Targeting Trop2 chimeric antigen receptor macrophage as well as preparation method and application thereof

Abstract

The invention discloses a Trop2 targeting chimeric antigen receptor macrophage as well as a preparation method and application thereof. The macrophage contains a specific CAR (chimeric antigen receptor) targeting Trop2 and a blocking agent for blocking an immunosuppression signal; the amino acid sequence of the Trop2 specific CAR targeting the breast cancer is as shown in SEQ ID NO. 1; the nucleotide sequence of the mRNA of the specific CAR of the targeted Trop2 is as shown in SEQ ID NO. 2. According to the invention, LNP is adopted to co-deliver CAR mRNA and TLR agonists, and macrophages are edited in vivo to generate CAR-macrophages expressing Trop2 in situ, so that a tedious engineering cell in-vitro preparation process is omitted, and potential whole-body toxic and side effects caused by immune cell system reinfusion are avoided.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a chimeric antigen receptor macrophage targeting Trop2 and a preparation method and application thereof. Background Art

[0002] Chimeric antigen receptor macrophage (CAR-M) therapy is a new treatment method that uses gene editing technology to modify the patient's macrophages and give them the ability to specifically clear tumor cells. Compared with chimeric antigen receptor T cell (CAR-T) therapy, which has been put into clinical treatment of malignant hematological tumors, CAR-macrophages have their unique advantages in the treatment of solid tumors, including good penetration and infiltration ability, the ability to present antigens to activate cellular immunity, etc., and have broader development prospects. However, it is still difficult for CAR-M therapy alone to overcome the immunosuppressive tumor microenvironment caused by tumor-associated macrophages (TAMs), and it is necessary to combine other drugs to jointly improve the anti-tumor ability of M1 macrophages. Although the first CAR-M therapy targeting HER2 showed that some M2 macrophages can be converted to M1 after macrophages are transduced with adenovirus vectors, the function of macrophages is often regulated by multiple signals at the same time, and it is difficult to overcome immunosuppression by the expression of CAR itself. Therefore, how to regulate the plasticity of macrophages in vivo, reprogram TAMs into M1 macrophages, and reverse the immunosuppressive tumor microenvironment is the key to improving the efficacy of CAR-M.

[0003] Breast cancer tissue highly expresses Trop2 (human trophoblast surface glycoprotein antigen 2), while normal tissue hardly expresses it. Therefore, Trop2 can be used as a specific marker for breast cancer and a target antigen for CAR-macrophages to recognize breast cancer cells. The construction of CAR-macrophages targeting Trop2 will achieve accurate recognition and phagocytosis of breast cancer cells and activate anti-tumor immune responses through antigen presentation. Many studies have shown that the application of TLR agonists in tumor immunotherapy is expected to turn "cold tumors" (non-immunogenic tumors) into "hot" (immunogenic), solve the problem of low response rate of single immune checkpoint inhibitors, and improve the effect of immunotherapy. This can not only help macrophages to repolarize, but also avoid problems caused by the immunosuppressive microenvironment.

[0004] Compared with plasmid DNA, mRNA can complete the translation of the encoded protein by binding to the ribosome in the cytoplasm, without entering the nucleus and inserting into the genome, and has higher translation efficiency and safety. Therefore, the present invention uses LNP (lipid nanoparticles) to co-deliver CAR mRNA and TLR agonists, and edits macrophages in vivo to generate CAR-macrophages expressing Trop2 in situ, omitting the cumbersome in vitro preparation process of engineered cells and avoiding potential systemic toxic side effects caused by the reinfusion of immune cell systems. At the same time, the TLR agonist released by LNP can reprogram TAMs in solid tumors to M1 type, stimulate NK and T cells, and change the tumor immunosuppressive microenvironment, thereby achieving the purpose of synergistic treatment of solid tumors. Summary of the invention

[0005] The present invention provides a chimeric antigen receptor (CAR) macrophage targeting Trop2 and a preparation method and application thereof. The present invention delivers mRNA and TNP agonist to transfect macrophages to form CAR-macrophages, thereby improving the anti-tumor effect of CAR-Trop2.

[0006] In order to solve the above technical problems, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a chimeric antigen receptor macrophage targeting Trop2, wherein the macrophage contains a specific CAR targeting Trop2 and a blocker for blocking immunosuppressive signals; the amino acid sequence of the Trop2-specific CAR targeting breast cancer is shown in SEQ ID NO.1.

