Mesenchymal stem cell with enhanced targeted tumor infiltration ability, and use thereof

Abstract

Provided are a mesenchymal stem cell (MSC) with an enhanced targeted tumor infiltration ability, and the use thereof. The MSC with an enhanced targeted tumor infiltration ability comprises a polynucleotide encoding an NKG2DL-targeting chimeric antigen receptor. The provided MSC with an enhanced targeted tumor infiltration ability can not only normally overexpress the NKG2DL-targeting chimeric antigen receptor, but also has a better solid tumor targeting property, thus being able to effectively break through solid tumor microenvironments and enter the interior of tumors. Compared to wild-type MSC cells, the MSC has a better retention ability at the tumor site, which enhances the survival ability thereof at the tumor site, thereby enabling the MSC to exert a more long-acting and sustained tumor inhibitory effect at the tumor site and further promoting the use of the MSC in the field of solid tumor treatment.

Description

Mesenchymal stem cells with enhanced tumor-targeted infiltration ability and application thereof

[0001] Cross-reference to related applications

[0002] The present application claims priority to the prior application with the patent application number 202410861582.6, the title of which is "Mesenchymal stem cells with enhanced tumor-targeted infiltration ability and application thereof", filed with the State Intellectual Property Office of China on June 28, 2024. The entire contents of the prior application are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to the technical field of mesenchymal stem cells, in particular to mesenchymal stem cells with enhanced tumor-targeted infiltration ability and application thereof. BACKGROUND

[0004] Mesenchymal stem cells are one of the most rapidly developing and most valued biotechnologies in the field of life sciences in recent years. Generally, stem cells can be divided into embryonic stem cells and adult stem cells, and mesenchymal stem cells belong to adult stem cells which are widely used. Adult stem cells exist in various tissues and organs of fetuses and adults, have a wide source, and do not involve ethical issues. As a typical adult stem cell, mesenchymal stem cells are highly expected in the process of disease treatment due to their excellent multi-directional differentiation potential, especially their low immunogenicity, which has obvious advantages in the field of cell therapy and transplantation. Mesenchymal stem cells have certain tumor homing ability, but the homing ability of pure mesenchymal stem cells is insufficient, and it is still a difficult problem in the development of cell therapy that mesenchymal stem cells break through the barrier of tumor microenvironment, realize close combination with tumor cells, and remain in tumors.

[0005] NKG2DL is a binding ligand of NKG2D, and the NKG2D ligand (NKG2DL) mainly includes major histocompatibility complex class I chain-related protein A / B (MICA / B) and human cytomegalovirus UL16 binding protein (ULBP). The NKG2DL is rarely expressed or only transiently expressed in healthy tissues, and is expressed only under stress conditions, and is usually expressed at a high level on the surface of various tumor cells of different origins. Generally, most of the cancers are solid tumors, and the specific targets on the surface of different solid tumors are often different, and the NKG2DL can be used as a target for immune cell targeted recognition. It has been reported in the prior art that the chimeric antigen receptor modified T cell targeting the NKG2D ligand can recognize the ligand on the surface of the tumor cell, and then transmit the activation signal through the adapter protein (DAP10 or DAP12), utilize the granzyme and perforin to cause the tumor cell lysis, so as to achieve the purpose of tumor treatment. However, the retention ability of the T cell in the solid tumor is low, which leads to poor performance of the T cell in the treatment of the solid tumor.

[0006] For mesenchymal stem cells, how to further improve the breakthrough ability of the mesenchymal stem cells to the tumor microenvironment barrier of the solid tumor and the sustained retention ability of the mesenchymal stem cells in the tumor is one of the problems to be solved by those skilled in the art. At present, effective technical breakthrough has not been achieved in the field. SUMMARY

[0007] In view of the deficiencies in the prior art, the present application provides a mesenchymal stem cell with enhanced tumor targeted infiltration ability and application thereof. The present application solves the problems of how to break through the tumor microenvironment barrier, achieve close combination with tumor cells, and remain in the tumor in the prior art.

[0008] In one aspect of the present application, an engineered mesenchymal stem cell is provided, wherein the engineered mesenchymal stem cell expresses a chimeric antigen receptor targeting NKG2DL or contains a polynucleotide encoding the chimeric antigen receptor targeting NKG2DL.

[0009] In one embodiment of the present application, a polynucleotide encoding a chimeric antigen receptor targeting NKG2DL is site-specifically integrated into the genome of the engineered mesenchymal stem cell, and the engineered mesenchymal stem cell expresses the chimeric antigen receptor.

[0010] In one embodiment of the present application, the site-specifically integrated gene site is a B2M locus.

[0011] In one embodiment of the present application, the chimeric antigen receptor comprises an NKG2DL binding domain and a signal transduction domain.

[0012] In one embodiment of the present application, the NKG2DL binding domain comprises an extracellular domain of NKG2D.

[0013] In an embodiment of the application, the NKG2D comprises any naturally occurring form of NKG2D or a NKG2D variant that retains its protein activity (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity within compared to native NKG2D). In an embodiment of the application, the NKG2D variant has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the entire sequence or a partial sequence (e.g., a 50, 100, 150, or 200 continuous amino acid portion) of the NKG2D compared to a naturally occurring form of NKG2D. In an embodiment of the application, the NKG2D has a NCBI Reference Sequence number of NP_031386.2. In some embodiments, the NKG2D has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the protein with NCBI Reference Sequence number of NP_031386.2 or a functional fragment thereof.

