Mesothelin chimeric antigen receptor, mRNA and application of mesothelin chimeric antigen receptor in preparation of medicine for treating solid tumor expressing mesothelin

Abstract

The invention relates to the technical field of biological medicine. The invention provides a novel, efficient and safe mesothelin chimeric antigen receptor and a coding mRNA thereof, and also provides a method for performing T cell engineering transformation by using the mesothelin chimeric antigen receptor mRNA, so as to treat tumors expressing mesothelin. According to the technical scheme, T cells are efficiently transfected in a non-virus mode, transient expression of CAR protein is achieved, and the safety of the CAR-T cells is enhanced; meanwhile, the limitation of the existing CAR-T cell therapy during solid tumor treatment is overcome, migration and infiltration of CAR T cells to tumor parts are improved, and the effect of treating solid tumors expressing MSLN is better enhanced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a mesothelin chimeric antigen receptor, mRNA and application thereof in preparation of a drug for treating solid tumors expressing mesothelin. BACKGROUND

[0002] Cancer, as a major cause of death worldwide, poses a serious threat to human health. In recent years, cancer immunotherapy, especially adoptive T cell therapy, has become a revolutionary approach to treating cancer after surgery, radiotherapy and chemotherapy. Immunotherapy aims to enhance the body's anti-tumor immunity or reduce the immune escape ability of tumor cells, use the body's immune system to recognize and eliminate cancer cells, and at the same time produce immune memory, so as to achieve a lasting anti-tumor effect.

[0003] Chimeric antigen receptor T cell (CAR-T) therapy is a major breakthrough in the field of tumor immunotherapy. Its principle is to use genetic engineering technology to make the patient's own T cells express synthetic chimeric antigen receptors (CARs), so that they can specifically recognize the surface antigens of tumor cells in a major histocompatibility complex (MHC) independent manner. Although CAR-T therapy has achieved great success in hematological malignancies, its application in solid tumor treatment still faces many challenges, and the effect is not satisfactory. The main limiting factors include: (1) immunosuppressive tumor microenvironment (TME): solid tumors often have a complex immunosuppressive microenvironment, rich in immunosuppressive cells (such as regulatory T cells, myeloid-derived suppressor cells MDSCs, M2 macrophages) and immunosuppressive molecules (such as TGF-β, IL-10, PD-L1), which together inhibit the proliferation, function and persistence of CAR-T cells; (2) insufficient tumor infiltration of CAR-T cells: the dense extracellular matrix and disordered vascular system of solid tumors form a physical barrier, limiting the effective migration and infiltration of CAR-T cells to the tumor site, affecting their anti-tumor activity; (3) antigen heterogeneity and antigen escape: the expression of target antigens on the surface of solid tumor cells may not be uniform, or antigen loss may occur during treatment, leading to tumor cells escaping the killing of CAR-T cells; (4) "on-target, off-tumor toxicity": many tumor-associated antigens (TAA) also exist at low levels in normal tissues, which may cause CAR-T cells to attack normal cells, triggering serious side effects such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS); (5) insufficient persistence of CAR-T cells: in the solid tumor microenvironment, CAR-T cells may undergo functional exhaustion, with a short survival time, limiting their long-term anti-tumor effect.

[0004] Based on the above, it is very important to develop a new, more efficient and effective technical solution that can overcome the above-mentioned defects. SUMMARY

[0005] The purpose of the present application is to provide a mesothelin chimeric antigen receptor, its encoding mRNA and application, which can overcome the limitations of existing CAR-T cell therapy for solid tumor treatment, thereby obtaining a more controllable, more easily expandable and safer tumor immunotherapy solution.

[0006] In order to achieve the above-mentioned application purpose, the present application provides the following technical solutions: The present application provides a mesothelin chimeric antigen receptor, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0007] As a preferred, the protein structure of the mesothelin chimeric antigen receptor includes a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain and an intracellular signal domain.