[0007] As a further description of the above scheme: the nucleotide sequence of the mRNA of the specific CAR targeting Trop2 is shown in SEQ ID NO.2.

[0008] Preferably, the blocker for blocking immunosuppressive signals is a cholesterol-TLR agonist conjugate.

[0009] In a second aspect, the present invention further provides a method for preparing a chimeric antigen receptor macrophage targeting Trop2, the method comprising the steps of preparing lipid nanoparticles and transfecting macrophages with lipid nanoparticles; The preparation steps of the lipid nanoparticles are as follows: Step (1) dissolving the mRNA of CAR targeting Trop2 in sodium citrate buffer to obtain an mRNA mixed solution; Step (2) ionizable lipid SM-102, cholesterol-TLR agonist conjugate, DMG-PEG 2000 , DSPC and DSPE-PEG-MAN were dissolved in ethanol to obtain a mixed lipid solution; Step (3) adding the mRNA mixture and the mixed lipid solution to the microfluidic device, and preparing lipid nanoparticles co-loaded with mRNA encoding a specific CAR targeting Trop2 and a cholesterol-TLR agonist conjugate by a microfluidic method.

[0010] Preferably, the synthesis method of the cholesterol-TLR agonist conjugate is as follows: The TLR agonist R848 is dissolved in an organic solvent, and then 0.8-1.2 molar times of cholesterol hemisuccinate, 0.8-1.2 molar times of 4-dimethylaminopyridine and 0.8-1.2 molar times of 1-ethyl-(3-dimethylaminopropyl)carbodiimide are added respectively; the reaction substrate is stirred at room temperature for 20-30 hours. After the reaction is completed, the crude product is purified by column chromatography to obtain a cholesterol-TLR agonist conjugate; The organic solvent is ethanol, dichloromethane, chloroform or methanol.

[0011] Preferably, the ionizable lipid SM-102, cholesterol-TLR agonist conjugate, DMG-PEG 2000 The molar ratio of DSPC and DSPE-PEG-MAN is 30-60:30-50:1-5:10-20:1-10; the concentration of the cholesterol-TLR agonist conjugate is 0.5-1 mg / mL.

[0012] Preferably, the concentration of mRNA in the mRNA mixture in step (1) is 3 to 10 μg / mL; The mass ratio of the mRNA in the mRNA mixed solution to the ionizable lipid SM-102 in the mixed lipid solution is 1:8-20.

[0013] As a further description of the above scheme: the lipid nanoparticles prepared in step (3) are transfected into macrophages in vivo or in vitro to obtain chimeric antigen receptor macrophages targeting Trop2.

[0014] The third aspect of the present invention also provides the use of the chimeric antigen receptor macrophage targeting Trop2 or the lipid nanoparticle in the preparation of an anti-tumor drug. Preferably, the tumor is breast cancer.

[0015] Compared with the prior art, the present invention has the following beneficial effects: the present invention adopts LNP to co-deliver CAR mRNA and TLR agonist, edits macrophages in vivo to generate CAR-macrophages expressing Trop2 in situ, omits the cumbersome in vitro preparation process of engineering cells, and avoids the potential systemic toxic side effects caused by the reinfusion of immune cell system.

