Mesenchymal stem cell capable of enhancing tumor killing ability and application of mesenchymal stem cell

Abstract

The invention provides a mesenchymal stem cell capable of enhancing tumor killing ability and an application of the mesenchymal stem cell. The mesenchymal stem cell expresses a chimeric antigen receptor targeting CD276 or contains nucleic acid for coding the chimeric antigen receptor targeting CD276. The CAR276-iMSCs prepared by the preparation method disclosed by the invention have better in-vivo durability, specifically enhanced tumor inhibition capability and solid tumor targeting capability, and specifically improved safety and stability.

Description

Technical Field

[0001] The present invention relates to the technical field of mesenchymal stem cells, and in particular to mesenchymal stem cells with enhanced tumor-killing ability and applications thereof. Background Art

[0002] In recent years, mesenchymal stem cells (MSCs) have been increasingly used in the treatment of tumors and autoimmune diseases. MSCs are adult stem cells with excellent proliferation and multidirectional differentiation potential, found in various tissues such as bone marrow, fat, and umbilical cord. However, due to irreconcilable differences in properties between MSCs from different sources, such as genetic background and epigenetic modifications, batch-to-batch cell variability and stability are significant. Furthermore, MSCs have a low in vivo survival rate. Even with various measures, their survival rate remains limited in vivo, limiting their transplantation efficacy and clinical application.

[0003] In addition, those skilled in the art are aware that CAR276 (which is a chimeric antigen receptor capable of targeting the CD276 target) is an artificial receptor that can specifically recognize the CD276 antigen and is used to target cells expressing CD276 molecules, such as malignant tumor cells, such as lung cancer, etc. A large number of related technologies for CAR-T cells or CAR-NK cells are disclosed in the prior art, but there are significant differences between CAR-MSC and CAR-T and CAR-NK cells. CAR-T cells often directly lyse tumor cells during treatment. Due to the rapid killing of tumor cells, some serious adverse reactions may occur, such as cytokine storms, the production of a large number of inflammatory factors (including IL-6, etc.), damage to the nervous system, neurotoxicity, and graft-versus-host disease. More seriously, there are occasional deaths caused by CAR-T cell therapy. While CAR-NK cells are much safer than CAR-T cells, generally not causing graft-versus-host disease or cytokine storms, and only secreting a small amount of IFN-γ, they have a shorter lifespan and lower potential unknown risks. Furthermore, CAR-NK cells do not require antigen presentation, can take effect more quickly, and have a broader spectrum of anti-tumor effects. However, the current problem is that CAR-T cells and CAR-NK cells have insufficient infiltration into solid tumors, making it difficult to penetrate the tumor microenvironment, and their killing ability in the tumor microenvironment is significantly reduced, resulting in a need for improvement in their effectiveness in treating solid tumors.

[0004] In response to the problems with CAR-T cells and CAR-NK cells in tumor treatment, mesenchymal stem cells have received increasing attention. MSCs inherently possess certain tumor tropism and tumor infiltration capabilities, making it easier for them to break through the barriers of the tumor microenvironment and effectively treat solid tumors. However, it is undeniable that this targeting ability is due to the inherent characteristics of MSCs themselves and is not strong. In addition, studies have shown that after gene editing of MSC cells, the cell genome is damaged to varying degrees. Compared with wild-type cells, the edited MSC cells have lower survival and persistence in the body.

[0005] Therefore, how to improve the survival rate and tumor suppression persistence of MSCs in vivo and enhance their tumor targeting to further improve the anti-tumor ability of MSC cells is one of the problems that technicians in this field need to solve. Summary of the Invention

[0006] In response to the shortcomings of the prior art, the present invention provides a mesenchymal stem cell with enhanced tumor-killing ability and its application, which solves the problems of low persistence or survival of MSCs in the body and low tumor targeting ability in the prior art.

[0007] In a first aspect, the present invention provides a mesenchymal stem cell, wherein the mesenchymal stem cell expresses a chimeric antigen receptor targeting CD276 or contains a nucleic acid encoding the chimeric antigen receptor targeting CD276.

[0008] In one embodiment of the present invention, compared with wild-type mesenchymal stem cells, the mesenchymal stem cells have the following characteristics:

[0009] (1) Improved survival rate;

[0010] (2) improved persistence of tumor suppression;

[0011] (3) enhanced tumor suppression ability;

[0012] (4) Improved tumor targeting.

[0013] In one embodiment of the present invention, the mesenchymal stem cells are derived from adult cells or stem cells.

[0014] In a preferred embodiment of the present invention, the mesenchymal stem cells are derived from pluripotent stem cells. Preferably, the pluripotent stem cells are selected from induced pluripotent stem cells.

[0015] In one embodiment of the present invention, the mesenchymal stem cells are derived from bone marrow, fat, muscle, heart, umbilical cord blood, or umbilical cord.

[0016] In one embodiment of the present invention, the chimeric antigen receptor targeting CD276 comprises: (a) an extracellular target portion; (b) a transmembrane domain; and (c) an intracellular region.

[0017] In one embodiment of the present invention, the extracellular targeting portion is used to recognize a target molecule on the surface of a tumor cell, wherein the target molecule is selected from CD276.

[0018] In one embodiment of the present invention, the extracellular target moiety comprises an antibody that specifically binds to the CD276 antigen.

[0019] In one embodiment of the present invention, the extracellular target portion includes any antibody in the prior art that can specifically bind to the CD276 antigen, such as the literature, H.Du, etc., Antitumor Responses in the Absence of Toxicity in Solid Tumors by Targeting B7-H3 via Chimeric Antigen Receptor TCells, Cancer Cell, 35 (2019) 221-237e228, which reported the use of CAR-T cells targeting CD276 (also known as B7-H3) to treat various tumors.

[0020] In one embodiment of the present invention, the antibody is selected from any one of the following: a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, an Fv, a scFv, a (scFv)2, an Ig NAR, a dsFv, a nanobody, a minibody, a monoclonal antibody, and a bifunctional antibody. In a preferred embodiment of the present invention, the antibody is a scFv fragment derived from an antibody that specifically binds to the CD276 antigen.