[0014] In an embodiment of the application, the signaling domain comprises a transmembrane domain and an intracellular domain, the intracellular domain comprising a signaling chain having an immunoreceptor tyrosine-based activation motif (ITAM).

[0015] In an embodiment of the application, the intracellular domain comprises, but is not limited to, CD3y, CD35, CD3s, CD3z, Ig a (CD79a), Ig b (CD79b), FcyRIII (CD16).

[0016] In an embodiment of the application, the intracellular domain further comprises an intracellular portion of a costimulatory domain, the intracellular portion of the costimulatory domain comprising CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27, and / or DAP10.

[0017] In an embodiment of the application, the transmembrane domain comprises, but is not limited to, an NKG2D transmembrane domain, a CD8a transmembrane domain, a CD28 transmembrane domain, an IgG4 transmembrane domain, a TNFR transmembrane domain, and a TLR transmembrane domain.

[0018] In an embodiment of the application, the chimeric antigen receptor further comprises a hinge region.

[0019] In an embodiment of the present application, the amino acid sequence of the chimeric antigen receptor is set forth in SEQ ID NO: 1, or an amino 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 to the sequence set forth in SEQ ID NO: 1.

[0020] In an embodiment of the present application, the mesenchymal stem cell is derived from an adult cell or a stem cell; preferably, the mesenchymal stem cell is derived from a pluripotent stem cell, more preferably, the pluripotent stem cell is selected from an induced pluripotent stem cell; preferably, the mesenchymal stem cell is derived from bone marrow, fat, muscle, heart, umbilical cord blood or umbilical cord.

[0021] In an embodiment of the present application, the engineered mesenchymal stem cell stably overexpresses NKG2D.

[0022] In an embodiment of the present application, the engineered mesenchymal stem cell has increased expression of NKG2D compared to a wild-type mesenchymal stem cell. In an embodiment of the present application, the engineered mesenchymal stem cell has enhanced tumor-targeted infiltration, tumor retention, tumor cell binding compared to a wild-type mesenchymal stem cell. In an embodiment of the present application, the tumor retention of the engineered mesenchymal stem cell is increased by at least 27% compared to a wild-type mesenchymal stem cell. In an embodiment of the present application, the tumor retention of the engineered mesenchymal stem cell is increased by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 57%, at least 60% compared to a wild-type mesenchymal stem cell.

[0023] In a second aspect of the present application, a composition comprising the engineered mesenchymal stem cell described above is provided.

[0024] In an embodiment of the present application, the composition comprises one or more other therapeutic agents.

[0025] In an embodiment of the present application, the other therapeutic agent comprises an anti-tumor agent, including but not limited to paclitaxel and its derivatives, docetaxel, camptothecin and its derivatives, etoposide, teniposide, doxorubicin hydrochloride, cyclophosphamide, dactinomycin, bleomycin, nanchamycin, doxorubicin, epirubicin, mitomycin, methotrexate, 5-fluorouracil, carboplatin, carmustine, lomustine, cisplatin, vinblastine, vincristine, tamoxifen, sulfanilamide, phenyl ethyl cholestanol.

[0026] In one embodiment of the application, the one or more additional therapeutic agents are administered in combination with the engineered mesenchymal stem cells. The combination administration includes sequential administration in any order or at any interval, such that the two or more therapeutic agents exert their biological activities at the same time. Preferably, the combination administration produces a synergistic therapeutic effect.

[0027] In one embodiment of the application, the composition further comprises a pharmaceutically acceptable excipient.

[0028] In one embodiment of the application, the excipient comprises a buffer selected from, for example, acetate, Tris, phosphate, citrate, and other organic acids; an antioxidant selected from, for example, ascorbic acid and methionine; a preservative selected from, for example, octadecyl dimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzotonic chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol; a protein selected from, for example, serum albumin, gelatin, or immunoglobulins; a hydrophilic polymer selected from, for example, polyvinylpyrrolidone; an amino acid such as glycine, glutamine, asparagine, histidine, arginine, or lysine; a chelating agent selected from, for example, EDTA; a sugar selected from, for example, glucose, mannose, dextrins, sucrose, mannitol, trehalose, or sorbitol; a surfactant selected from, for example, polysorbates; a metal complex selected from, for example, zinc-protein complexes; a non-ionic surfactant selected from, for example, Tween or polyethylene glycol (PEG); a liposome, an albumin microsphere, a polyester, a micelle, a sustained-release matrix, and the like.

[0029] In a third aspect of the application, a method for preparing the engineered mesenchymal stem cell described above is provided, comprising introducing the nucleic acid encoding the chimeric antigen receptor into the mesenchymal stem cell to obtain the engineered mesenchymal stem cell.

[0030] In one embodiment of the application, the method comprises introducing the nucleic acid encoding the chimeric antigen receptor into the induced pluripotent stem cell and inducing differentiation to obtain the engineered mesenchymal stem cell.