[0008] As a preferred, the sequence of the signal peptide is shown in SEQ ID NO. 2; the antigen binding domain includes a heavy chain variable region shown in SEQ ID NO. 3 and a light chain variable region shown in SEQ ID NO. 4; the sequence of the hinge region is shown in SEQ ID NO. 6; the sequence of the transmembrane domain is shown in SEQ ID NO. 7; the sequence of the intracellular signal domain is shown in SEQ ID NO. 8.

[0009] As a preferred, the heavy chain variable region and the light chain variable region are connected by a connecting peptide, and the sequence of the connecting peptide is shown in SEQ ID NO. 5.

[0010] The present application provides an mRNA encoding the mesothelin chimeric antigen receptor, and the structure of the mRNA includes a 5'UTR, an MSLN-CAR coding region, a 3'UTR and a polyA tail; the nucleic acid sequence of the MSLN-CAR coding region is shown in SEQ ID NO. 12.

[0011] As a preferred, the nucleic acid sequence of the 5'UTR is shown in SEQ ID NO. 11; the nucleic acid sequence of the 3'UTR is shown in SEQ ID NO. 13; and the nucleic acid sequence of the polyA tail is shown in SEQ ID NO. 14.

[0012] The present application provides a vector containing the mRNA.

[0013] The present application provides an engineered immune effector cell containing the mesothelin chimeric antigen receptor, the mRNA or the vector.

[0014] Preferably, the immune effector cells include T cells.

[0015] The present invention also provides the use of the mRNA or the engineered immune effector cells in the preparation of drugs for treating solid tumors expressing mesothelin.

[0016] The present invention also provides a nucleic acid drug for treating solid tumors expressing mesothelin, comprising a lipid delivery carrier and the mRNA.

[0017] By adopting the above technical solution, the present invention has the following beneficial effects: This invention provides a novel, highly efficient, and safe chimeric antigen receptor (CAR) mRNA targeting mesothelin (MSLN), which efficiently transfects T cells via a non-viral method to achieve transient expression of the CAR protein and enhance the safety of CAR-T cells. At the same time, it overcomes the limitations of existing CAR-T cell therapies, especially for the treatment of solid tumors, such as high preparation costs, complex manufacturing processes, risks of viral integration, and potential serious side effects, thus providing a more controllable, scalable, and safer tumor immunotherapy regimen.

[0018] The technical solution described in this invention has high safety and high specific anti-tumor activity. It can improve the production efficiency and accessibility of CAR-T cells, enhance T cell function, and has the potential to overcome the challenges of solid tumors, as detailed below: (1) By employing mRNA technology, the risks of random gene integration mediated by viral vectors and potential insertional oncogenicity are avoided. Due to the transient expression characteristics of CAR mRNA, the expression time of CAR protein on the surface of T cells is limited, which greatly reduces the incidence and duration of serious adverse reactions such as cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and off-target toxicity. This provides a built-in safety mechanism that allows for rapid discontinuation of treatment in the event of adverse reactions.

[0019] (2) The scFv in the CAR protein can specifically recognize and bind to tumor cells expressing MSLN, guiding CAR-T cells to precisely kill tumors and reduce damage to normal tissues. MSLN is highly expressed in a variety of solid tumors, while its expression level is low in normal tissues, providing a larger safety window for CAR-T cell therapy. MSLN CAR T cells have been shown in in vitro experiments to effectively kill MSLN-positive tumor cells and release high levels of pro-inflammatory cytokines (such as IL-2, IFN-γ, and TNF-α).

[0020] (3) mRNA is prepared by in vitro transcription (IVT) technology, which has a much shorter production cycle than viral vectors, enabling rapid and large-scale production with minimal batch-to-batch variation. This is of great significance for patients with rapidly progressing diseases, reduces production costs and technical barriers, and improves the accessibility of CAR-T cells.