[0016] The nano drug delivery system provided by the present invention has unique advantages. First, the present invention adopts the addition of DSPE-PEG-MAN (distearoylphosphatidylethanolamine polyethylene glycol mannose) in the LNP formulation to achieve recognition and active targeting of mannose receptors on macrophages. Secondly, the addition of ionizable lipids can promote the lysosomal escape of gene drugs. In addition, the small molecule drug R848 is conjugated with cholesterol and added to the LNP system, which can not only regulate the hydrophobicity of R848, which is conducive to its encapsulation by LNP, but also avoid the systemic toxicity caused by intravenous injection of R848 alone. These designs provide significant advantages for the combined drug to exert synergistic effects and overcome the immunosuppressive microenvironment through multiple mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the structure of chimeric antigen receptor (CAR); Figure 2 H for TLR agonist (R848)-cholesterol conjugate 1 -NMR spectrum; A is H of R848 1 -NMR spectrum; B is the H of TLR agonist (R848)-cholesterol conjugate 1 -NMR spectrum; Figure 3 are the particle size, potential, PDI and encapsulation efficiency of M-LNP / CAR mRNA+TLRa; A is the particle size of M-LNP / CAR mRNA+TLRa; B is the PDI of M-LNP / CAR mRNA+TLRa; C is the Zeta potential of M-LNP / CAR mRNA+TLRa; D is the encapsulation efficiency of M-LNP / CAR mRNA+TLRa; Figure 4 A is an electron micrograph of M-LNP / CAR mRNA and M-LNP / CAR mRNA+TLRa; A is an electron micrograph of M-LNP / CAR mRNA; B is an electron micrograph of M-LNP / CAR mRNA+TLRa; Figure 5 is the transduction efficiency of LNP / CAR mRNA +TLRa on macrophages and T cells in vivo; A is the number of macrophages in the peripheral blood of mice and the proportion of expressing CAR; B is the number of T cells in the peripheral blood of mice and the proportion of expressing CAR; Figure 6 is the transduction efficiency of M-LNP / CAR mRNA +TLRa on macrophages and T cells in vivo; A is the number of macrophages in the peripheral blood of mice and the proportion of expressing CAR; B is the number of T cells in the peripheral blood of mice and the proportion of expressing CAR; Figure 7Cytotoxicity of M-LNP / CAR mRNA (A) and M-LNP / CAR mRNA+TLRa (B) on BMDMs; Figure 8 is the transfection efficiency of M-LNP / CAR mRNA+TLRa in human macrophages (THP-1 induced); Fig. 9 The phagocytic ability of THP-1 (A) and BMDM (B) cells to tumor cells after transfection with M-LNP / CAR mRNA+TLRa; Fig.10 The killing ability of different LNPs on tumor cells; Fig.11 is the biodistribution of M-LNP / CAR mRNA+TLRa in vivo; Fig.12 is the total number of CAR-M cells in vivo after injection of M-LNP / CAR mRNA+TLRa; Fig.13 The therapeutic effect of M-LNP / CAR mRNA+TLRa in vivo; A is the mouse tumor volume curve after administration; B is the mouse tumor picture on the 17th day after administration; C is the tumor growth curve of tumor-bearing mice in 6 administration groups; Fig.14 are cytokines produced by M-LNP / CAR mRNA+TLRa in vivo; A is the IFN-γ content in plasma on the 5th day after tail vein injection of LNP / GFP, LNP / CAR mRNA, M-LNP / CAR mRNA, LNP / CAR mRNA+TLRa and M-LNP / CAR mRNA+TLRa; B is the IL-10 content in mouse plasma on the 5th day after administration; C is the TNF-α content in mouse plasma on the 5th day after administration; Fig.15 is the in vivo safety of M-LNP / CAR mRNA+TLRa; A is the concentration of alanine aminotransferase (ALT) in the serum of each group of mice after administration; B is the concentration of aspartate aminotransferase (AST) in the serum of each group of mice after administration; C is the concentration of urea nitrogen (BUN) in the serum of each group of mice after administration; D is the concentration of creatinine (CREA) in the serum of each group of mice after administration; E is the concentration of serum uric acid (UA) in the serum of each group of mice after administration. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solution. In the following embodiments, unless otherwise specified, the reagents used are purchased from a reagent company.

[0019] Example 1 Construction of Trop2-specific CAR mRNA targeting breast cancer The CAR extracellular domain was designed using the triple-negative breast cancer specific target protein Tumor-associated calcium signal transducer 2 (Trop2) as the specific antigen.

[0020] like Figure 1 As shown, the chimeric antigen receptor comprises a CD8 front-end signal peptide, a specific anti-breast cancer cell Trop2 monoclonal antibody G15 (Anti-Trop2-scFv), a myc tag gene, a CD8α hinge region, a CD8 transmembrane region, and a CD3ζ intracellular domain.

[0021] The specific construction process is as follows: the amino acid sequence of each element is found through NCBI, the sequence is optimized, and then the sequence is concatenated in the order of CD8 front-end signal peptide, Anti-Trop2-scFv, CD8α hinge region, CD8 transmembrane region and CD3ζ intracellular domain, and a Trop2-specific CAR targeting breast cancer is constructed. Its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence of mRNA is shown in SEQ ID NO.2 (due to the WIPO system, U in the mRNA nucleotide sequence is replaced by T). The mRNA of the CAR is synthesized by Kaituo Biotechnology Co., Ltd.

