A chimeric antigen receptor targeting TROP2, a drug delivery body and its application

Abstract

The present invention provides a chimeric antigen receptor targeting TROP2, a drug delivery body and its application, belonging to the technical field of hybrid peptides, wherein the amino acid sequence of the chimeric antigen receptor targeting TROP2 is shown in SEQ ID NO: 1 in the sequence table; the chimeric antigen receptor targeting TROP2 is obtained by sequentially connecting the following modules: CD8 signal peptide, TROP2 single-chain antibody, CD8 hinge region, CD8 transmembrane region, CD28 and CD226 costimulatory region, CD3ζ intracellular region, T2A, optimized RISR-RIAD peptide. The CAR-TROP2-iNEs containing the optimized RISR-RIAD peptide prepared by the present invention can improve the killing efficiency of SK-OV-3 cell line and LOVO cell line after in vitro transfection of T cells, with a killing efficiency of 79.6% for SK-OV-3 cells and 87.3% for LOVO cells.

Description

Technical Field

[0001] The invention relates to a chimeric antigen receptor targeting TROP2, a drug delivery body and application thereof, and belongs to the technical field of hybrid peptides. Background Art

[0002] TROP2 is a cancer-associated antigen that is expressed at very low levels in normal tissues and overexpressed in a variety of malignant tumors. It is mainly expressed in various human epithelial cancers, including breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, and ovarian cancer. The expression level of TROP2 is related to the malignancy of the disease. TROP2 overexpression can promote tumor cell growth, proliferation, and metastasis. This differential expression of TROP2 protein makes it a new target for researchers to develop antibody drugs.

[0003] Although CAR-T therapy has become increasingly mature, it still faces the problems of individualization and long waiting time for patients. In actual application, it is limited by the patient's blood quality, immune rejection and other reasons, and the treatment effect is not as good as expected, resulting in the sales of listed CAR-T products not meeting expectations. Therefore, it is necessary to optimize the delivery method of chimeric antigen receptors, reduce patient waiting time, and reduce production costs.

[0004] Studies have shown that protein kinase A (PKA) has the function of inhibiting T cells, and RISR-RIAD peptide can block the role of protein PKA in immune regulation. Therefore, RISR-RIAD peptide enhances the function of CAR-T cells by inhibiting the PKA signaling pathway in the tumor microenvironment, thereby improving the transport and anti-tumor activity of modified CAR-T cells.

[0005] There are currently no reports on the delivery of TROP2 chimeric antigen receptors in the form of nanoparticles to form CAR-T cells in vivo. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a chimeric antigen receptor targeting TROP2, a drug delivery body and its application, to achieve the following invention objectives: to deliver CAR-TROP2 into the body in the form of mRNA-iNEs to form CAR-T cells, thereby improving the anti-tumor effect of CAR-TROP2.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A chimeric antigen receptor targeting TROP2, wherein the amino acid sequence of the chimeric antigen receptor targeting TROP2 is shown in SEQ ID NO: 1 in the sequence table; the chimeric antigen receptor targeting TROP2 is obtained by sequentially connecting the following modules: CD8 signal peptide, TROP2 single-chain antibody, CD8 hinge region, CD8 transmembrane region, CD28 and CD226 costimulatory regions, CD3ζ intracellular region, T2A, and optimized RISR-RIAD peptide.

[0009] The nucleotide sequence of the chimeric antigen receptor targeting TROP2 is shown in SEQ ID NO: 2 in the sequence listing.

[0010] A drug delivery body targeting TROP2, wherein the drug delivery body is a nanoemulsion preparation of mRNA targeting TROP2; the mRNA is transcribed from the chimeric antigen receptor.

[0011] The nanoemulsion preparation is prepared from an mRNA solution and a liposome mixture; the liposome mixture consists of C12-200, DOPE, vitamin E, and DMG-PEG2000, with a molar ratio of 35:16:46.5:2.5.

[0012] The ratio of positive charges in the liposome mixture to negative charges in the mRNA solution was 15:1.