[0031] In one embodiment of the application, the method comprises site-specifically integrating the nucleic acid encoding the chimeric antigen receptor into the genome of the mesenchymal stem cell by a gene editing system.

[0032] In one embodiment of the application, the site of the site-specific integration is the B2M locus.

[0033] In an embodiment of the present application, the gene editing system is selected from the group consisting of Cre-lox system, Zinc Finger Nucleases (ZFNs), CRISPR-Cas or Transcription Activator-Like Effector Nucleases (TALENs), preferably CRISPR-Cas or TALENs; more preferably CRISPER / Cas system.

[0034] In an embodiment of the present application, the CRISPER / Cas system is selected from the group consisting of CRISPR-Cas9, CRISPR-Cas12a (Cpf1), CRISPR-Cas13, CRISPR-Cas14, CRISPR-CasX, CRISPR-CasY, preferably CRISPR-Cas9.

[0035] In an embodiment of the present application, the preparation method comprises: introducing a targeting vector containing a nucleic acid encoding a chimeric antigen receptor and a CRISPER / Cas system into mesenchymal stem cells, and site-specifically integrating the nucleic acid encoding the chimeric antigen receptor into the B2M gene locus by homologous recombination.

[0036] In an embodiment of the present application, the introduction is selected from the group consisting of transformation, transfection, heat shock, electroporation, transduction, microinjection.

[0037] In an embodiment of the present application, the introduction is a non-viral method.

[0038] In a fourth aspect of the present application, the use of the engineered mesenchymal stem cells and the composition in the preparation of a diagnostic, prophylactic and therapeutic anti-tumor drug is provided.

[0039] In an embodiment of the present application, the tumor includes but is not limited to ovarian cancer, such as serous ovarian cancer, endometrioid ovarian cancer, clear cell ovarian cancer, mucinous ovarian cancer; uterine cancer, such as endometrial cancer, uterine sarcoma; lung cancer, such as non-small cell lung cancer, small cell lung cancer; Merkel cell carcinoma, skin cancer, breast cancer, malignant soft tumor, neuroendocrine tumor, brain tumor, pharyngeal cancer, laryngeal cancer, thyroid cancer, esophageal cancer, gastric cancer, colon cancer, liver cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, bone tumor, preferably at least one selected from the group consisting of breast cancer, uterine cancer, ovarian cancer, lung cancer, Merkel cell carcinoma, neuroendocrine tumor, brain tumor.

[0040] In an embodiment of the present application, the tumor is selected from the group consisting of tumors expressing NKG2DL molecules. In an embodiment of the present application, the tumor highly expresses NKG2DL molecules.

[0041] In one embodiment of the present application, the anti-tumor drug is administered via intravenous, intramuscular, intraperitoneal, intracerebral, subcutaneous, intraspinal, intrathecal, oral, topical, or inhalation routes.

[0042] In one embodiment of the present application, the anti-tumor drug is used to treat a mammal (e.g., a human) having a tumor or who is likely to develop a tumor. For example, the engineered mesenchymal stem cells described in the present application are used as the only active agent to treat a mammal (e.g., a human) having a tumor or who is likely to develop a tumor. In some cases, the engineered mesenchymal stem cells described in the present application can be administered in combination with one or more other therapeutic agents to treat a mammal (e.g., a human) having a tumor or who is likely to develop a tumor.

[0043] In a fifth aspect of the present application, there is provided use of the engineered mesenchymal stem cell or the chimeric antigen receptor targeting NKG2DL in the manufacture of a medicament for improving tumor infiltration ability of mesenchymal stem cells or improving survival ability of mesenchymal stem cells at a tumor site.

[0044] In a sixth aspect of the present application, there is provided a method for improving tumor infiltration ability of mesenchymal stem cells or survival ability of mesenchymal stem cells at a tumor site, comprising causing the mesenchymal stem cells to express a chimeric antigen receptor targeting NKG2DL.

[0045] Compared with the prior art, the present application has the following beneficial effects:

[0046] 1、The present application uses iPSCs as seed cells of MSCs cells, improves the stability and uniformity of MSC cells, and integrates the gene encoding NKG2D-CAR into the B2M gene locus of iPSCs cells in a non-viral manner, then induces the positive clone after site-specific integration into MSCs to prepare B2M-NKG2D-CAR-iMSC. Through in vivo and in vitro analysis and detection, B2M-NKG2D-CAR-iMSC not only can normally overexpress the chimeric antigen receptor targeting NKG2DL, but also has better solid tumor targeting property, can effectively break through the solid tumor microenvironment and enter the inside of the tumor. The in vivo and in vitro experimental results prove that after gene editing, B2M-NKG2D-CAR-iMSC has better retention ability at the tumor site than wild-type MSC cells, enhances the survival ability at the tumor site, so that the mesenchymal stem cells can play a more long-acting and sustained anti-tumor effect at the tumor site, and further promote the application of MSC cells in the field of solid tumor treatment.

