nanoparticles for targeted delivery of CAR genes in vivo. The nanoparticles are the same as those in Example 2, except that they contain only lipid nanoparticles that encapsulate tumor vascular endothelial cell mRNA (VEGFR2 CAR mRNA).

[0077] Comparative Example 9

[0078] This invention provides nanoparticles for targeted delivery of CAR genes in vivo. The nanoparticles are the same as those in Example 2, except that they contain only lipid nanoparticles that encapsulate tumor cell mRNA (TRP1 CAR mRNA or CLDN18.2 CAR mRNA).

[0079] Experimental Example 1

[0080] This experiment tested the spleen targeting ability of the in vivo targeted delivery mRNA nanoparticles prepared in Example 1. The test results are as follows: Figure 2 As shown. Specifically, it includes the following steps:

[0081] (1) The nanoparticles prepared in Example 1 and Comparative Examples 1-2 were injected into the tail vein, and each mouse was injected with LNP containing 8 μg mRNA.

[0082] (2) Six hours later, each mouse was injected intraperitoneally with D-luciferin (150 mg / kg). Fifteen minutes later, the mice were dissected and the main organs (heart, liver, spleen, lung and kidney) were removed. The luciferase activity was measured by a small animal optical 3D in vivo imaging system.

[0083] The fluorescence distribution of the liver and spleen was statistically analyzed, and the results are shown in Table 1. The quantitative fluorescence analysis of the spleen was performed, and the results are shown in Table 2.

[0084]

[0085] from Figure 2 As shown in Table 1, LNPs doped with DOPS facilitated the transfer of mRNA expression from the liver to the spleen, achieving spleen-specific expression. Table 2 shows that when DOPS was doped at a molar ratio of 10% (Example 1), the fluorescence intensity in the spleen was significantly increased, indicating improved spleen delivery efficiency.

[0086] Experiment Example 2

[0087] This experiment compared the in vitro transfection efficiency of different anionic lipids (DOPS, DOPG, and DOPA) on T cells, and the test results are shown in Table 3. The specific steps included:

[0088] (1) Jurkat cells were prepared at a ratio of 1×10 5 A concentration of 100 μl / ml was spread in 96-well plates, 100 μl per well, and incubated overnight.

[0089] (2) Add Example 1, Comparative Example 3 and Comparative Example 4 to 96-well plates (200 ng per well) and incubate with Jurkat cells for 24 hours.

[0090] (3) The expression of luciferase in cells was detected using the Bright-Glo™ luciferase reporter gene detection system.

[0091]

[0092] As shown in Table 3, compared with other anionic lipids DOPG and DOPA, liposomes with added DOPS have a stronger transfection capacity for T cells.

[0093] Experimental Example 3

[0094] This experiment tested the morphology, particle size, particle size distribution index (PDI), and mRNA encapsulation efficiency of the in vivo edited CAR-T tumor-targeting drug prepared in Example 2. Morphology was determined by cryo-electron microscopy, particle size and particle size distribution index were measured by dynamic light scattering, and mRNA encapsulation efficiency was determined using the RiboGreen kit. The test results are shown in Table 4. The cryo-electron microscopy morphology is shown in... Figure 3 As shown.

[0095]

[0096] From Table 4 and Figure 3 As can be seen, the in vivo edited CAR-T tumor-targeting drugs obtained by the preparation method of this invention have uniform particle size, complete spherical structure, excellent dispersibility, and high mRNA loading efficiency.

[0097] Experiment Example 4

[0098] This invention determined the effect of Example 1 on spleen CD3 in vivo. + CD4 + CD8 + The transfection efficiency of T cells was measured by flow cytometry, and the test results are shown in Table 5. The specific steps included:

[0099] (1) Take 15 mice and divide them into 5 groups of 3 mice each;

[0100] (2) PBS, nanoparticles prepared in Example 2 and Comparative Examples 5-7 were injected via tail vein (20 μg mRNA / animal).

[0101] (3) After 24 hours, the mice were sacrificed, and the spleens were ground and split open to prepare cell suspension. A certain amount of cell suspension was taken, centrifuged, and then resuspended with flow cytometry antibody (1:100 dilution) and incubated in the dark for 20 minutes.

[0102] (4) After washing twice with PBS, resuspend in 500 μl PBS and perform detection.

[0103]

[0104] As shown in Table 5, the transfection efficiency of DOPS-LNP (Comparative Example 6) on spleen T cells was higher than that of MC3-LNP (Comparative Example 7), indicating that the doping of DOPS can improve the transfection efficiency of T cells. At the same time, the transfection efficiency of Example 2 was further improved compared with Comparative Examples 5-6, which shows that the in vivo edited CAR-T tumor-targeting drug obtained by the preparation method of the present invention has the highest transfection efficiency for T cells.