[0021] In one embodiment of the present invention, the intracellular region includes an intracellular signaling domain and / or a costimulatory domain. The intracellular signaling domain is a cytoplasmic functional domain derived from the T cell receptor CD3ζ or selected from any one or more of the following cytoplasmic functional domains: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b and CD66d. The costimulatory domain is selected from any one or more of the following cytoplasmic functional domains: 2B4, HVEM, ICOS, LAG3, DAP10, DAP12, CD27, CD28, CD137, CD134, CD30, CD40, CD278, glucocorticoid-induced tumor necrosis factor receptor (GITR), lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C and B7-H3.

[0022] In one embodiment of the present invention, the intracellular region includes the cytoplasmic functional region derived from CD137 and the cytoplasmic functional region of CD3ζ.

[0023] In one embodiment of the present invention, the transmembrane domain is selected from any one or several of the following transmembrane regions: α or β chain of T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, and CD154.

[0024] In one embodiment of the present invention, the chimeric antigen receptor targeting CD276 further comprises (d) a hinge region, wherein the hinge region is used to connect the extracellular target portion and the transmembrane domain. Preferably, the hinge region is selected from the hinge region comprising CD8α.

[0025] In one embodiment of the present invention, the amino acid sequence of the chimeric antigen receptor targeting CD276 is as shown in SEQ ID NO:6, or an amino acid sequence that is at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.9% identical to the sequence shown in SEQ ID NO:6.

[0026] In one embodiment of the present invention, the nucleic acid is included in an expression vector for expressing the chimeric antigen receptor targeting CD276.

[0027] In one embodiment of the present invention, the expression vector is a plasmid or a viral vector. In a specific embodiment of the present invention, the viral vector comprises a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector. Preferably, the lentiviral vector is a lentiviral vector.

[0028] In one embodiment of the present invention, the expression vector further comprises a nucleic acid sequence encoding a tag protein. Preferably, the tag protein comprises any one of HIS, Flag, GST, Myc, eGFP, eCFP, eYFP, and mCherryeGFP.

[0029] In one embodiment of the present invention, the nucleic acid comprises a nucleic acid sequence encoding the chimeric antigen receptor targeting CD276.

[0030] In one embodiment of the present invention, the nucleic acid sequence is as shown in SEQ ID NO: 1, or a nucleic acid sequence that is at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.9% identical to the sequence shown in SEQ ID NO: 1.

[0031] In a second aspect, the present invention provides a composition comprising the above-mentioned mesenchymal stem cells.

[0032] In one embodiment of the present invention, the composition further comprises a second therapeutic agent, which comprises a chemotherapeutic drug, an immunotherapeutic drug, a hormone drug and / or a biological therapeutic drug.

[0033] In one embodiment of the present invention, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0034] In one embodiment of the present invention, the composition is administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, topically, or by direct injection or infusion.

[0035] In a third aspect, the present invention provides use of the above-mentioned mesenchymal stem cells or the above-mentioned composition in the preparation of a drug for treating anti-tumor.

[0036] In one embodiment of the present invention, the tumor is selected from small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, brain cancer, testicular cancer, bile duct cancer, gallbladder cancer, gastric cancer, melanoma, leukemia and various types of head and neck cancer.

[0037] In one embodiment of the present invention, the tumor is selected from tumors that highly express CD276 molecules, preferably lung cancer.

[0038] In a fourth aspect, the present invention provides a method for constructing mesenchymal stem cells, comprising the following steps:

[0039] S1: Preparation and culture of mesenchymal stem cells;

[0040] S2: transferring a nucleic acid encoding a chimeric antigen receptor targeting CD276 into the mesenchymal stem cells.

[0041] In one embodiment of the present invention, step S1 includes inducing the differentiation of iPSCs into mesenchymal stem cells in vitro and expanding the culture.

[0042] In one embodiment of the present invention, step S2 comprises cloning the nucleic acid encoding the chimeric antigen receptor targeting CD276 into an expression vector, and then transferring the expression vector into mesenchymal stem cells.

[0043] In one embodiment of the present invention, the transfer comprises transfection or transduction.

[0044] In one embodiment of the present invention, the nucleic acid encoding the chimeric antigen receptor targeting CD276 is site-specifically integrated into the genome of the mesenchymal stem cells.

[0045] In one embodiment of the present invention, the nucleic acid encoding the chimeric antigen receptor targeting CD276 is site-specifically integrated into the gene locus or rDNA region of the mesenchymal stem cells.

[0046] In one embodiment of the present invention, the locus is selected from any one of the AAVS1 locus, the B2M locus, the CCR5 locus, and the CIITA locus.

[0047] In one embodiment of the present invention, the engineered mesenchymal stem cells are analyzed by functional assays, cytotoxicity assays and / or in vivo activity.

[0048] In one embodiment of the present invention, the engineered mesenchymal stem cells are analyzed by flow cytometry, mass cytometry, DNA sequencing, or a combination thereof.

[0049] In a fifth aspect, the present invention provides the use of the chimeric antigen receptor targeting CD276 in the preparation of any one or more of the following products:

[0050] (1') Improve the survival rate of mesenchymal stem cells; (2') Improve the tumor suppression persistence of mesenchymal stem cells; (3') Improve the tumor targeting of mesenchymal stem cells; (4') Improve the tumor suppression ability of mesenchymal stem cells.

[0051] In a sixth aspect, the present invention provides a method for improving the survival rate of mesenchymal stem cells, comprising the following steps:

[0052] S1': Preparation and culture of mesenchymal stem cells;

[0053] S2': transferring a nucleic acid encoding a chimeric antigen receptor targeting CD276 into the mesenchymal stem cells.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] 1. The present invention discovered for the first time that the chimeric antigen receptor CAR276 is used to modify MSC cells, which improves the in vivo survival rate and tumor suppression persistence of MSC cells, and improves the persistence of mesenchymal stem cells in the body, enabling them to exert a continuous and long-term tumor suppression effect, thereby greatly improving the transplantation effect and clinical application of MSC cells.