[0013] The application of the chimeric antigen receptor targeting TROP2 in the preparation of anticancer drugs.

[0014] The drug delivery body is used in the preparation of anticancer drugs.

[0015] Compared with the prior art, the present invention achieves the following beneficial effects:

[0016] The CAR-TROP2-iNEs containing the optimized RISR-RIAD peptide prepared by the present invention can improve the killing efficiency of SK-OV-3 cell lines and LOVO cell lines after in vitro transfection of T cells, with the killing efficiency of SK-OV-3 cells being 79.6% and the killing efficiency of LOVO cells being 87.3%.

[0017] The CAR-TROP2-iNEs containing the optimized RISR-RIAD peptide prepared by the present invention can form CAR-T cells in vivo when used to treat a rectal cancer mouse model, and can significantly inhibit the growth of the tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The structural diagram of the extracellular domain of the CAR designed with the specific marker TROP2 as the specific antigen;

[0019] A is the structural diagram of CAR-TROP2; B is the structural diagram of CAR-TROP2-2;

[0020] Figure 2 The bar graph is the evaluation of the toxicity of CAR-TROP2-iNEs and CAR-TROP2-2-iNEs on T cells;

[0021] Figure 3 It is a graph showing the efficiency of CAR-TROP2-iNEs and CAR-TROP2-2-iNEs transfection into T cells;

[0022] A is the efficiency diagram of CAR-TROP2-iNEs transfection of T cells; B is the efficiency diagram of CAR-TROP2-2-iNEs transfection of T cells;

[0023] Figure 4 This is a bar graph showing the killing efficiency of T cells transfected with CAR-TROP2-iNEs and CAR-TROP2-2-iNEs against tumor cells;

[0024] Figure 5 This is a graph showing the tumor growth curve of the mouse tumor model. DETAILED DESCRIPTION

[0025] Example 1 Construction of mRNA containing CAR-TROP2

[0026] The CAR extracellular domain was designed using the specific marker TROP2 as the specific antigen. Figure 1 As shown, the structure of CAR-TROP2 includes CD8 signal peptide, TROP2 single-chain antibody, CD8 hinge region, CD8 transmembrane region, CD28 and CD226 co-stimulatory region, CD3ζ intracellular region, T2A, and optimized RISR-RIAD peptide. The amino acid sequence of CAR-TROP2 is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2.

[0027] The optimized RISR-RIAD peptide is based on the optimized RISR-SIAD peptide, with the 8th amino acid optimized from E to R and the 18th amino acid optimized from Y to A.

[0028] The optimized RISR-RIAD peptide in the structure of CAR-TROP2 is replaced with the pre-optimized RISR-RIAD peptide to obtain the structure of CAR-TROP2-2; the amino acid sequence of the CAR-TROP2-2 is shown in SEQ ID NO: 3, and the nucleotide sequence is shown in SEQ ID NO: 4.

[0029] Linearized DNA templates were obtained through enzyme digestion experiments and purification, and transcription was completed under the action of T7 polymerase. Commercial reagents were further used for capping and tailing. Finally, the mRNA of CAR-TROP2 and CAR-TROP2-2 was purified using the MEGAclear kit to obtain purified mRNA, and its concentration was adjusted to 1.0 mg / mL.

[0030] Example 2 Preparation of nanoemulsion (iNEs) formulation

[0031] 1. Prepare 1 mL of liposome mixture according to the molar ratio and final concentration of each substance in Table 1.

[0032] Table 1 Components in the liposome mixture

[0033]

[0034] 2. According to the ratio of positive charge (N) of C12-200 to negative charge (P) of purified mRNA in 1 mL of liposome mixture of 15:1, the two mRNAs were diluted with 10 mM citric acid buffer at pH = 4 to prepare 3 mL of mRNA solution with a concentration of 0.17 mg / mL.