[0047] 2、The application first discovers that the chimeric antigen receptor targeting NKG2DL is more suitable for site-specific integration in mesenchymal stem cells, i.e., the B2M gene locus. The application discovers that the chimeric antigen receptor targeting NKG2DL (such as NKG2D-CAR) cannot be site-specifically integrated at will. When the site-specific integration site is selected improperly, the expression of NKG2D-CAR on the cell surface is not ideal, and even not expressed. Based on this, the application particularly selects the B2M gene locus as the site-specific integration site of NKG2D-CAR, and overcomes the problem that NKG2D-CAR cannot be effectively expressed at other sites (such as the AAVS1 site and the rDNA region site).

[0048] 3、The application improves the breakthrough ability of mesenchymal stem cells to the tumor microenvironment barrier of solid tumors, and greatly increases the sustained retention ability of mesenchymal stem cells in the tumor site. Compared with wild-type mesenchymal stem cells, the mesenchymal stem cells provided in some embodiments of the application have a 57% increase in retention signal at the thirteenth day observed in the mouse tumor site.

[0049] 4、The application first discovers that the chimeric antigen receptor targeting NKG2DL can be used to modify MSC cells by site-specific integration. Unlike the virus modification method commonly used in the prior art, the application uses a non-viral method in the entire cell preparation process, reduces the potential risks of immune response, off-target and insertion mutation, and further improves the safety in the cell therapy process. BRIEF DESCRIPTION OF DRAWINGS

[0050] Fig. 1 is a schematic diagram of the plasmid vector NKG2D-CD8TM-4-1BB-CD3 zeta core element and site-specific integration in embodiment 1 of the application.

[0051] Fig. 2 is an expression verification diagram of the plasmid vector NKG2D-CD8TM-4-1BB-CD3 zeta in embodiment 2 of the application.

[0052] Fig. 3 is an electropherogram of PCR products of iPSCs clones for identifying site-specific integration across the upstream homologous arm and Sanger sequencing results of the AAVS1 site in embodiment 3 of the application.

[0053] Fig. 4 is a flow-sorted NKG2D-positive iPSCs clone (a) and electropherograms of PCR products of the B2M site across the upstream homologous arm (b) and Sanger sequencing results (c) in embodiment 3 of the application.

[0054] Fig. 5 is an electropherogram of PCR products of iPSCs clones for identifying site-specific integration of the rDNA region site (a) and a column chart of qPCR for confirming NKG2D site-specific integration (b) in embodiment 3 of the application.

[0055] Figure 6 is a flow cytometry identification of the expression of the 3 site-specific integration clones of NKG2D in Example 3 of the present application.

[0056] Figure 7 is a morphology of B2M-NKG2D-CAR-iMSC in Example 4 of the present application.

[0057] Figure 8 is a surface marker identification of B2M-NKG2D-CAR-iMSC in Example 4 of the present application.

[0058] Figure 9 is a flow cytometry identification of the expression of NKG2D of B2M-NKG2D-CAR-iMSC in Example 4 of the present application.

[0059] Figure 10 is a flow cytometry representative graph and statistical graph of the cell interaction experiment of B6-iMSCs and B2M-NKG2D-CAR-iMSC with A549, MCF7, and H460 in Example 5 of the present application.

[0060] Figure 11 is a flow chart of the animal experiment in Example 6 of the present application.

[0061] Figure 12 is a statistical graph of the DiR 750 signal of the in vivo imaging of mice in Example 6 of the present application.

[0062] Figure 13 is a graph and statistical graph of the DiR 750 signal of tumor imaging in Example 6 of the present application. DETAILED DESCRIPTION

[0063] The technical solutions of the present application will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustrative and explanatory of the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope of protection intended by the present application.

[0064] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0065] iPSCs, iPSC or ips (Induced pluripotent stem cells), refer to induced pluripotent stem cells, which are reprogrammed from terminally differentiated somatic cells to pluripotent stem cells by introducing transcription factors, thereby restoring the terminally differentiated somatic cells to totipotency. They can be used interchangeably in this text. The induced pluripotent stem cells used in the specific embodiments of the present application are not strictly limited and can be commercially available products or reprogrammed by known methods disclosed in the prior art.

[0066] iMSC or iMSCs, refers to mesenchymal stem cells formed after induced differentiation using pluripotent stem cells (e.g., induced pluripotent stem cells) as seed cells, which are used interchangeably in the detailed description of the present application.

[0067] Mesenchymal stem cells, also referred to as MSC or MSCs, can be derived from bone marrow, fat, muscle, heart, umbilical cord blood or umbilical cord, or induced differentiation from pluripotent stem cells. MSCs as described herein include, but are not limited to, MSCs derived from fat, MSCs derived from bone marrow, MSCs derived from placental tissue, MSCs derived from dental pulp tissue, MSCs derived from umbilical cord, blood mesenchymal stem cells (MSCs), Wharton's jelly mesenchymal stem cells (MSCs), dermal mesenchymal stem cells (MSCs), olfactory mucosal mesenchymal stem cells (MSCs), peripheral blood mesenchymal stem cells (MSCs), amniotic membrane mesenchymal stem cells (MSCs).