[0021] (4) The optimized intracellular signaling domain (including CD3ζ and the complex co-stimulation domain) can effectively activate T cells, promote their proliferation and persistence, thereby improving the efficacy of MSLN CAR T cells in the solid tumor microenvironment. Attached Figure Description

[0022] Figure 1 The hydrodynamic diameter diagram of each nanoparticle in Example 3 of the present invention is shown. Figure 1 In the diagram, A represents the hydrodynamic diameter map of MSLN CAR mRNA@LNP, B represents the hydrodynamic diameter map of MSLN CAR mRNA@LNP (new), and C represents the hydrodynamic diameter map of Blank LNP. Figure 2 The Zeta potential diagrams for each nanoparticle in Example 3 of this invention are shown. Figure 3 This is a standard curve of fluorescence signal intensity versus mRNA concentration in Example 3 of the present invention; Figure 4 Flow cytometry images of various nanoparticle groups in Example 4 of this invention ( Figure 4 In the diagram, A represents the flow cytometry plot of Blank LNP, B represents the flow cytometry plot of MSLN CAR mRNA@LNP, and C represents the flow cytometry plot of MSLN CAR mRNA@LNP (new). Figure 5 This is a graph showing the transfection efficiency results of each nanoparticle group in Example 4 of the present invention; Figure 6 The diagram shows the CCK8 proliferation results of each nanoparticle group in Example 5 of this invention. Figure 7 The flow cytometry apoptosis results of each nanoparticle group in Example 5 of this invention are shown. Detailed Implementation

[0023] This invention provides a mesothelin chimeric antigen receptor, the amino acid sequence of which is shown in SEQ ID NO.1, specifically as follows: MALPVTTLALPLAALLHAARPQVQLQQPGAELVKPGASMKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGMIHPNSDNTIYYEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAIIITPVVPKFDYWGQGTTLTVSSGGGSGGSSSGGGGSGGIVMTQSHQFMSTSVGDRVSVTCKASHDVGTSVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQS EDLADYFCQQYSSYPLTFGAGTKLELKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0024] In this invention, the mesothelin chimeric antigen receptor is a novel third-generation chimeric antigen receptor whose protein structure includes a signal peptide, an antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain from the N-terminus to the C-terminus.

[0025] In this invention, the sequence of the signal peptide (SP) is shown in SEQ ID NO.2, specifically MALPVTTLALPLAALLHAARP. The signal peptide of this invention guides the correct folding and transport of CAR proteins within the cell to the cell surface.

[0026] In this invention, the antigen-binding domain is composed of a single-chain variable fragment (scFv) against mesothelin, including a heavy chain variable region (VH) and a light chain variable region (VL). The sequence of the heavy chain variable region of this invention is shown in SEQ ID NO.3, specifically QVQLQQPGAELVKPGASMKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGMIHPNSDNTIYYEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAIIITPVVPKFDYWGQGTTLTVSS; the sequence of the light chain variable region is shown in SEQ ID NO.4, specifically IVMTQSHQFMSTSVGDRVSVTCKASHDVGTSVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPLTFGAGTKLELK. The scFv described in this invention can specifically recognize and bind to mesothelin antigen, which is highly expressed on the surface of tumor cells.

[0027] In this invention, the heavy chain variable region and the light chain variable region are preferably linked by a flexible scFv linker peptide, the sequence of which is shown in SEQ ID NO.5, specifically GGGSGGSSSGGGGSGG. The scFv linker peptide of this invention endows scFv with sufficient flexibility, enabling it to efficiently bind to target antigens.

[0028] In this invention, the sequence of the hinge domain is shown in SEQ ID NO.6, specifically IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP. This region, located between the scFv and the transmembrane domain, provides molecular flexibility and ensures that the scFv can effectively access the target antigen, avoiding steric hindrance.

[0029] In this invention, the sequence of the transmembrane domain (TM) is shown in SEQ ID NO.7, specifically FWVLVVVGGVLACYSLLVTVAFIIFWV. This region, as described in this invention, is responsible for anchoring the CAR to the T cell membrane and transmitting signals.