[0056] 2. The CAR276-iMSCs cells prepared by the present invention have specifically enhanced tumor suppression ability and targeting ability for solid tumors. It has been verified that CAR276-iMSCs cells have the best tumor suppression ability and targeting ability both in vitro and in vivo. In particular, after the chimeric antigen receptor targeting CD276 molecules is used to modify MSCs in the present invention, its in vivo persistence is better, which further improves the sustained inhibitory effect of MSC cells on tumors, promotes the further application of MSC cells in the field of tumor cell therapy technology, and provides a new MSC solution for tumor treatment (especially cancers with high expression of CD276 molecules, such as lung cancer).

[0057] 3. The present invention uses cells at the iPSC stage as seed cells for producing MSC cells, thereby improving the stability and uniformity of MSC cells.

[0058] 4. The CAR276-iMSCs cells prepared by the present invention have specifically improved safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Schematic diagram of the core component structure of the pLVCAR276 lentiviral vector.

[0060] Figure 2 Part of the Sanger sequencing map.

[0061] Figure 3 Bright field images and GFP fluorescence images of the cell morphology of iMSCs and CAR276-iMSCs.

[0062] Figure 4 Representative flow cytometry results for CAR276 and GFP expression in iMSCs and CAR276-iMSCs. The horizontal axis (R2-A) represents CAR276 expression, and the vertical axis (B1-A) represents eGFP expression. The left column of flow cytometry plots represents the unstained control group, and the right column of flow cytometry plots represents the stained experimental group.

[0063] Figure 5 Representative flow cytometry images of the cell interaction experiments between iMSCs and CAR276-iMSCs and A549 and MCF7 cells.

[0064] Figure 6 Representative flow cytometry images of apoptosis in solid tumor cells after co-culture of iMSCs and CAR276-iMSCs with A549 and MCF7.

[0065] Figure 7 Flowchart of animal experiments.

[0066] Figure 8 This is the volume growth curve of A549 subcutaneous tumor.

[0067] Figure 9 This is an immunofluorescence detection image of A549 subcutaneous tumor sections.

[0068] Figure 10 Statistical diagram of DiR 750 signal in vivo imaging of mice.

[0069] Figure 11 The figure shows the statistical graph of mouse body weight. DETAILED DESCRIPTION

[0070] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0071] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0072] Technical terms

[0073] iPSCs, iPSC or ips (Induced pluripotent stem cells), refers to induced pluripotent stem cells and can be used interchangeably in this article.

[0074] iMSCs or iMSCs refer to MSCs formed by induced differentiation of pluripotent stem cells (such as induced pluripotent stem cells) as seed cells.

[0075] Mesenchymal stem cells, also known as MSCs, can be differentiated from progenitor cells in fat, bone marrow, umbilical cord blood, or dental pulp, or induced to differentiate from pluripotent stem cells.

[0076] In the cell differentiation process of the specific embodiment of the present invention, mesenchymal progenitor cells refer to iMSC cells, and the two can be used interchangeably in the specific embodiment of the present invention.

[0077] An antibody that specifically binds to the CD276 antigen refers to an antibody that specifically binds to the CD276 antigen and immunologically recognizes the CD276 antigen, so that the binding of CAR to the antigen triggers an immune response.

[0078] Chimeric Antigen Receptor (CAR): A chimeric molecule comprising an antigen binding portion (e.g., a single domain antibody or scFv) and a signaling domain (e.g., a signaling domain from a T cell receptor (e.g., CD3ζ)). Typically, CAR consists of an antigen binding portion (also known as an extracellular target portion), a transmembrane domain, and an intracellular domain (also known as an intracellular region). The intracellular domain typically includes a signaling chain having an immunoreceptor tyrosine activation motif (ITAM), such as CD3ζ. In some cases, the intracellular domain also includes an intracellular portion of at least one costimulatory domain, such as CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27, and / or DAP10, etc.

[0079] CAR276 is a chimeric antigen receptor that can target the CD276 target. It can specifically recognize the CD276 antigen and is used to target cells expressing the CD276 molecule.

[0080] Induced pluripotent stem cells (iPSCs) are reprogrammed into pluripotent stem cells by introducing transcription factors into terminally differentiated somatic cells, thereby restoring them to a totipotent state. MSCs derived from iPSCs are considered an ideal method for producing MSCs, as they exhibit excellent consistency, minimal variability between batches, and excellent stability.

[0081] In the specific implementation manner of the present invention, certain quantities are represented by nen, where n is a specific number, for example, 1e4 represents 10,000.

[0082] Overexpression in the present invention means that compared with wild-type cells (ie, unmodified cells), the modified cells show a higher expression level.

[0083] Example 1 Construction of CAR276-iMSC cells

[0084] 1. Preparation of iMSCs from iPSCs

[0085] In view of the increasing use of mesenchymal stem cells (MSCs) in the field of regenerative medicine and cell therapy, this embodiment purposefully induced the differentiation of iPSCs (stem cell bank of the Chinese Academy of Sciences, DYR0100) in vitro to produce iMSCs. The differentiation process can be referred to the prior art literature (Z.Wang, H.Chen, P.Wang, M.Zhou, G.Li, Z.Hu, Q.Hu, J.Zhao, X.Liu, L.Wu, D.Liang, Site-Specific Integration of TRAIL in iPSC-DerivedMesenchymal Stem Cells for Targeted Cancer Therapy, Stem Cells TranslationalMedicine, 11 (2022) 297-309). In this embodiment, iPSCs were differentiated into mesenchymal stem cells and expanded into culture using the STEMdiffTMMesenchymal Progenitor Kit (STEM CELL, Cat. No. 05240).

[0086] 2. Preparation of CAR276-iMSC Cells

[0087] In this example, iMSCs overexpressing CAR276 (CAR276-iMSCs) were established by direct transduction of iMSCs with lentivirus. The specific preparation method of CAR276-iMSCs is as follows:

[0088] 1) Prepare CAR276 lentivirus (i.e., LVCAR276).