[0068] Chimeric antigen receptor (CAR): a chimeric molecule comprising an antigen binding moiety (e.g., a single domain antibody or scFv) and a signaling domain (e.g., a signaling domain from a T cell receptor (e.g., CD3 zeta)). Typically, a CAR is composed of an antigen binding moiety (also known as an extracellular targeting moiety), a transmembrane domain, and an intracellular domain (also known as an intracellular region). The antigen binding moiety can be any suitable antigen binding domain. In some cases, the antigen binding moiety includes an antibody or fragment thereof that targets a target antigen. The antigen can be any target antigen, such as an antigen expressed on the surface of a cell. In preferred embodiments, the target antigen is an antigen on a tumor cell, such as a NKG2DL. The tumor cell can be a cell of a solid tumor or a blood cancer, for example. In certain specific embodiments, the antigen binding moiety includes, but is not limited to, an antigen binding fragment (Fab), an antibody heavy chain (VH) variable region, a light chain (VL) variable region, and a single chain variable fragment (scFV), a polypeptide, a receptor, a ligand, and a cytokine. For example, the antigen binding moiety described herein includes an NKG2D receptor that targets a NKG2DL. The transmembrane domain includes, but is not limited to, an NKG2D transmembrane domain, a CD8a transmembrane domain, a CD28 transmembrane domain, an IgG4 transmembrane domain, a TNFR transmembrane domain, and a TLR transmembrane domain. For example, the transmembrane domain described herein includes a CD8TM. The intracellular domain typically includes a signaling chain with an immunoreceptor tyrosine-based activation motif (ITAM), such as CD3 zeta. 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. For example, the intracellular portion of the costimulatory domain described herein includes 4-1BB.

[0069] CAR-MSC refers to mesenchymal stem cells that can express CAR, and the CAR gene is introduced into MSC by genetic engineering means, so that MSC expresses CAR that specifically recognizes target antigens, thereby combining the migration ability of MSC and the specific killing function of CAR to treat one or several diseases in the target. In an embodiment of the present application, the CAR-MSC is a mesenchymal stem cell expressing a chimeric antigen receptor targeting or capable of binding to an antigen on a tumor cell. In an embodiment of the present application, the CAR-MSC is a mesenchymal stem cell expressing a chimeric antigen receptor targeting or capable of binding to NKG2DL.

[0070] NKG2D, also known as KLRK1, is a transmembrane protein belonging to the CD94 / NKG2 family of C-type lectin-like receptors. In humans, it is expressed by NK cells, gd T cells, and CD8+ ab T cells. NKG2D recognizes self-inducing proteins from the MIC and RAET1 / ULBP families, which appear on the surface of stressed, malignant, and infected cells. The NKG2D described in the present application includes any naturally occurring form of NKG2D or NKG2D variants that maintain its protein activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to native NKG2D). In some embodiments, the NKG2D variant has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the entire sequence or a partial sequence (e.g., a 50, 100, 150, or 200 continuous amino acid portion) of NKG2D compared to the naturally occurring form of NKG2D. In some embodiments, the NKG2D has a NCBI Reference Sequence number of NP_031386.2. In some embodiments, the NKG2D has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the protein with NCBI sequence reference sequence number NP_031386 or a functional fragment thereof.

[0071] NKG2DL, also known as NKG2DLs, is the binding ligand of NKG2D. NKG2DLs are mainly divided into two categories, one is MHC class I molecule associated genes A and B (i.e., MICA and MICB), and the other is UL-16 binding protein (i.e., ULBP1-6). NKG2DLs have limited expression in healthy tissues, but there is extensive expression in solid tumors.

[0072] In the present application, the NKG2D-CAR is a chimeric antigen receptor capable of targeting NKG2DL targets, which can specifically recognize and bind NKG2DL, and is used for targeting cells with high expression of NKG2DL. In a specific embodiment of the present application, the NKG2D-CAR is NKG2D-CD8TM-4-1BB-CD3zeta.

[0073] In the present application, B2M-NKG2D-CAR-iMSC refers to the mesenchymal stem cells obtained by site-specific integration of the chimeric antigen receptor targeting NKG2DL targets into the B2M gene locus through gene editing.

[0074] iPSC-derived MSCs are considered an ideal way to prepare MSCs, which have excellent consistency, less cell difference between different batches, and good stability.

[0075] Overexpression in the present application refers to the modified cells showing higher expression levels compared to wild-type cells (i.e. unmodified cells).

[0076] In a specific embodiment of the present application, a gene editing tool is used to target the gene expressing NKG2D-CAR (also referred to as "polynucleotide sequence") into the genome of the target cell by site-specific integration. Preferably, the target cell is iPSCs, which are then induced to differentiate into mesenchymal stem cells to obtain mesenchymal stem cells with enhanced tumor targeting infiltration ability.

[0077] B2M locus: refers to the location of the Beta-2-Microglobulin (β2 microglobulin) gene in the human genome. The B2M locus is located on chromosome 15, and this gene encodes Beta-2-Microglobulin, a small protein that is usually associated with major histocompatibility complex (MHC I) molecules on the surface of most human cells.