[0022] Example 2 Preparation of TLR agonist-cholesterol conjugate 0.16 mmol of TLR agonist (R848) was dissolved in 2 mL of anhydrous dichloromethane, and then 1.1 molar times of cholesterol hemisuccinate, 1.1 molar times of 4-dimethylaminopyridine, and 1.1 molar times of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added. The reaction was stirred at room temperature for 24 h; the reaction was monitored by thin layer chromatography.

[0023] After the reaction was complete, the crude product was purified by column chromatography using a 5% methanol: dichloromethane gradient to obtain the TLR-7 / 8 agonist amphiphilic compound as a white solid. 1 The TLR agonist-cholesterol conjugate R848-Chol (TLRa) was obtained by H-NMR structural determination.

[0024] Example 3 Preparation and characterization of M-LNP / CAR mRNA+TLRa A method for preparing lipid nanoparticles (M-LNP / CAR mRNA+TLRa) co-loading mRNA encoding Trop2-specific CAR targeting breast cancer and a TLR agonist-cholesterol conjugate, comprising the following steps: (1) Dilute 20 μL of CAR mRNA (1 mg / mL) to 3 mL using sodium citrate buffer at pH = 4 to obtain an mRNA mixture; (2) Ionizable lipid SM-102, cholesterol-TLR agonist conjugate, DMG-PEG 2000 , DSPC and DSPE-PEG-MAN (purchased from Xi'an Ruixi Biotechnology Co., Ltd.) were dissolved in 1 mL of ethanol at a molar ratio of 45:38.5:1.5:10:2 to obtain a mixed lipid solution; (3) 3 mL of mRNA mixture and 1 mL of mixed lipid solution were added to the microfluidic device, wherein the mass ratio of mRNA in the mRNA mixture and ionizable lipid SM-102 in the mixed lipid solution was 1:14.5. Lipid nanoparticles (M-LNP / CAR mRNA+TLRa) co-loaded with mRNA encoding Trop2-specific CAR targeting breast cancer and TLR agonist-cholesterol conjugate were prepared by microfluidic method.

[0025] The encapsulation efficiency of M-LNP / CAR mRNA+TLRa was determined using the RiboGreen kit, and the hydrated particle size and surface potential were determined using a Malvern particle size analyzer. Figure 3 shown.

[0026] Depend on Figure 3 It can be seen that M-LNP / CAR mRNA+TLRa can efficiently encapsulate mRNA, with an encapsulation efficiency of 96.2±1.25%; the hydrated particle size of M-LNP / CAR mRNA+TLRa is 112±4.0nm, the particle size is uniform, and it is weakly positively charged.

[0027] The content of R848 in LNP was determined by HPLC: the lipid membrane of the prepared M-LNP / CAR mRNA+TLRa was destroyed with acetonitrile, filtered with a 0.2 μm organic membrane, and detected by Agilent 1260 infinity high performance liquid chromatograph and C18 detection column. The column temperature was set at 25 °C, the flow rate was 0.5 mL / min, and the mobile phase was 70% methanol solution. With anhydrous methanol as the solvent, 0.001 mg / mL, 0.002 mg / mL, 0.005 mg / mL, 0.01 mg / mL and 0.015 mg / mL solutions were prepared with R848 standard, and the peak area of ​​the drug at a wavelength of 320 nm was detected by high performance liquid chromatography to calculate the content of R848.

[0028] It was calculated that the content of TLR agonist (R848-Chol) encapsulated in M-LNP / CAR mRNA+TLRa was 0.047 mg / mL.

[0029] Take 1 drop of diluted M-LNP / CAR mRNA+TLRa and drop it on the copper grid. After drying for 3-5 minutes, drop a drop of phosphotungstic acid solution for negative staining. After the sample is dry, observe it under a transmission electron microscope.

[0030] Depend on Figure 4 It can be seen that M-LNP / CAR mRNA+TLRa is spherical in shape with a particle size of about 100 nm, and the particle size of M-LNP / CAR mRNA without integrated cholesterol-TLR agonist conjugate is about 100 nm; the particle size difference between the two is not much.

[0031] Example 4 Screening of lipid nanoparticle formulations The lipid nanoparticle formulations were screened according to the formulations in Table 1. The numbers in Table 1 represent molar multiples, and the lipid nanoparticle formulations were preliminarily screened based on particle size and PDI.