[0089] First, the nucleotide sequence of CAR276 (SEQ ID NO.1) was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and cloned into a lentiviral vector plasmid using molecular cloning methods. The resulting vector plasmid was designated pLVCAR276 (its nucleotide sequence is shown in SEQ ID NO.2). The core component structure of the pLVCAR276 lentiviral vector plasmid is shown in FIG. Figure 1 shown.

[0090] The vector was verified by Sanger sequencing (eg Figure 2). CAR276 is followed by a T2A-eGFP sequence. The T2A sequence (its nucleotide sequence is shown in SEQ ID NO.3) is responsible for encoding a 2A connecting peptide (its amino acid sequence is shown in SEQ ID NO.4) to ensure the expression of the subsequent eGFP sequence; the eGFP sequence is responsible for encoding a GFP protein (its nucleotide sequence is shown in SEQ ID NO.5) to indicate the expression of the CAR276 protein (its amino acid sequence is shown in SEQ ID NO.6).

[0091] 293T cells were then transfected with the pLVCAR276 to obtain the CAR276 lentivirus (i.e., LVCAR276). In this example, the CAR276 lentivirus was prepared using previously disclosed methods, steps, and processes. For specific steps, reference can be made to paragraph

[0089] of the Detailed Instructions section of the prior art document CN109735558A (for the production of LV particles). The produced LV vector is designated LVCAR276.

[0092] The obtained LVCAR276 was used to transduce iMSCs cells. Specifically, 1.5e6 iMSCs were seeded into 10 cm dish, LVCAR276 was added after 24 hours, and polybrene 8 μg / mL was added and incubated for 6 hours. Then, the medium was replaced with a new one and cultured as normal. After 3 days of culture, the expression of GFP was observed under a microscope (as shown in Figure 2). Figure 3 Considering that the eGFP sequence and the CAR276 sequence are connected by a T2A sequence and are the same peptide chain during protein translation, the eGFP signal indicates the expression of the CAR276 molecule.

[0093] 3. Identification of CAR276-iMSCs

[0094] Flow cytometry was used to detect the expression of CAR276. We used recombinant fluorescently labeled recombinant B7-H3 protein (Recombinant Human B7-H3 Fc Chimera Alexa 647Protein, R&D Systems, AFR1027) to detect CAR276 molecules. Figure 4 As shown in the figure, CAR276-iMSCs have obvious signals and coexist with eGFP signals, while iMSCs have no signals, which further indicates that CAR276 molecules are successfully expressed on CAR276-iMSCs.

[0095] Example 2 CAR276-iMSCs enhance tumor adhesion and tumor cell apoptosis in vitro

[0096] 1. The adhesion ability of CAR276-iMSCs to tumor cells was assessed by in vitro co-culture. Tumor cell lines: MCF7 breast cancer cell line and A549 non-small cell lung cancer cell line were purchased from Wuhan Punosai Life Science Co., Ltd., and A549-Luc2 non-small cell lung cancer cell line was purchased from Zhejiang Meisen Cell Technology Co., Ltd. Analysis confirmed that all of these cell lines expressed the CD276 molecule.

[0097] Specifically, in order to detect whether CAR276 can enhance the targeting of iMSCs to CD276-positive solid tumor cells in vitro, a fluorescently labeled cell interaction experiment was performed using flow cytometry in this example. Tumor cells were co-cultured with iMSCs cells or CAR276-iMSCs cells. Before co-culture, tumor cells were labeled with CellTrace FarRed (Invitrogen, C34564), and iMSCs cells and CAR276-iMSCs cells were labeled with CellTrace Violet (Invitrogen, C34557). The labeling method was carried out according to the instructions. The specific steps are as follows:

[0098] 1) First, iMSCs cells or CAR276-iMSCs cells were cultured with TrypLE TM Select (Gibco, 12563-011) was digested into single cells, inoculated into 12-well plates at a number of 150,000 / mL / well, and the cells were shaken using the cross-shaking method.

[0099] 2) After 12 hours, discard the MSC culture medium in the 12-well plate filled with iMSCs. Add TrypLE TM Solid tumor cells MCF7 or A549 cells digested with Select (Gibco, 12563-011) were inoculated onto the plated iMSCs at a number of 100,000 / mL / well, and the cells were shaken evenly using the cross-shaking method.

[0100] 3) Incubate in a cell culture incubator at 37°C and 5% CO2 for 90 minutes.

[0101] 4) Discard the culture medium in the 12-well plate and wash once with 1× DPBS. Add 1× DPBS to the 12-well plate again and place it on a horizontal shaker at room temperature at 130 rpm / min for 3 minutes to remove weakly adherent solid tumor cells.

[0102] 5) Using TrypLE TM Select (Gibco, 12563-011) to digest and collect cells. Digest for 15 seconds at room temperature. Ensure consistent pipetting force and frequency when collecting cells.

[0103] 6) Centrifuge at 350 g for 5 min at room temperature, discard the supernatant, and gently resuspend the cells in 200 μL of Cell Staining Buffer (BD, 554657) for flow cytometry analysis.

[0104] Representative flow cytometry results are shown in Figure 2. Figure 5 As shown. The CellTrace Violet / Far Red double-positive cell population was regarded as iMSCs / solid tumor cell aggregates, and its percentage was calculated. The results showed that the degree of interaction between CAR276-iMSCs and non-small cell lung cancer A549 cells was significantly higher than that of iMSCs. Figure 5 The interaction degree between CAR276-iMSCs and breast cancer cells MCF7 was also significantly higher than that of iMSCs. Figure 5 The results showed that CAR276 enhanced the targeting of iMSCs to CD276-positive solid tumor cells A549 and MCF7.