[0078] Site-specific integration: refers to the insertion or integration of all or part of a desired sequence (e.g., a target sequence) into a desired site or locus (e.g., a target sequence) within the genome. Methods of site-specific integration are well known to those skilled in the art. For example, they include calcium phosphate-mediated integration: by combining an exogenous gene with a calcium phosphate carrier (such as CaPO4), the exogenous gene is integrated into the cell using methods of electric shock or ultraviolet light activation. Transposon-mediated integration: using transposons (such as Tn7, Tn5) to integrate exogenous genes into the chromosome of the cell. CRISPR / Cas9-mediated integration: using the CRISPR / Cas9 system to site-specifically integrate exogenous genes into the chromosome of the cell. Direct DNA ligation: using DNA ligase to directly ligate exogenous genes to specific locations on the chromosome of the cell. In some embodiments, site-specific integration is performed using the CRISPR / Cas9 system.

[0079] In some embodiments of the present application, the integration is carried out by gene editing. Any gene editing technology known in the art can be used in the present application as long as it can achieve the site-specific integration of the polynucleotide of interest, such as the Cre-Lox system, Zinc Finger Nucleases (ZFN), CRISPR or TALEN, preferably CRISPR or TALEN, or TALEN / TALENickase-mediated gene editing.

[0080] Methods of determining sequence identity or similarity are well known in the art. See, e.g., Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M., and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). Preferred methods of determining identity are those that give the greatest match between the sequences tested. Methods of determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG suite of programs (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith Waterman algorithm can also be used to determine identity.

[0081] "Targeting vector" refers to a vector used in gene targeting technology. Gene targeting technology is a method to specifically insert exogenous DNA into a predetermined position of the genome by homologous recombination. The basic components of a targeting vector usually include homology arms, a promoter, a gene of interest, a selection marker gene. The homology arms are usually composed of DNA fragments homologous to the sequences flanking the target locus, usually ranging from 500bp to several kilobases on each side. In an embodiment of the present application, the homology arms are homologous to the sequence in the B2M locus. In an embodiment of the present application, the gene of interest is a nucleic acid encoding a chimeric antigen receptor NKG2D-CAR. In an embodiment of the present application, the nucleic acid encoding a chimeric antigen receptor NKG2D-CAR has a sequence as set forth in SEQ ID NO: 2, or a nucleotide 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 to the sequence set forth in SEQ ID NO: 2.

[0082] Construction of Example 1 targeting vector carrying NKG2D-CD8TM-4-1BB-CD3 zeta (the "targeting vector" in the present application is also referred to as "plasmid")

[0083] The structure design of NKG2D-CAR in the embodiment of the application is NKG2D-CD8TM-4-1BB-CD3 zeta, wherein each part adopts a known sequence disclosed in the prior art (see Prior Art GMP-compliant manufacturing of NKG2D CAR memory T cells using CliniMACS prodigy. Letc. Frontiers in Immunology, 2019). Meanwhile, three target sites of AAVS1, B2M and rDNA region are selected in the embodiment to construct the corresponding targeting vectors. The polynucleotide sequence expressing NKG2D-CD8TM-4-1BB-CD3 zeta is respectively integrated into the AAVS1, B2M site using the CRISPR / Cas9 gene editing tool (Genscript Biotechnology Co., Ltd., Z03702), and the polynucleotide sequence expressing NKG2D-CD8TM-4-1BB-CD3 zeta is integrated into the rDNA region using the TALEN nickase editing tool (see Prior Art “TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus”, Wu Y, Gao T, Wang X, et al, Biochemical & Biophysical Research Communications, 2014, 446(1): 261-266). Specifically, the amino acid sequence of the NKG2D-CD8TM-4-1BB-CD3 zeta fusion protein in the embodiment of the application is as shown in SEQ ID NO: 1, and the nucleotide sequence thereof is as shown in SEQ ID NO: 2. The NKG2D-CD8TM-4-1BB-CD3 zeta fusion gene sequence is connected to the homologous arm skeleton of the three sites of AAVS1, B2M and rDNA synthesized in advance by enzyme digestion and homologous recombination (the homologous arm of the targeting vector of AAVS1, B2M and rDNA region is synthesized by Shengong Bioengineering (Shanghai) Co., Ltd., and the nucleotide sequence of the homologous arm skeleton of AAVS1, B2M and rDNA region after synthesis is as shown in SEQ ID NO: 3 (AAVS1), SEQ ID NO: 4 (B2M) and SEQ ID NO: 5 (rDNA region)), and an EF1 alpha promoter is added upstream of the gene. The vector is transformed into E. coli, and ampicillin is selected to obtain positive clones. Taking the AAVS1 gene locus corresponding to the targeting vector as an example, the main elements and the site-specific integration process are shown in FIG. 1.

[0084] Example 2 Targeting vector transient transfection of 293T cells for expression

[0085] 1. Endotoxin-free plasmid extraction: The NKG2D-CD8TM-4-1BB-CD3 zeta plasmid targeting vector was extracted using the OMEGA kit E.Z.N.A. Endo-free Plasmid mini Kit II (catalog number: D6950), and the specific extraction steps were performed according to the kit instructions.

[0086] 2. Transfection of 293T cells

[0087] ① Trypsinize the cells and count them, take 180,000 cells and inoculate them into a 24-well plate, add 500 μl of medium, so that the confluence reaches 60%-80% after 24 hours.

[0088] ② The transfection reagent is Polyplus's transfection reagent (catalog number: 1011000046), according to the method in the instructions, 50 μl of buffer is used to resuspend 250 ng of plasmid, mixed evenly, then 1 μl of reagent is added, mixed well, and incubated at room temperature for 15 min.