[0030] In this invention, the sequence of the intracellular signaling domain (ICD) is shown in SEQ ID NO.8, specifically: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. This region, as described in this invention, is responsible for activating T cells and mediating their anti-tumor function.

[0031] In this invention, the intracellular signaling domain preferably includes a 4-1BB (CD137) co-stimulatory signaling domain and a CD3ζ signaling domain. The sequence of the 4-1BB (CD137) co-stimulatory signaling domain is shown in SEQ ID NO.9, specifically KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL; this 4-1BB (CD137) co-stimulatory signaling domain provides a co-activation signal, enhancing T cell proliferation, survival, and cytokine secretion. The sequence of the CD3ζ signaling domain is shown in SEQ ID NO.10, specifically RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR; this CD3ζ signaling domain provides the main signal for T cell activation, initiating downstream signaling pathways and inducing cytotoxic responses.

[0032] The present invention also provides an mRNA encoding the mesothelin chimeric antigen receptor, the structure of which includes a 5'UTR, an MSLN-CAR coding region, a 3'UTR, and a polyA tail.

[0033] In this invention, the nucleic acid sequence of the 5'UTR is shown in SEQ ID NO.11, specifically GGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGGCGCC. The 5' untranslated region (5'UTR) of this invention is located upstream of the encoding mRNA sequence and plays a crucial regulatory role in the translation efficiency and stability of mRNA. The 5'UTR of this invention is obtained through optimization and can enhance mRNA expression.

[0034]

[0035] In this invention, the nucleic acid sequence of the 3'UTR is shown in SEQ ID NO.13, specifically GCTGGAGCCTCGGTGGCCTAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCC. The 3' untranslated region (3'UTR) in this invention is located downstream of the encoding mRNA sequence and regulates mRNA stability and translation efficiency. The 3'UTR in this invention is optimized to increase the expression level of the CAR protein.

[0036] In this invention, the nucleic acid sequence of the poly(A) tail is shown in SEQ ID NO.14, specifically: GTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA. The poly(A) tail of this invention is located after the 3' UTR, providing mRNA stability and efficient translation.

[0037] The present invention also provides a vector comprising the mRNA described above.

[0038] In this invention, the vector can be used to transfect T cells in vitro or in vivo.

[0039] The present invention provides an engineered immune effector cell comprising the mesothelin chimeric antigen receptor, the mRNA, or the vector.

[0040] In this invention, the immune effector cells include T cells.

[0041] In this invention, the engineered immune effector cells can transiently express MSLN CAR protein and specifically recognize and kill tumor cells expressing MSLN.

[0042] The present invention also provides the use of the mRNA or the engineered immune effector cells in the preparation of drugs for treating solid tumors expressing mesothelin.

[0043] In this invention, the solid tumors include, but are not limited to, mesothelioma, pancreatic cancer, ovarian cancer, non-small cell lung cancer, squamous cell carcinoma, adenocarcinoma, triple-negative breast cancer, and thymic carcinoma.

[0044] The present invention also provides a nucleic acid drug for treating solid tumors expressing mesothelin, comprising a lipid delivery carrier and the mRNA.

[0045] In this invention, the lipid delivery carrier is preferably any one or more of ionizable lipids, neutral lipids, cholesterol, and long-circulating lipids modified with MAL.

[0046] In this invention, the ionizable lipid is preferably SM-102; the neutral lipid is preferably DSPC; and the long-circulating lipid modified with MAL is preferably DMG-PEG-maL.

[0047] In this invention, the lipid delivery carrier is preferably composed of ionizable lipids, neutral lipids, cholesterol, and MAL-modified long-circulating lipids in a molar ratio of 50:10:(38-39):1.5. The molar ratio is further preferably 50:10:(38.2-38.8):1.5, and even more preferably 50:10:38.5:1.5.