[0105] Experimental Example 5

[0106] This invention evaluates the efficacy of the in vivo edited CAR-T tumor-targeting drug obtained in Example 1 using B16F10 melanoma mice, specifically including the following steps:

[0107] (1) Take 24 mice bearing B16F10 melanoma and divide them into 4 groups of 6 mice each;

[0108] (2) The nanoparticles prepared in Example 2 and Comparative Examples 8-9 were injected via tail vein. The injection method was as follows:

[0109] The control group was injected with 150 μL of physiological saline, while the other three groups were injected with 150 μL of the in vivo edited CAR-T tumor-targeting drugs described in Examples 1 and Comparative Examples 1-2. Specifically, Example 2 contained 25 μg of mRNA (VEGFR2 CAR mRNA 20 μg; TRP1 CAR mRNA 5 μg), Comparative Example 8 contained 20 μg of mRNA (VEGFR2 CAR mRNA), and Comparative Example 9 contained 5 μg of mRNA (TRP1 CAR mRNA). Tumor volume was measured in mice at 5, 7, 9, 11, 13, 15, and 17 days post-inoculation, and the average volume was recorded. Tumor volume was calculated by multiplying the length by the square of the width and dividing by 2. A tumor volume greater than 2000 mm² was considered normal. 3 Mice were subsequently judged dead; changes in body weight and survival time were observed, and the number of days with a survival rate of 50% and 0% was recorded; tumor volume (mm) was also recorded. 3 The test results are shown in Table 6, and the survival rate (d) test results are shown in Table 7.

[0110]

[0111] As shown in Table 6, compared with the blank group and the single-target tumor blood vessel or tumor cell group (Comparative Examples 8-9), the in vivo edited CAR-T tumor-targeting drug provided in Example 2 of this invention can significantly improve the efficacy and effectively inhibit tumor growth in mice.

[0112]

[0113] As shown in Table 7, compared with the blank group and comparative examples 8-9, the in vivo edited CAR-T tumor-targeting drug provided in Example 2 of this invention can significantly increase the survival time of mice.

[0114] Experimental Example 6

[0115] This invention uses CT26-mCLDN18.2 colon cancer mice to evaluate the efficacy of the in vivo edited CAR-T tumor-targeting drug obtained in Example 3. The procedure is the same as in Experiment 5. From day 9 to day 25 after tumor inoculation, the tumor volume of the mice was measured every two days and the average volume was recorded. The tumor volume (mm3) test results are shown in Table 8, and the survival rate (d) test results are shown in Table 9.

[0116]

[0117]

[0118] As shown in Tables 8 and 9, the in vivo edited CAR-T tumor-targeting drugs and treatment regimens provided by this invention can significantly inhibit tumor growth and improve mouse survival in both tumor models, demonstrating the effectiveness and universality of this invention.

[0119] Experimental Example 7

[0120] This invention uses CT26-mCLDN18.2 colon cancer mice to evaluate the ability of the in vivo edited CAR-T tumor-targeting drug prepared in Example 3 to promote immune cell infiltration into tumors, specifically including the following steps:

[0121] (1) Twenty-four mice bearing CT26-mCLDN18.2 colon cancer were divided into four groups of six mice each.

[0122] (2) The nanoparticles prepared in Example 3 and Comparative Examples 8-9 were injected via tail vein. The injection method was as follows:

[0123] On days 9, 13, and 17 post-tumor inoculation, 150 μL of the in vivo edited CAR-T tumor-targeting drugs obtained in Examples 3, 8, and 9 were injected via the tail vein, while the control group received an equal volume of saline. On day 18, tumors from the sacrificed mice were harvested for further analysis. CD45, CD3, and CD8 markers were used to statistically analyze tumor-infiltrating T cells.

[0124] Specifically, equal masses of tumor tissue were taken, ground, and tumor cells were extracted. After antibody staining, flow cytometry was performed, and the results are shown in Table 10.