[0089] ③ The above mixture is evenly added dropwise to the cell culture system.

[0090] ④ Incubate at 37°C, 5% CO2, 100% saturated humidity for 12 hours, then replace the 293T complete medium and continue to culture. After 48 hours of transfection, the cells are collected for flow cytometry analysis to detect NKG2D expression.

[0091] 3. Flow cytometry detection of NKG2D positive expression

[0092] Trypsinize the cells for 2 min, resuspend the cells in 293T complete medium, centrifuge at 1000 rpm for 5 min to remove the supernatant, and wash once with DPBS. Then resuspend the cells in 100 μl of DPBS containing 1 μl of PE-cy7 NKG2D flow cytometry antibody (Biolegend, #320812), stain at room temperature for 30 min, and wash once with DPBS. Then resuspend in 200 μl of DPBS and detect the NKG2D positive proportion. The flow cytometry results are shown in Figure 2, where (a) is the control group, 293T without transfection. Figures b / c / d in Figure 2 are the transfection results of NKG2D-CD8TM-4-1BB-CD3 zeta targeting vectors at AAVS1 / B2M / rDNA sites. The vertical axis SSC-H represents the lateral scattering value, and the horizontal axis represents the fluorescence intensity after antibody staining. From the flow cytometry results, it can be seen that the three targeting vectors synthesized in Example 1 can successfully express NKG2D.

[0093] Example 3 Single cell nuclear transfer of human iPSCs with targeting vectors

[0094] 1. Nuclear transfer of AAVS1, B2M sites

[0095] Single cell nuclear transfer of iPSCs was performed using Thermo's Neon TM Transfection kit.

[0096] ① 2h before nuclear transfer, replace fresh mTeSR-Plus medium (purchased from STEMCELL, item number: 100-0276) in iPSCs culture wells, and add 10μM Y27632.

[0097] ② Aspirate the mTeSR-Plus medium, add an appropriate amount of TrypLE Select enzyme (purchased from Thermo Fisher, item number: 12563029), and digest the cells at 37°C for 5 minutes. When the cells are observed to round and gradually detach, aspirate the TrypLE Select and add 3-5mL of mTesR-Plus medium.

[0098] ③ Blow off the detached cells with a large tip and resuspend them into a single cell suspension. After cell counting, centrifuge at 90g for 5 minutes at room temperature.

[0099] ④ After aspirating the supernatant, resuspend the cells with 1x DPBS. Dilute the cells to 1.5x10 6 Dilute the number of iPSCs cells in each tube into a 15mL centrifuge tube and centrifuge again.

[0100] ⑤ While centrifuging, prepare Cas9-RNP. In a 1.5mL EP tube, add 16.7μl buffer R and 1μl Cas9 protein (25pmol) (purchased from Jinser Biological Technology Co., Ltd., item number: Z03702), 2.3μl sgRNA (50pmol) (synthesized by Jinser Biological Technology Co., Ltd., sequence as SEQ ID NO: 6 (AAVS1), SEQ ID NO: 7 (B2M)), and incubate at 37°C for 15 minutes to form RNP.

[0101] ⑥ Aspirate the 1x DPBS supernatant in the centrifuge tube, resuspend the iPSCs with 100μl buffer R, mix with RNP, and then add 4μg of nuclear transfer plasmid (targeting vector synthesized in Example 1) and mix.

[0102] ⑦ Perform nuclear transfer according to the nuclear transfer program 14.

[0103] 8. The iPSCs after nucleofection were seeded into the well plates pre-coated with Matrigel (purchased from Corning, Cat# 354277) and added with 10 mM Y27632 (purchased from STEMCELL, Cat# 72304), and then placed in a 37°C 5% CO2 saturated humidity incubator for culture with mTesR-Plus medium after cross shaking.

[0104] 9. The fresh medium was replaced and added with 10 mM Y27632 12 hours after nucleofection, and the cell adhesion was observed under a microscope. Then, the fresh medium was replaced every day.

[0105] 2. rDNA site nucleofection

[0106] The iPSCs were subjected to single cell nucleofection using the LONZA Amaxa Human Stem Cell Nucleofector Starter Kit nucleofection kit. The kit was pre-warmed at room temperature for 30 minutes before nucleofection.

[0107] 1. 2 hours before nucleofection, the iPSCs culture well was replaced with fresh mTeSR-Plus medium (purchased from STEMCELL, Cat# 100-0276) and added with 10 mM Y27632.

[0108] 2. The mTeSR-Plus medium was aspirated, and an appropriate amount of TrypLE Select enzyme (purchased from Thermo Fisher, Cat# 12563029) was added to digest the cells at 37°C for 5 minutes. When the cells were observed to be rounded and gradually detached, the TrypLE Select was aspirated, and 3-5 mL of mTesR-Plus medium was added.

[0109] 3. The detached cells were blown down with a large tip and resuspended into a single cell suspension. After cell counting, the cells were centrifuged at 90g for 5 minutes at room temperature.

[0110] 4. After aspirating the supernatant, the cells were resuspended with 1x DPBS. The cells were counted to be 1.5x10 6 The number of iPSCs cells in each tube was divided into a 15 mL centrifuge tube and centrifuged again.