[0048] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1

[0050] A mesothelin chimeric antigen receptor, wherein the MSLN CAR mRNA is composed of a 5'UTR, an MSLN-CAR coding region, a 3'UTR, and a polyA tail. The nucleic acid sequence of the 5'UTR is shown in SEQ ID NO.11; The nucleic acid sequence of the MSLN-CAR coding region is shown in SEQ ID NO.12; The nucleic acid sequence of the 3'UTR is shown in SEQ ID NO.13; The nucleic acid sequence of the polyA tail is shown in SEQ ID NO.14.

[0051] Example 2

[0052] Preparation of lipid nanoparticles loaded with MSLN CAR mRNA.

[0053] Ionizable lipids SM-102 (5.92 mg), DMG-PEG-mAL (0.68 mg), DSPC (1.32 mg), and cholesterol (2.48 mg) were dissolved in anhydrous ethanol and stirred until a clear solution A was formed. MSLN CAR mRNA (novel) (prepared in Example 1 above) was dissolved in 20 mM Tris / sodium acetate buffer at pH 4.0 to form solution B. Solution A and solution B were mixed using a microfluidic mixer to obtain LNPs. Subsequently, the LNPs were ultrafiltered in a 100 kDa ultrafiltration centrifuge tube, and the buffer system was replaced with Tris-HCl (pH 7.0) containing 8% sucrose. After ultrafiltration and centrifugation, the LNPs were passed through a 0.22 μm filter to prepare MSLN CAR mRNA@LNP (novel).

[0054] Meanwhile, the MSLN CAR mRNA disclosed in patent application number 202411973281.9 was set as the comparative group. Using the same preparation process as described above, only the MSLN CAR mRNA (new) was replaced with MSLN CAR mRNA to prepare MSLNCAR mRNA@LNP.

[0055] Example 3

[0056] Characterization comparison of MSLN CAR mRNA @LNP.

[0057] The liposome nanoparticles loaded with MSLN CAR mRNA were dynamically characterized by light scattering using a PSS Nicomp Z3000 instrument. The hydrodynamic diameter, concentration, and zeta potential of the nanoparticles were detected.

[0058] Experimental Procedure: MSLN CAR mRNA @LNP (new) nanoparticles and MSLN CAR mRNA LNP were suspended in PBS buffer, respectively. Unloaded nanoparticles (Blank LNP, prepared in the same manner as in Example 2, but without RNA loading) were used as a control. These were added to the sample cell, which was then placed on the Zetasizer instrument's sample stage, ensuring the sample completely covered the measurement optical path. The required measurement parameters were selected, including the hydrodynamic diameter (e.g., [missing information]). Figure 1 ), Zeta potential (e.g.) Figure 2 The instrument automatically measures and saves the data for subsequent analysis.

[0059] The mRNA content in the lipid nanoparticles prepared in Example 2 was detected using the Quant-it™ RiboGreen RNA assay kit, and the encapsulation efficiency was calculated. 6 μL of the RNA standard provided in the kit was added to 294 μL of TE buffer to prepare a 2 μg / mL RNA stock solution. The solutions were diluted with TE buffer to 0 / 10 / 25 / 50 / 100 / 250 / 500 / 750 / 1000 ng / mL, and 100 μL of each solution was added to a 96-well plate. 1 μL of Ribogreen dye was diluted with TE buffer to 100 μL in each well and added to the sample. The plates were incubated at room temperature for 5 min. Using a microplate reader, the excitation wavelength was set to 485 nm and the emission wavelength to 528 nm. Detection was performed, and the data were recorded. A standard curve of fluorescence signal intensity versus mRNA concentration and a linear regression equation (F = 7036.3C + 181711, R0) were plotted. 2 =0.9976), such as Figure 3 .

[0060] 50 μL of the lipid nanoparticles prepared in Example 2 were mixed with 50 μL of TE buffer and added to a 96-well plate. 1 μL of Ribogreen dye was diluted to 100 μL with TE buffer in each well and added to the sample. The plate was incubated at room temperature for 5 min. The concentration of free RNA (Cfree) was measured using a microplate reader. 50 μL of the nanoparticles provided in Example 2 were mixed with 50 μL of TE-Triton 100 (1xTE buffer: Triton = 49:1) and added to a 96-well plate. 1 μL of Ribogreen dye was diluted to 100 μL with TE buffer in each well and added to the sample. The plate was incubated at room temperature for 5 min. The total RNA concentration (Ctotal) was measured using a microplate reader.