[0105] 2. Ability of CAR276-iMSCs to induce tumor cell apoptosis in vitro

[0106] iMSCs and CAR276-iMSCs were co-cultured with tumor cells A549 and MCF7 at a ratio of 5:1 for 72 hours to evaluate whether CAR276 can enhance the pro-apoptotic effect of iMSCs on CD276-positive solid tumor cells. Annexin V-APC (Biolegend, Cat. No. 640920) and Zombie NIR (Biolegend, Cat. No. 423105) were used to detect tumor cell apoptosis. In the process of tumor cell death induced by iMSC cells, its cell membrane will change. In the early stage, phosphatidylserine (PS) inside the cell membrane will be exposed and recognized by Annexin V antibodies; in the later stage, the integrity of the cell membrane will be destroyed, and Zombie NIR dye will enter the cell more. Therefore, Annexin V-positive cells are considered to be apoptotic cells, and cells that are Annexin V-positive and Zombie NIR-positive (represented as Annexin V+ / NIR+ in the figure) are late apoptotic cells. The experimental results are as shown in the figure. Figure 6 As shown in Figure 2, CAR276-iMSCs significantly induced more A549 cells to enter late apoptosis than iMSCs (11.7% and 3.92%, respectively). Figure 6The total apoptosis is significantly higher as shown in the middle bar graph. A similar phenomenon was observed for MCF7 cells, that is, CAR276-iMSCs induced more MCF7 cells to enter late apoptosis than iMSCs (15.8% and 8.42%, respectively). Figure 6 The total apoptosis rate, as shown in the middle bar graph, was also significantly higher. This indicates that CAR276-iMSCs significantly induced tumor cell apoptosis compared to iMSCs, suggesting that CAR276 can enhance the apoptotic effect of iMSCs on CD276-positive solid tumor cells. This result was unexpected, considering that iMSCs themselves are not tumor-killing cells.

[0107] Example 3 Enhanced tumor-targeted infiltration and anti-tumor effects of CAR276-iMSCs in vivo

[0108] 1. Establishment of CD276-positive human mouse xenograft tumor model

[0109] Specifically, this example established a subcutaneous tumor model of nude mice (BALB / c nude, purchased from Slack Genda) with human lung cancer cell line A549 cells. Figure 7 As shown: On day 0, 5 million A549-Luc2 cells were subcutaneously injected into the axilla of mice. On day 16, iMSCs were first stained with DiR 750, and then 2 million iMSCs were injected into the mice via the tail vein. This was a single iMSC injection. On day 38, the mice were sacrificed, and tumor tissue and internal organs were collected. Tumor volume, body weight, in vivo fluorescence imaging, and tumor weight were measured during the experiment.

[0110] In the specific operation, 5e6 A549-Luc2 cells (purchased from Zhejiang Meisen Cell Technology Co., Ltd.) were first transplanted into the subcutaneous tissue of nude mice by subcutaneous injection (recorded as Day 0), and then the progress of A549 cell transplants at the injection site was observed. When the tumor volume reached about 100mm 3 At day 16 (in this example, it was day 16), the mice were divided into three groups, one group was injected with 2e6 iMSCs cells for treatment, recorded as the iMSCs treatment group (iMSCs); one group was injected with 2e6 CAR276-iMSCs cells for treatment, recorded as the CAR276-iMSCs treatment group (CAR276-iMSCs); and one group was not injected with iMSCs cells, recorded as the untreated group (Untreated).

[0111] Before injection, iMSCs cells and CAR276-iMSCs cells were stained with the fluorescent dye DiR 750 (IVISenseDiR750, 125964, PerkinElmer), and the specific labeling method was carried out according to the instructions. The maximum excitation wavelength and maximum emission wavelength of DiR 750 are 748nm and 780nm, respectively. In this embodiment, it is used to monitor the content of iMSCs in the body and the migration to the tumor site (i.e., targeting). In this embodiment, iMSCs were injected into mice through the tail vein. The animal experiment flow chart is as follows Figure 7 shown.

[0112] 2. Monitoring of mouse tumor volume

[0113] In this example, the tumor volume of mice was monitored and counted. Figure 8 The mouse tumor volume growth curve shows that the tumor volume in the iMSCs group grew slower than that in the untreated group, and the tumor volume in the CAR276-iMSCs group was significantly smaller than that in both the untreated and iMSCs groups. This suggests that iMSCs themselves have a certain effect in inhibiting tumor progression, and that overexpression of CAR276 can further enhance the tumor inhibitory effect of iMSCs.

[0114] 3. Cell Infiltration Detection

[0115] This example further analyzed the infiltration of iMSCs and CAR276-iMSCs cells in mouse tumor tissues. Figure 9 As shown. Specifically, immunofluorescence detection of tumor tissue sections revealed that no obvious DiR 750 fluorescence signal was found in the tumor tissues of the Untreated group and the iMSCs group; whereas there was a DiR 750 fluorescence signal in the tumor tissue of the CAR276-iMSCs group. This shows that the content of iMSCs cells in the tumor is already very low, while CAR276-iMSCs still has a high content. This means that CAR276 enhances the in vivo tumor targeting and tumor infiltration ability of iMSCs, and this solid tumor targeting of CAR276-iMSCs still exists 3 weeks after one injection (20230315-20230406), and it has enhanced survival persistence in the tumor microenvironment.

[0116] Example 4 CAR276-iMSCs enhance in vivo survival and tumor suppression persistence

[0117] In this example, the PerkinElmer IVIS Lumina III in vivo imaging system was used to perform in vivo imaging of mice at specific time points to monitor and record the DiR 750 fluorescence signal, which reflects the progress of the injected iMSCs cells in the mice. Figure 10 As shown. Among them, the signal value of the Untreated group was at the background level, while both the iMSCs group and the CAR276-iMSCs group had obvious signals, and the signals of iMSCs cells and CAR276-iMSCs gradually decreased over time. Importantly, the experimental results unexpectedly found that the initial level of the signal of CAR276-iMSCs after injection was comparable to that of the iMSCs group, but its signal decreased more slowly, indicating that the survival rate of CAR276-iMSCs was significantly higher than that of iMSCs, and the time lasted for 16 days or even longer, which means that CAR276-iMSCs can exert its efficacy more sustainably in the body, and CAR276-iMSCs has better persistence in tumor suppression in the body.

[0118] Since MSCs can be attacked by the mouse's immune cells in the mouse body, leading to their death, the results show that after adding CAR276, the immune cells in the mouse body are less aggressive towards the gene-edited CAR276-iMSCs than towards wild-type MSCs. CAR276 enhances the in vivo survival rate and tumor suppression persistence of iMSCs.