[0032] Table 1

[0033] This application screened several LNP formulations, and selected LNPs with a particle size of <200 nm and a PDI of <0.2 for further screening. Several formulations with potential delivery were selected to test whether they could express mRNA on macrophages, and the results are shown in Table 2. The results showed that the transfection efficiency of the LNP6 formulation was higher, so the LNP6 formulation was preferred for preparing the LNP delivery system.

[0034] Table 2

[0035] LNP / CAR mRNA + TLRa without targeting ligand (DSPE-PEG-MAN) was prepared using the LNP6 formula and injected into tumor-bearing mice. The efficiency of CAR expression in vivo was detected by flow cytometry. Figure 5 shown.

[0036] Depend on Figure 5 The results showed that 74% of macrophages expressed CAR, but also transduced some T cells (10.4%).

[0037] In order to improve the expression efficiency, the present invention then modified the mannose targeting ligand on the surface of LNP, that is, DSPE-PEG-MAN was added to the LNP formulation, and the molar ratio of ionizable lipid SM-102 to DSPE-PEG-MAN was 22.5:1; the targeting of macrophages was enhanced. After injection into the body, the expression efficiency was detected. Figure 6 shown.

[0038] Depend on Figure 6The results showed that 96% of macrophages expressed CAR, and only a small number of cells transduced T cells, about 1.3%. That is, adding DSPE-PEG-MAN to the LNP formulation can increase the targeting of LNP to macrophages.

[0039] Example 5 Cytotoxicity Study of M-LNP / CAR mRNA+TLRa 1. Extraction, isolation and culture of bone marrow-derived macrophages (BMDMs) Balb / C mice were euthanized and femurs and tibias were obtained. The bone marrow was washed and the cells were collected by centrifugation after passing through a 70 μm cell strainer. DMEM medium containing 10 ng / mL M-CSF was used to culture for seven days to induce differentiation into macrophages.

[0040] 2. Cytotoxicity study of M-LNP / CAR mRNA+TLRa BMDMs were seeded in 96-well plates (5×10 3 10 cells / well), cultured overnight to allow attachment, and then added with the M-LNP / CAR mRNA+TLRa culture medium solution prepared in Example 3 (1000 ng / mL, in terms of mRNA), and the BMDMs cell viability was detected at different time points of co-incubation. The results are as follows Figure 7 shown.

[0041] Depend on Figure 7 It can be seen that M-LNP / CAR mRNA+TLRa has no obvious cytotoxicity to macrophages and has good safety.

[0042] Example 6 In vitro macrophage transfection experiment of M-LNP / CAR mRNA+TLRa Human THP-1 cells were induced into macrophages using PMA and seeded in 24-well plates (6×10 4 After 24 hours, the cells were divided into two groups and M-LNP / CAR mRNA+TLRa (100 ng / well, calculated as mRNA) and an equal volume of PBS solution were added to each group. After incubation for 12 hours, the cells were collected and the transfection efficiency was detected. The results are shown in Figure 8 shown.

[0043] Depend on Figure 8 It can be seen that the expression of CAR protein in macrophages treated with M-LNP / CAR mRNA+TLRa can reach 11.3%±1.7% (n=3), indicating that M-LNP / CAR mRNA+TLRa can successfully co-deliver mRNA.

[0044] Example 7 Investigation of the in vitro tumor phagocytosis ability of M-LNP / CAR mRNA+TLRa 1. According to the method described in Example 3, lipid nanoparticles loaded with mRNA encoding Trop2-specific CAR targeting breast cancer (LNP / CAR mRNA without integrating DSPE-PEG-MAN and cholesterol-TLR agonist conjugates and M-LNP / CAR mRNA without integrating cholesterol-TLR agonist conjugates) and lipid nanoparticles loaded with Trop2-specific CAR mRNA and TLR agonist-cholesterol conjugates (without integrating DSPE-PEG-MAN) LNP / CAR mRNA+TLRa and M-LNP / CAR mRNA+TLRa) were prepared respectively.

[0045] 2. Human macrophages (THP-1 induced) and BMDMs cells labeled with fluorescent dye CFSE were seeded in 24-well plates (6×10 4 Cells / well), 24 hours later, divided into four groups, added with PBS, LNP / CAR mRNA without integration of DSPE-PEG-MAN and cholesterol-TLR agonist conjugate, M-LNP / CAR mRNA (without cholesterol-TLR agonist conjugate), LNP / CAR mRNA+TLRa (without integration of DSPE-PEG-MAN) and M-LNP / CAR mRNA+TLRa (100 ng / well, calculated as mRNA), respectively. After incubation for 12 hours, DiD-labeled tumor cells (6×10 4 cells / well) and continued to culture for 12 hours. The cells were collected and the phagocytosis of each group was measured using flow cytometry. The results are shown in Fig. 9 shown.