[0061] MSLN CAR mRNA@LNP: Encapsulated RNA concentration (C_encapsulated) = C_total - C_free = 451.23 - 28.96 = 422.27 ng / μL

[0062] Encapsulation efficiency (%) = C_encapsulation / C_total 100% = 93.58%

[0063] MSLN CAR mRNA@LNP (new): Encapsulated RNA concentration (C_encapsulated) = C_total - C_free = 449.28 - 21.61 = 427.67 ng / μL

[0064] Encapsulation efficiency (%) = C_encapsulation / C_total 100% = 95.19%

[0065] In summary, it can be seen that after preparing LNPs using MSLN CAR mRNA (new), the particle size, potential, and encapsulation efficiency were consistent with those of the control mRNA, and MSLN CAR mRNA (new) did not affect the physical properties of LNPs.

[0066] Example 4

[0067] Transfection efficiency of MSLN CAR mRNA loaded with LNP was studied.

[0068] Mouse T cell extraction: Mouse T cells were isolated from wild-type male mice aged 10-14 weeks (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). The T cells were activated using CD3 / CD28 Dynabeads (Gibco 11453D) and expanded using 100 units / mL of recombinant mouse interleukin-2 (R&D system 402-ML). The T cells were cultured in RPMI 1640 (Gibco 6124654) containing 10% fetal bovine serum (Gibco B210805RP), 4 mM glutamine (Invitrogen 25030081), penicillin / streptomycin (Invitrogen 15140122), 1 mM sodium pyruvate (Invitrogen 11360079), and 50 μM mercaptoethanol (Gibco 21985023).

[0069] Mouse T cells were incubated with lipid nanoparticles prepared in Example 2. The treatment groups included: mouse T cells + LNP (liposomes without nucleic acid loading); mouse T cells + MSLN CAR mRNA@LNP; and mouse T cells + MSLN CAR mRNA@LNP (novel).

[0070] Flow cytometry was used to detect the percentage of T cells transfected with MSLN CAR. The isolated T cells were washed with phosphate-buffered saline (PBS). The cell pellet was resuspended in an appropriate amount of PBS and centrifuged (300-500g, 10 min), discarding the supernatant. The washing step was repeated 2-3 times to remove impurities, cell debris, and residual culture medium components. The washed T cells were counted using an automated cell counter. The cell concentration was adjusted to 1×10⁻⁶ cells according to the flow cytometry requirements. 5 ~1×10 6Cells / mL range. Cells were digested with 0.25% trypsin without EDTA, and after digestion, they were collected and centrifuged at 1500 rpm for 5 min. The supernatant was discarded, and the cells were collected. Cells were resuspended twice in pre-chilled PBS, centrifuged at 1500 rpm for 5 min, and washed. 5 μL of His-tagged recombinant MSLN was added, mixed well, and incubated at room temperature for 15 min in the dark. 10 μL of Rabbit-anti-His-PE was added, mixed well, and incubated at room temperature for 15 min in the dark. Flow cytometry analysis was then performed (e.g., ...). Figure 4 ).

[0071] By calculating the results for His+ and CD8+, the proportion of His+ and CD8+ T cells in the total CD8+ T cells was further calculated.

[0072] The results show that, Figure 5 LNPs carrying MSLN CARs can significantly improve the transfection efficiency of T cells, while the transfection efficiency of MSLN CAR mRNA@LNP (new) is significantly higher than that of MSLN CAR mRNA@LNP.

[0073] Example 5

[0074] The killing function of T cells incubated with LNP-mRNA in vitro against mouse gastric cancer cells (MFC cells, purchased from the ATCC cell bank).