[0035] 3. Add mRNA solution and liposome mixture to the microfluidic device, wherein the volume ratio of the mRNA solution and the liposome mixture is 3:1, and prepare mRNA-iNEs nanoformulation by microfluidic method. The iNEs prepared by CAR-TROP2 are called CAR-TROP2-iNEs, and the iNEs prepared by CAR-TROP2-2 are called CAR-TROP2-2-iNEs.

[0036] The encapsulation efficiency of CAR-TROP2-iNEs and CAR-TROP2-2-iNEs was determined using the Quant-iT™ RiboGreen® RNA Reagent and Kit (purchased from Invitorgen), and the particle size and PDI were determined using a Malvern particle size analyzer. The results are shown in Table 2.

[0037] Table 2 Physicochemical properties of iNEs

[0038]

[0039] Example 3 Cytotoxicity and in vitro transfection experiments of CAR-TROP2-iNEs and CAR-TROP2-2-iNEs in T cells

[0040] (1) Preparation of activated T cells

[0041] 50 mL of peripheral blood donated by volunteers was collected and PBMC (peripheral blood mononuclear cells) was isolated using TBD sample density separation solution (purchased from Tianjin Haoyang Huake Biotechnology Co., Ltd.). The separated cells were counted and the number of cells was 1×10 6 cells / mL, added to a medium (purchased from Corning, catalog number: 88-551-CM) containing 1000IU / mL recombinant interferon α2a (purchased from Shenyang Sansheng Pharmaceutical). Cultured in a 37°C, 5% CO2 incubator for 24 hours, added 1500IU / mL (final concentration) of recombinant interleukin 2 (purchased from Shenyang Sansheng Pharmaceutical) and 50ng / mL (final concentration) of CD3 monoclonal antibody (purchased from Tongli Haiyuan) and continued to culture for 24 hours to obtain activated T cells.

[0042] (2) Cytotoxicity assay

[0043] Activated T cells were seeded in 96-well plates (5×10 3 After overnight culture, CAR-TROP2-iNEs (5.5 ng / well in terms of mRNA) and CAR-TROP2-2-iNEs (5.5 ng / well in terms of mRNA) were added, and T cell viability was detected at 0 h, 6 h, 12 h, 18 h, and 24 h of co-incubation. The results are shown in Figure 2 shown.

[0044] Depend on Figure 2 It can be seen that CAR-TROP2-iNEs and CAR-TROP2-2-iNEs have no obvious cytotoxicity and have good safety.

[0045] (3) In vitro transfection experiment

[0046] Activated T cells were seeded in 24-well plates (6 × 10 4 The cells were divided into three groups 24 hours later and CAR-TROP2-iNEs (55 ng / well, calculated as mRNA), CAR-TROP2-2-iNEs (55 ng / well, calculated as mRNA) and an equal volume of PBS solution were added, respectively. After 24 hours of incubation, the cells were collected and the transfection efficiency was detected using TROP2 flow cytometry antibody (purchased from Abcam, catalog number: ab214488). The results are shown in Figure 3 shown.

[0047] Depend on Figure 3 It can be seen that the expression rate of CAR in T cells was 36.7% when CAR-TROP2-iNEs were transfected into T cells, and the expression rate of TROP2 in T cells was 38.2% when CAR-TROP2-2-iNEs were transfected into T cells, indicating that CAR-T cells were successfully obtained after CAR-TROP2 was transfected into T cells.

[0048] Example 4 Evaluation of in vitro tumor killing ability

[0049] Activated T cells were seeded in 24-well plates (6 × 10 4 PBS, CAR-TROP2-iNEs (55 ng / well, calculated as mRNA), and CAR-TROP2-2-iNEs (55 ng / well, calculated as mRNA) were added to the cells respectively. After incubation for 24 h, luciferase-labeled ovarian cancer tumor SK-OV-3 cells (6×10 4 Cells / well) were added and cultured for 4 h. The cells were collected and the killing of tumor cells in each group was determined using an ELISA instrument. The killing ability of the colorectal cancer LOVO cell line was detected in the same way.