[0119] Further statistics on the weight of mice showed that there was no significant difference in the weight of mice in the CAR276-iMSCs group compared with the iMSCs group and the Untreated group, and there was no significant difference in the weight of mice in the iMSCs group compared with the Untreated group (e.g. Figure 11 This indicates that the injected iMSCs had no adverse effects on mice, and overexpression of CAR276 did not cause a significant negative effect on the safety of iMSCs.

[0120] In a specific embodiment of the present invention, it was found that CAR276-iMSCs had stronger tumor cell interactions than iMSCs during in vitro co-culture, and CAR276 enhanced the ability of non-tumor killer iMSCs to induce tumor cell apoptosis. Experiments have shown that the presence of CAR276 enhances the anti-tumor potential of iMSCs. In in vivo experiments, CAR276-iMSCs were found to have a stronger tumor inhibitory effect than iMSCs and a stronger ability to infiltrate tumors; and unexpectedly, the effect persisted better in vivo; and like iMSCs, there were no significant side effects. CAR276 directly enhances the anti-tumor potential of iMSCs cells themselves. It has been verified that CAR276-iMSCs cells have the best anti-tumor ability both in vitro and in vivo.

[0121] In summary, iPSC-derived iMSCs are a promising source of MSCs, exhibiting modest tumor-suppressive effects and exhibiting no significant safety issues in mice. Furthermore, iMSCs overexpressing CAR276 exhibit enhanced in vivo persistence, tumor-suppressive capacity, and targeting ability. CAR276-iMSCs have been shown to possess optimal anti-tumor activity both in vitro and in vivo.

[0122] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

[0123] SEQ ID NO: 1 CAR276 nucleotide sequence

[0124] ATGGCCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTTCTGCACGCCGCCAG

[0125] GCCCGGCAGCCAGGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGGTGAAGCCCGGCGC

[0126] CTCCGTGAAGCTGAGCTGCAAGACCAGCGGCTACACCTTCACCAACTACGACATCAAC

[0127] TGGGTGCGCCAGCGGCCCGGCCAGGGCCTGGAGTGGATCGGCTGGATCTTCCCCGGCG

[0128] ACGGCAGCACCCAGTACAACGAGAAGTTCAAGGGCAAGGCCACCCTGACGACCGACA

[0129] CCAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGGAGCGAGGATACAGCCG

[0130] TGTATTTTTGTGCTAGACAGACAACCGCCACTTGGTTTGCCTATTGGGGCCAGGGAACC

[0131] CTCGTGACAGTGTCTAGTGGCGGAGGCGGCTCTGGAGGCGGAGGCAGCGGAGGCGGC

[0132] GGGTCTGAAATTGTGATGACCCAGAGTCCAGCTACTCTGTCTGTGTCTCCTGGAGAAAG

[0133] AGTGACCCTGAGCTGTAGAGCCAGCCAGTCTATTTCTGATTATCTCTATTGGTATCAGC

[0134] AGAAGAGTCATGAATCTCCTAGACTGCTGATTAAGTATGCTAGCCAGTCTATTAGCGG

[0135] AATTCCAGCCCGATTTTCTGGAAGCGGAAGTGGCTCTGAGTTTACACTGACAATTAATT

[0136] CTGTGGAGCCTGAAGATGTGGGAGTGTATTATTGTCAGAATGGCCATAGCTTTCCACTG

[0137] ACATTTGGACAGGGAACAAAGCTGGAACTGAAGAGAACAACAACACCTGCCCCTAGA

[0138] CCACCTACACCTGCCCCAACTATTGCTTCTCAGCCTCTGAGCCTGAGACCTGAAGCCTG

[0139] TAGACCTGCAGCTGGAGGAGCTGTGCATACAAGAGGGCTGGATTTTGCCTGTGATGAT

[0140] ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTAT

[0141] CACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTT

[0142] ATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAG

[0143] AAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCG

[0144] CGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGG

[0145] AGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGA

[0146] GAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGG

[0147] AGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATG

[0148] GCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCA

[0149] GGCCCTGCCCCCTCGC

[0150] SEQ ID NO:2 pLVCAR276 full sequence

[0151] CTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGT

[0152] CTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATC

[0153] TACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATA

[0154] GGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTA

[0155] GATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATA

[0156] ATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTA

[0157] GAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCA

[0158] AACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACT

[0159] CTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGT

[0160] GTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTC

[0161] TGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTG

[0162] GACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGT

[0163] GCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGA

[0164] GCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAG

[0165] CGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTA

[0166] TCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTC

[0167] GTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTG

[0168] GCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGAT

[0169] AACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGC

[0170] GCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCC

[0171] CCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGC

[0172] GGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCT

[0173] TTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCA

[0174] CACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGG

[0175] GAACAAAAGCTGGAGCTGCAAGCTTAATGTAGTCTTATGCAATACTCTTGTAGTCTTGC

[0176] AACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCA

[0177] TGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGG

[0178] GTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATAATTGTATTTAA

[0179] GTGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAG

[0180] CTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCT

[0181] TCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCT

[0182] TTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAA

[0183] GGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCA

[0184] AGAGGCGAGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAG

[0185] AAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGA

[0186] TGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATA

[0187] GTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACAT

[0188] CAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGA

[0189] AGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATA

[0190] GAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGGACAAAACAAAGT

[0191] AAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAG

[0192] GGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGA

[0193] GTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGG

[0194] AATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCG

[0195] TCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGA

[0196] ACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGG

[0197] CATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAG

[0198] CTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAA

[0199] TGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACACGACCTGGATGGAG

[0200] TGGGACAGAGAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGC

[0201] AAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTT

[0202] TGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTTATAAAATTATTCATAATGATA

[0203] GTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTGCTGTACTTTCTATAGTGAATAGAGT

[0204] TAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCC

[0205] GACAGGCCCGAGGAATAGAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCAT

[0206] TCGATTAGTGAACGGATCTCGACGGTATCGGTTAACTTTTAAAAAAAAGGGGGGGATT

[0207] GGGGGGTACAGTGCAGGGAAGAATAGTAGACATAATAGCAACAGACATACAAACT

[0208] AAAGAATTCAAAAAACAAATTCAAAAATTCAAAATTTTTATCGATCACGAGACTAGCC

[0209] TCGAGAAGCTTGATCGATGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCA

[0210] CAGTCCCCGAGAAGTTGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGT

[0211] GGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGG

[0212] TGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGG

[0213] TTTGCCGCCAGAACACAGGTGTCGTGACGCGGATCCGCCACCATGGCCCTGCCCGTGA

[0214] CCGCCCTGCTGCTGCCCCTGGCCCTGCTTCTGCACGCCGCCAGGCCCGGCAGCCAGGTG

[0215] CAGCTGGTGCAGAGCGGCGCCGAGGTGGTGAAGCCCGGCGCCTCCGTGAAGCTGAGCT

[0216] GCAAGACCAGCGGCTACACCTTCACCAACTACGACATCAACTGGGTGCGCCAGCGGCC

[0217] CGGCCAGGGCCTGGAGTGGATCGGCTGGATCTTCCCCGGCGACGGCAGCACCCAGTAC

[0218] AACGAGAAGTTCAAGGGCAAGGCCACCCTGACGACCGACACCAGCACCAGCACCGCC

[0219] TACATGGAGCTGAGCAGCCTGAGGAGCGAGGATACAGCCGTGTATTTTTGTGCTAGAC

[0220] AGACAACCGCCACTTGGTTTGCCTATTGGGGCCAGGGAACCCTCGTGACAGTGTCTAG

[0221] TGGCGGAGGCGGCTCTGGAGGCGGAGGCAGCGGAGGCGGCGGGTCTGAAATTGTGAT

[0222] GACCCAGAGTCCAGCTACTCTGTCTGTGTCTCCTGGAGAAAGAGTGACCCTGAGCTGT

[0223] AGAGCCAGCCAGTCTATTTCTGATTATCTCTATTGGTATCAGCAGAAGAGTCATGAATC

[0224] TCCTAGACTGCTGATTAAGTATGCTAGCCAGTCTATTAGCGGAATTCCAGCCCGATTTT

[0225] CTGGAAGCGGAAGTGGCTCTGAGTTTACACTGACAATTAATTCTGTGGAGCCTGAAGA

[0226] TGTGGGAGTGTATTATTGTCAGAATGGCCATAGCTTTCCACTGACATTTGGACAGGGAA

[0227] CAAAGCTGGAACTGAAGAGAACAACAACACCTGCCCCTAGACCACCTACACCTGCCCC

[0228] AACTATTGCTTCTCAGCCTCTGAGCCTGAGACCTGAAGCCTGTAGACCTGCAGCTGGAG

[0229] GAGCTGTGCATACAAGAGGGCTGGATTTTGCCTGTGATGATATCTACATCTGGGCGCCC

[0230] TTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACG

[0231] GGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACT

[0232] ACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGT

[0233] GAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAG

[0234] AACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACA

[0235] AGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGG

[0236] AAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTG

[0237] GGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCA

[0238] GTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCgag

[0239] ggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggcccaATGGTGAGCAAGGGCGAGGagctgttcac

[0240] cggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccaccta

[0241] cggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtg

[0242] cttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaag

[0243] gacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaag

[0244] gaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatca

[0245] aggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggcc

[0246] ccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggag

[0247] ttcgtgaccgccgccgggatcaCTCTCGGCATGGACGAGCtgtacaagtaaGTCGACCTCGAGGGAATTCC

[0248] GATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGT

[0249] TGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTC

[0250] CCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGA

[0251] GTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCC

[0252] CCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCC

[0253] TCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCT

[0254] CGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATG

[0255] GCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTC

[0256] GGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTC

[0257] CGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCAT

[0258] CGGGAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTT

[0259] AGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGAC

[0260] AAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAG

[0261] CTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCT

[0262] TCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCT

[0263] TTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATT

[0264] TATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTA

[0265] TAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCAC

[0266] TGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGC

[0267] TATCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCC

[0268] GCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTG

[0269] AGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGGACGTACCCAATTCG

[0270] CCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTG

[0271] GGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCT

[0272] GGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAA

[0273] TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACG

[0274] CGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCC

[0275] TTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTT

[0276] AGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATG

[0277] GTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCC

[0278] ACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGT

[0279] CTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCT

[0280] GATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGG

[0281] CACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAA

[0282] ATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAG

[0283] GAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG

[0284] CCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGT

[0285] TGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAG

[0286] TTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCG

[0287] CGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCT

[0288] CAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGA

[0289] CAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTT

[0290] ACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGG

[0291] GATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACG

[0292] ACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAAC

[0293] TGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGAT

[0294] AAAGTTGCAGGACCACTTCTGCG

[0295] SEQ ID NO:3 T2A nucleotide sequence:

[0296] gagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggccca

[0297] SEQ ID NO:4 T2A amino acid sequence:

[0298] EGRGSLLTCGDVEENPGP

[0299] SEQ ID NO:5 eGFP nucleotide sequence:

[0300] ATGGTGAGCAAGGGCGAGGagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaa

[0301] gttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccct

[0302] ggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgcca

[0303] tgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacac

[0304] cctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccac

[0305] aacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgc

[0306] cgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaa

[0307] gaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcaCTCTCGGCATGGACGAGCtgt

[0308] acaagtaa

[0309] Amino acid sequence of SEQ ID NO:6 CAR276:

[0310] MALPVTALLLPLALLLHAARPGSQVQLVQSGAEVVKPGASVKLSCKTSGYTFTNYDINWV

[0311] RQRPGQGLEWIGWIFPGDGSTQYNEKFKGKATLTTDTSTSTAYMELSSLRSEDTAVYFCAR

[0312] QTTATWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERVTLSCRA

[0313] SQSISDYLYWYQQKSHESPRLLIKYASQSISGIPARFSGSGSGSEFTLTINSVEPEDVGVYYC

[0314] QNGHSFPLTFGQGTKLELKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF

[0315] ACDDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE

[0316] EEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR

[0317] KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL

[0318] PPR

Claims

1. A mesenchymal stem cell, characterized in that: The mesenchymal stem cells express a chimeric antigen receptor targeting CD276 or contain a nucleic acid encoding the chimeric antigen receptor targeting CD276.