[0125]

[0126] As shown in Table 10, compared with the blank group and the group that only injected lipid nanoparticles encoding mRNA targeting tumor cells (Comparative Example 9), the addition of lipid nanoparticles encoding mRNA targeting tumor vascular endothelial cells (Example 3 and Comparative Example 8) can significantly increase the number of cytotoxic T cells infiltrating the tumor. This indicates that targeting tumor blood vessels can effectively promote the infiltration of immune cells, and the combination of targeting tumor blood vessels and tumor cells can synergistically kill tumor cells and enhance the anti-tumor efficacy.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A lipid nanoparticle, characterized in that, It consists of cationic lipids, anionic lipids, other lipids, linkers, and antibodies that target T cells; The anionic lipid is DOPS; the cationic lipid is MC3; The other lipids consist of the following components: cholesterol, phospholipids, and PEG lipids; The phospholipid is DSPC, and the PEG lipid is DMG-PEG2000; The anionic lipids account for 10% of the total lipid concentration. The molar ratio of the cationic lipid MC3 to the other lipids is 1~2:1~2; The linker is an anti-rat IgG2α antibody; The targeting antibody is a CD5 antibody.

2. The lipid nanoparticles according to claim 1, characterized in that, The molar ratio of the cationic lipid MC3 to the other lipids is 1:1; The other lipids also include reactive PEG lipids; The ratio of cationic lipids, cholesterol, phospholipids, PEG lipids and reactive PEG lipids in the lipid nanoparticles is 45~55:36~40:9~12:1.3~1.7:0.4~0.

6.

3. The lipid nanoparticles according to claim 2, characterized in that, The ratio of cationic lipids, cholesterol, phospholipids, PEG lipids and reactive PEG lipids in the lipid nanoparticles is 50:38:10:1.5:0.

5.

4. The lipid nanoparticles according to claim 2, characterized in that, The molar ratio of the linker to the reactive PEG lipid is 1:4~6.5; The mass ratio of the targeting antibody to the linker is 1:

1.

5. The use of the lipid nanoparticles according to any one of claims 1-4 in the preparation of a drug delivery carrier or in the preparation of a drug, wherein the drug is a gene therapy drug.

6. A drug delivery carrier, characterized in that, The drug delivery carrier contains lipid nanoparticles as described in any one of claims 1-4.

7. A targeted drug for in vivo CAR-T editing, characterized in that, The targeted drug includes: The lipid nanoparticles according to any one of claims 1-4; or the drug delivery carrier according to claim 6; Nucleic acid for encoding CAR protein, the nucleic acid being loaded into lipid nanoparticles.

8. The targeted drug according to claim 7, characterized in that, The CAR protein includes proteins that target tumor vascular endothelial cells and tumor-targeting proteins. The proteins that target tumor vascular endothelial cells include VEGFR2, TAM1, and CLEC14A; The tumor-targeting proteins include one or more of TRP1, CLDN18.2, HER2, GPC3, and MSLN; The ratio of VEGFR2 to the tumor-targeting protein is 3~5:1; The amino acid sequence encoding the CAR molecule targeting VEGFR2 is shown in SEQ ID NO.1, the amino acid sequence encoding the CAR molecule targeting TRP1 is shown in SEQ ID NO.2, and the amino acid sequence encoding the CAR molecule targeting CLDN18.2 is shown in SEQ ID NO.

3.

9. The targeted drug according to claim 8, characterized in that, The protein that targets tumor vascular endothelial cells is VEGFR2, and the ratio of VEGFR2 to the tumor-targeting protein is 4:

1.

10. The targeted drug according to any one of claims 7-9, characterized in that, The nucleic acid used to encode the CAR protein is mRNA or a CRISPR-Cas system.

11. The targeted drug according to any one of claims 7-9, characterized in that, The nucleic acid used to encode the CAR protein is mRNA; The mass ratio of the lipid nanoparticles to the mRNA is 4:

1.

12. The use of the lipid nanoparticles according to any one of claims 1-4 in the preparation of a drug for treating tumors, wherein the lipid nanoparticles encapsulate VEGFR2 CAR mRNA and TRP1 CAR mRNA, and the tumor is melanoma; or The lipid nanoparticles encapsulate VEGFR2 CAR mRNA and CLDN18.2 CAR mRNA, and the tumor is colon cancer.

13. The use of the drug delivery carrier of claim 6 in the preparation of a drug for treating tumors, wherein the drug delivery carrier encapsulates VEGFR2 CAR mRNA and TRP1 CAR mRNA, and the tumor is melanoma; or The drug delivery vector contains VEGFR2 CAR mRNA and CLDN18.2 CAR mRNA, and the tumor is colon cancer.

14. The use of the targeted drug according to any one of claims 7-10 in the preparation of a tumor treatment drug, wherein the targeted drug encapsulates VEGFR2 CAR mRNA and TRP1 CAR mRNA, and the tumor is melanoma; or The targeted drug contains VEGFR2 CAR mRNA and CLDN18.2 CAR mRNA, and the tumor is colon cancer.

Images