[0046] Depend on Fig. 9 It can be seen that M-LNP / CAR mRNA+TLRa showed the highest phagocytic efficiency, indicating that M-LNP / CAR mRNA+TLRa further induced macrophages to clear tumors and had a powerful tumor removal effect.

[0047] Example 8 In vitro killing of tumor cells by M-LNP / CAR mRNA+TLRa After THP-1 was transfected with different LNPs (induced into macrophages), they were added to MBA-MB-231 breast cancer cell culture. After 24 hours of co-incubation, the supernatant was aspirated for LDH determination to detect its ability to kill tumor cells. Fig.10 shown.

[0048] The results showed that M-LNP / CAR-mRNA+TLRa had the strongest killing ability after transducing macrophages in vivo.

[0049] Example 9 Evaluation of in vivo tissue distribution of M-LNP / CAR mRNA+TLRa Balb / C mice were inoculated with 4T1-Trop2 tumor cells in situ, and tumors grew in the mice 7 days later. The M-LNP / CAR mRNA+TLRa prepared in Example 3 of the present invention was labeled with the near-infrared dye DiD, and then injected through the tail vein. The tissues and organs were taken out at 24h, 48h and 72h, and the in vivo tissue distribution of M-LNP / CAR mRNA+TLRa was detected by an ELISA instrument ( Fig.11 ).

[0050] Depend on Fig.11 The results showed that M-LNP / CAR mRNA+TLRa could be effectively distributed in tumors in vivo.

[0051] Example 10 M-LNP / CAR mRNA+TLRa produces CAR-macrophages targeting Trop2 in vivo Balb / C female mice were inoculated with 4T1-Trop2 tumor cells in situ, and on the 7th day, LNP / GFP (LNP without DSPE-PEG-MAN and cholesterol-TLR agonist conjugates encapsulated mRNA expressing only GFP), LNP / CAR mRNA, M-LNP / CAR mRNA, LNP / CAR mRNA+TLRa, M-LNP / CAR mRNA+TLRa (0.3 mg / kg, in terms of mRNA) and TLRa+M-LNP / CAR mRNA (TLRa was injected first, then M-LNP / CARmRNA was injected) prepared in Example 3 of the present invention were injected into the tail vein, 5 mice in each group. 0.1 mL of peripheral blood was collected from mice on the 2nd, 5th and 16th days, and the collected peripheral blood was separated using monocyte separation solution, and the number of CAR-M cells was detected by flow cytometry ( Fig.12 ), and the total number of CAR-M cells in peripheral blood was calculated.

[0052] Depend on Fig.12 It can be seen that M-LNP / CAR mRNA+TLRa achieved the transformation of macrophages into CAR-M cells in tumor-bearing mice.

[0053] Example 11 In vivo pharmacodynamic evaluation of M-LNP / CAR mRNA+TLRa 4T1-Trop2 cells in the logarithmic growth phase were digested and injected into the fourth mammary gland of Balb / C female mice to establish an orthotopic breast cancer mouse model. Seven days later, the mice were randomly divided into five groups (n=5) and injected with LNP / GFP, LNP / CAR mRNA, M-LNP / CAR mRNA, LNP / CAR mRNA+TLRa and M-LNP / CAR mRNA+TLRa (0.3 mg / kg, in terms of mRNA) through the tail vein. The mice were given the drug once every three days for three times, and the tumor size of the mice was monitored every three days since the day of modeling. The results are shown in Figure 2. Fig.13 shown.

[0054] Depend on Fig.13 It can be seen that M-LNP / CAR mRNA+TLRa showed the best anti-tumor effect, and the tumor volume was significantly smaller than that of other groups after treatment.

[0055] Example 12 Cytokines produced by M-LNP / CAR mRNA+TLRa in vivo like Fig.14 As shown, the IFN-γ and TNF-α secreted in the plasma of the M-LNP / CAR mRNA+TLRa group were significantly higher than those of the ordinary LNP / CAR mRNA, and had a stronger killing effect.