[0050] The results are as follows Figure 4 As shown, the killing efficiency of CAR-TROP2-iNEs-transfected T cells on SK-OV-3 cells was 79.6%, and the killing efficiency on LOVO cells was 87.3%. The killing efficiency of CAR-TROP2-2-iNEs-transfected T cells on SK-OV-3 cells was 51.4%, and the killing efficiency on LOVO cells was 65.2%. Compared with T cells transfected with CAR-TROP2-2-iNEs, the killing efficiency of CAR-TROP2-iNEs-transfected T cells on the two tumor cells was significantly improved. This shows that the optimized RISR-RIAD peptide can significantly improve the killing efficiency of CAR-TROP2-iNEs-transfected T cells on tumor cells.

[0051] Example 5 In vivo efficacy evaluation of CAR-TROP2-iNEs

[0052] LOVO cells in the logarithmic growth phase were digested and injected subcutaneously into C57BL / 6 mice to establish a colorectal cancer mouse model. The tumor size was measured to be 95-100 mm 3 After successful modeling, the mice were randomly divided into three groups (n=10) and injected with PBS, CAR-TROP2-iNEs (0.3 mg / kg, calculated as mRNA), and CAR-TROP2-2-iNEs (0.3 mg / kg, calculated as mRNA) through the tail vein, respectively. The mice were given the drug once a week and twice a week (once every other week). The tumor size of the mice was monitored every 3 days within 30 days after the day of administration, and the tumor growth curve was drawn. The results are shown in the figure. Figure 5 shown.

[0053] Depend on Figure 5 It can be seen that after 30 days of administration, the average tumor volume of mice in the PBS control group was 1465mm 3The mean tumor volume of mice in the CAR-TROP2-iNEs group was 335 mm 3 The mean tumor volume of mice in the CAR-TROP2-2-iNEs group was 984 mm 3 Both CAR-TROP2-iNEs and CAR-TROP2-2-iNEs had significant inhibitory effects on mouse tumors, but the inhibitory effect of CAR-TROP2-iNEs containing the optimized RISR-RIAD peptide on tumors was more significant than that of CAR-TROP2-2-iNEs containing the RISR-RIAD peptide before optimization, indicating that the optimized RISR-RIAD peptide can improve the tumor-killing effect of CAR-TROP2-iNEs.

Claims

1. A chimeric antigen receptor targeting TROP2, characterized in that: The amino acid sequence of the chimeric antigen receptor targeting TROP2 is shown in SEQ ID NO: 1; the chimeric antigen receptor targeting TROP2 is obtained by sequentially connecting the following modules: CD8 signal peptide, TROP2 single-chain antibody, CD8 hinge region, CD8 transmembrane region, CD28 and CD226 co-stimulatory region, CD3ζ intracellular region, T2A, and optimized RISR-RIAD peptide.

2. A chimeric antigen receptor targeting TROP2 according to claim 1, characterized in that: The nucleotide sequence of the chimeric antigen receptor targeting TROP2 is shown in SEQ ID NO:

2.

3. A drug delivery system targeting TROP2, characterized in that: The drug delivery body is a nanoemulsion preparation of mRNA targeting TROP2; the mRNA is transcribed from the chimeric antigen receptor according to claim 1.

4. The drug delivery device according to claim 3, characterized in that: The nanoemulsion preparation is prepared from an mRNA solution and a liposome mixture; the liposome mixture consists of C12-200, DOPE, vitamin E, and DMG-PEG2000, with a molar ratio of 35:16:46.5:2.

5.

5. The drug delivery device according to claim 4, characterized in that: The ratio of positive charges in the liposome mixture to negative charges in the mRNA solution was 15:

1.

6. Use of T cells transfected with a chimeric antigen receptor targeting TROP2 according to any one of claims 1 to 2 in the preparation of drugs against ovarian cancer and / or colorectal cancer.

7. Use of T cells transfected by the drug delivery body according to any one of claims 3 to 5 in the preparation of drugs against ovarian cancer and / or colorectal cancer.

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