2. The mesenchymal stem cell according to claim 1, wherein: Compared with wild-type mesenchymal stem cells, the mesenchymal stem cells have the characteristics shown in at least one of the following (1)-(4): (1) Improved survival rate; (2) improved persistence of tumor suppression; (3) enhanced tumor suppression ability; (4) Improved tumor targeting.

3. The mesenchymal stem cell according to claim 1, wherein: The mesenchymal stem cells are derived from adult cells or stem cells; Preferably, the mesenchymal stem cells are derived from pluripotent stem cells, more preferably, the pluripotent stem cells are selected from induced pluripotent stem cells; Preferably, the mesenchymal stem cells are derived from bone marrow, fat, muscle, heart, umbilical cord blood or umbilical cord.

4. The mesenchymal stem cell according to claim 1, wherein: The chimeric antigen receptor targeting CD276 comprises: (a) an extracellular target portion; (b) a transmembrane domain; (c) an intracellular region; The extracellular targeting portion includes an antibody that specifically binds to the CD276 antigen; Preferably, the antibody is selected from any one of the following: Fab fragment, Fab' fragment, F(ab)'2 fragment, Fv, scFv, (scFv)2, Ig ​​NAR, dsFv, nanobody, minibody, monoclonal antibody, bifunctional antibody; more preferably, the antibody is a scFv fragment derived from an antibody that specifically binds to the CD276 antigen; The intracellular region includes an intracellular signaling domain and / or a costimulatory domain; Preferably, the intracellular signal transduction domain is a cytoplasmic functional region derived from the T cell receptor CD3ζ or is selected from any one or more of the following cytoplasmic functional regions: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b and CD66d; the costimulatory domain is selected from any one or more of the following cytoplasmic functional regions: 2B4, HVEM, ICOS, LAG3, DAP10, DAP12, CD27, CD28, CD137, CD134, CD30, CD40, CD278, glucocorticoid-induced tumor necrosis factor receptor (GITR), lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C and B7-H3; More preferably, the intracellular region includes the cytoplasmic functional region derived from CD137 and CD3ζ; Preferably, the transmembrane domain is selected from any one or several of the following transmembrane regions: α or β chain of T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154; Preferably, the chimeric antigen receptor targeting CD276 further comprises (d) a hinge region, wherein the hinge region is used to connect the extracellular target portion and the transmembrane domain; more preferably, the hinge region is selected from the hinge region comprising CD8α.

5. The mesenchymal stem cell according to claim 4, wherein: The amino acid sequence of the chimeric antigen receptor targeting CD276 is shown in SEQ ID NO.6, or an amino acid sequence that is at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.9% identical to the sequence shown in SEQ ID NO:

6.

6. The mesenchymal stem cell according to claim 1, wherein: The nucleic acid is included in an expression vector for expressing the chimeric antigen receptor targeting CD276; Preferably, the expression vector is a plasmid or a viral vector; more preferably, the viral vector includes a lentiviral vector, an adenoviral vector, an adeno-associated viral vector or a retroviral vector; further preferably, a lentiviral vector; Preferably, the expression vector further comprises a nucleic acid sequence encoding a tag protein; more preferably, the tag protein comprises any one of HIS, Flag, GST, Myc, eGFP, eCFP, eYFP, and mCherryeGFP; Preferably, the nucleic acid comprises a nucleic acid sequence encoding the chimeric antigen receptor targeting CD276 as claimed in claim 4 or 5; Preferably, the nucleic acid sequence is as shown in SEQ ID NO: 1, or a nucleic acid sequence having at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.9% identity with the sequence shown in SEQ ID NO:

1.

7. A composition, characterized in that: The composition comprises the mesenchymal stem cells according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier or excipient.

8. Use of the mesenchymal stem cells according to any one of claims 1 to 6 or the composition according to claim 7 in the preparation of an anti-tumor drug; Preferably, the tumor is selected from tumors expressing CD276 molecules, preferably lung cancer.

9. A method for constructing the mesenchymal stem cells according to any one of claims 1 to 6, comprising the following steps: S1: Preparation and culture of mesenchymal stem cells; S2: transferring a nucleic acid encoding a chimeric antigen receptor targeting CD276 into the mesenchymal stem cells; Preferably, the step S1 comprises inducing the differentiation of iPSCs into mesenchymal stem cells in vitro and expanding the culture; Preferably, step S2 comprises cloning a nucleic acid encoding a chimeric antigen receptor targeting CD276 into an expression vector, and then transferring the expression vector into mesenchymal stem cells; Preferably, the introducing comprises transfection or transduction; Preferably, the nucleic acid encoding the chimeric antigen receptor targeting CD276 is site-specifically integrated into the genome of the mesenchymal stem cells; Preferably, the nucleic acid encoding the chimeric antigen receptor targeting CD276 is site-specifically integrated into the gene locus or rDNA region of the mesenchymal stem cells; Preferably, the locus is selected from any one of the AAVS1 locus, the B2M locus, the CCR5 locus, and the CIITA locus.

10. Use of the chimeric antigen receptor targeting CD276 in mesenchymal stem cells according to any one of claims 1 to 6 in the preparation of any one or more of the following products: (1') Improve the survival rate of mesenchymal stem cells; (2') Improve the tumor suppression persistence of mesenchymal stem cells; (3') Improve the tumor targeting of mesenchymal stem cells; (4') Improve the tumor suppression ability of mesenchymal stem cells.

11. A method for improving the survival rate of mesenchymal stem cells, comprising the following steps: S1': Preparation and culture of mesenchymal stem cells; S2': transferring a nucleic acid encoding a chimeric antigen receptor targeting CD276 into the mesenchymal stem cells.

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