[0075] The T cells treated in Example 4 (denoted as LNP_T, LNP-mRNA-CAR_T, and LNP-mRNA(nov)-CAR_T, respectively) were co-cultured with mouse gastric cancer MFC cells for 48 hours. Cell proliferation in each group was detected using CCK8 assay, and apoptosis levels in each group of MFC cells were detected using flow cytometry combined with Annexin V-FITC / PI double staining kit.

[0076] CCK8 proliferation results showed ( Figure 6 LNP-mRNA-treated mouse T cells showed significant inhibitory effects on MFC cells, and the inhibitory effect of novel LNP-mRNA was significantly higher than that of the drug-treated LNP-mRNA. Flow cytometry apoptosis results showed... Figure 7 LNP-mRNA treatment significantly increased the apoptosis level of MFC cells in mouse T cells. Among them, LNP-mRNA (new) increased the killing activity of T cells against MFC cells by more than 10% compared with LNP-mRNA treatment, showing a significantly better effect.

[0077] In summary, the present invention provides a novel, highly efficient, and safe chimeric antigen receptor mRNA targeting mesothelin, which efficiently transfects T cells via a non-viral method, achieving transient expression of the CAR protein and enhancing the safety of CAR-T cells. Simultaneously, it overcomes the limitations of existing CAR-T cell therapies, particularly for solid tumor treatment, such as high preparation costs, complex manufacturing processes, viral integration risks, and potential serious side effects, thus offering a more controllable, scalable, and safer tumor immunotherapy approach. The MSLN CAR mRNA@LNP(novel) prepared in this invention exhibits high transfection efficiency for MSLNs; and the LNP-mRNA(novel)-CAR_T shows stronger killing activity against MFC cells, resulting in better anti-tumor efficacy in the solid tumor microenvironment.

[0078] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A mesothelin chimeric antigen receptor, characterized in that, The amino acid sequence of the mesothelin chimeric antigen receptor is shown in SEQ ID NO.

1.

2. The mesothelin chimeric antigen receptor according to claim 1, characterized in that, The protein structure of the mesothelin chimeric antigen receptor includes a signal peptide, an antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular signal domain. The sequence of the signal peptide is shown in SEQ ID NO.2; The antigen-binding domain includes a heavy chain variable region as shown in SEQ ID NO.3 and a light chain variable region as shown in SEQ ID NO.4; The sequence of the hinge region is shown in SEQ ID NO.6; The sequence of the transmembrane domain is shown in SEQ ID NO.7; The sequence of the intracellular signaling domain is shown in SEQ ID NO.

8.

3. The mesothelin chimeric antigen receptor according to claim 2, characterized in that, The heavy chain variable region and the light chain variable region are linked by a linker peptide, the sequence of which is shown in SEQ ID NO.

5.

4. An mRNA encoding the mesothelin chimeric antigen receptor of claim 1, characterized in that, The structure of the mRNA includes a 5'UTR, an MSLN-CAR coding region, a 3'UTR, and a polyA tail; The nucleic acid sequence of the MSLN-CAR coding region is shown in SEQ ID NO.

12.

5. The mRNA according to claim 4, characterized in that, The nucleic acid sequence of the 5'UTR is shown in SEQ ID NO.11; The nucleic acid sequence of the 3'UTR is shown in SEQ ID NO.13; The nucleic acid sequence of the polyA tail is shown in SEQ ID NO.

14.

6. A carrier, characterized in that, The vector contains the mRNA as described in claim 4 or 5.

7. An engineered immune effector cell, characterized in that, It comprises the mesothelin chimeric antigen receptor of claim 1, the mRNA of claim 4, or the vector of claim 6.

8. The immune effector cell according to claim 7, characterized in that, The immune effector cells include T cells.

9. The use of the mRNA of claim 4 or the engineered immune effector cells of claim 7 in the preparation of a drug for treating solid tumors expressing mesothelin.

10. A nucleic acid drug for treating solid tumors expressing mesothelin, characterized in that, Includes a lipid delivery carrier and the mRNA as described in claim 4 or 5.

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