[0056] Example 13 In vivo safety evaluation of M-LNP / CAR mRNA+TLRa Balb / C female mice were orthotopically inoculated with 4T1-Trop2 tumor cells and then injected with PBS (control group), LNP / GFP, LNP / CAR mRNA, M-LNP / CAR mRNA, LNP / CAR mRNA+TLRa, and M-LNP / CAR mRNA+TLRa (0.3 mg / kg, in terms of mRNA) via tail vein, once every three days for three times. The peripheral blood of mice was collected the day after the third administration, and the kinase level was determined. The results are shown in Fig.15 shown.

[0057] Depend on Fig.15 It can be seen that compared with the PBS group, the kinase levels in the M-LNP / CAR mRNA+TLRa group had no significant difference and were all within the normal range, proving that it has good biosafety ( Fig.15 ).

[0058] The above are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modification, equivalent replacement or improvement without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims

1. A chimeric antigen receptor macrophage targeting Trop2, characterized in that: The macrophage contains a specific CAR targeting Trop2 and a blocker for blocking immunosuppressive signals; the amino acid sequence of the Trop2-specific CAR targeting breast cancer is shown in SEQ ID NO.

1.

2. The chimeric antigen receptor macrophage targeting Trop2 according to claim 1, characterized in that The nucleotide sequence of the mRNA of the specific CAR targeting Trop2 is shown in SEQ ID NO.

2.

3. The chimeric antigen receptor macrophage targeting Trop2 according to claim 1, characterized in that The blocker for blocking immunosuppressive signals is a cholesterol-TLR agonist conjugate.

4. A method for preparing a chimeric antigen receptor macrophage targeting Trop2 according to claim 2, characterized in that: The method comprises the steps of preparing lipid nanoparticles and transfecting macrophages with the lipid nanoparticles; The preparation steps of the lipid nanoparticles are as follows: Step (1) dissolving the mRNA of CAR targeting Trop2 in sodium citrate buffer to obtain an mRNA mixed solution; Step (2) ionizable lipid SM-102, cholesterol-TLR agonist conjugate, DMG-PEG 2000 , DSPC and DSPE-PEG-MAN were dissolved in ethanol to obtain a mixed lipid solution; Step (3) adding the mRNA mixture and the mixed lipid solution to the microfluidic device, and preparing lipid nanoparticles co-loaded with mRNA encoding a specific CAR targeting Trop2 and a cholesterol-TLR agonist conjugate by a microfluidic method.

5. The method for preparing a chimeric antigen receptor macrophage targeting Trop2 according to claim 4, characterized in that: The synthesis method of the cholesterol-TLR agonist conjugate is as follows: The TLR agonist R848 is dissolved in an organic solvent, and then 0.8-1.2 molar times of cholesterol hemisuccinate, 0.8-1.2 molar times of 4-dimethylaminopyridine and 0.8-1.2 molar times of 1-ethyl-(3-dimethylaminopropyl)carbodiimide are added respectively; the reaction substrate is stirred at room temperature for 20-30 hours. After the reaction is completed, the crude product is purified by column chromatography to obtain a cholesterol-TLR agonist conjugate; The organic solvent is ethanol, dichloromethane, chloroform or methanol.

6. The method for preparing a chimeric antigen receptor macrophage targeting Trop2 according to claim 4, characterized in that: The ionizable lipid SM-102, cholesterol-TLR agonist conjugate, DMG-PEG 2000 The molar ratio of DSPC and DSPE-PEG-MAN is 30-60:30-50:1-5:10-20:1-10; the concentration of the cholesterol-TLR agonist conjugate is 0.5-1 mg / mL.

7. The method for preparing a chimeric antigen receptor macrophage targeting Trop2 according to claim 4, characterized in that: The concentration of mRNA in the mRNA mixture in step (1) is 3 to 10 μg / mL; The mass ratio of the mRNA in the mRNA mixed solution to the ionizable lipid SM-102 in the mixed lipid solution is 1:8-20.

8. The method for preparing a chimeric antigen receptor macrophage targeting Trop2 according to any one of claims 4 to 7, characterized in that: The lipid nanoparticles prepared in step (3) are transfected into macrophages in vivo or in vitro to obtain chimeric antigen receptor macrophages targeting Trop2.

9. Use of the chimeric antigen receptor macrophage targeting Trop2 according to claim 1, or the lipid nanoparticles according to any one of claims 4 to 7 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that: The tumor is breast cancer.

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