[0111] 5. Centrifuge while preparing nucleofection solution, add 82 μΐ of Solution 2 and 18 μΐ of Supplement 1 (both components of Amaxa Human Stem Cell Nucleofector Starter Kit) into a 1.5 mL EP tube, and add 5 μg of NKG2D-CD8TM-4-1BB-CD3ζ targeting plasmid and 5 μg of each of nucleic acid tool enzymes TALEN, TALENickases into the tube, mix well, and let stand at room temperature for 5 min.

[0112] 6. Resuspend iPSCs with nucleofection solution containing mixed plasmids, and transfer them into a LONZA nucleofection cup, making sure that no bubbles are generated in the cup during the transfer.

[0113] 7. Tighten the cap of the nucleofection cup and place it into the nucleofector, and select nucleofection program B-016 for nucleofection.

[0114] 8. Immediately after nucleofection, add 0.5 ml of pre-warmed mTeSR-Plus at 37 °C into the nucleofection cup, and mix gently with a disposable plastic tip to neutralize the cells, and incubate at 37 °C for 5 min.

[0115] 9. Uniformly seed the nucleofected iPSCs dropwise into a 12-well plate pre-coated with Matrigel (purchased from Corning, Cat. No. 354277) using a disposable plastic tip, and add 10 μΜ Y27632 (purchased from STEMCELL, Cat. No. #72304), shake crossly, and then place the plate in a 37 °C 5% CO2saturated humidity incubator for culture with mTeSR-plus medium.

[0116] 10. Replace fresh medium and add 10 μΜ Y27632 12 h after nucleofection, and observe the cell adhesion under a microscope. Then, replace fresh medium every day.

[0117] 3. Screening of rDNA locus site G418-resistant iPSCs clones

[0118] 1. Replace fresh medium for the nucleofected iPSCs every day, and observe the morphology and density of the cell clones.

[0119] 2. Replace mTeSR-Plus medium containing G418 (Geneticin) at a final concentration of 50 μg / mL every day for screening of the cells 5-7 days after nucleofection when the cell density reaches 60-70%.

[0120] ③After 4-5 days of G418 screening, cells without NEO gene integration expression (unsuccessfully targeted cells) gradually died off, and only a small number of resistant clones could survive.

[0121] ④Stop adding G418 screening for 2-3 days, and wait for the G418-resistant clones to recover growth, and obtain the cells after G418 screening, which can be used for subsequent single cell inoculation.

[0122] 4, AAVS1 and B2M site NKG2D positive cell flow sorting

[0123] ①At 10-14 days after nucleofection, when the transient plasmid is almost completely lost, NKG2D flow sorting is performed. 107 cells (4 6cm dishes, 80% confluence) are prepared for sorting before sorting.

[0124] ②Discard the culture medium, wash with DPBS, add 2ml TryPLE 37℃ digestion for 4-6min, take it out under a microscope at 4min to observe the digestion. When the cells are round and gradually fall off under the microscope, aspirate the TrypLE Select.

[0125] ③Blow the cells with medium, resuspend and count.

[0126] ④After centrifugation at 1000rpm for 5min, aspirate the supernatant and wash with DPBS once.

[0127] ⑤Discard the supernatant, resuspend with DPBS to 1x10 7 / ml, add 10μl PE-Cy7 NKG2D flow cytometry antibody, and stain for 30min.

[0128] ⑥After staining, centrifuge at 1000rpm for 5min, aspirate the supernatant, and wash with DPBS once.

[0129] ⑦Resuspend with 1ml DPBS, and sort on the machine. The NKG2D positive rate is about 1%, and 10w iPSCs are obtained. In order to exclude the residual transient plasmid leading to NKG2D expression, the sorted clones continue the subsequent clone selection steps, and PCR identification of site-specific integration clones.

[0130] 5, Single cell inoculation and picking of single clones

[0131] ①The rDNA region after G418 screening and the cells sorted by AAVS1 and B2M sites were inoculated with single cells, and positive clones were picked. Specifically, the above cells were first digested with 1-2mL TrypLE Select enzyme at room temperature for 5min, and when the cells were round and gradually fell off under the microscope, the TrypLE Select was aspirated.

[0132] ②Add 3-5 mL mTesR-Plus medium, blow off the cells with a large tip and resuspend into a single cell suspension, and then count the cells and centrifuge at 90g for 5 min at room temperature.

[0133] ③Take 1000 cells and inoculate them in a 6cm dish coated with Matrigel, and then add 3 mL of mTesR-Plus medium containing 10% CloneR additive (purchased from STEMCELL, item number: #05888), and then follow the instructions for subsequent medium replacement. The rDNA site cells can continue to be screened with G418 after the removal of CloneR to improve the positive rate.

[0134] ④Mechanically pick the single cell clones in the 6cm dish with a small tip and inoculate them in a 48-well plate coated with Matrigel, and then culture them with mTesR-Plus medium, and then shake them and place them in a 37°C 5% CO2 saturated humidity incubator.

[0135] ⑤Culture the single cell clones for 4-5 days, and then transfer them from the 48-well plate to two 48-well plates, one line for cell expansion culture and the other line for cell crude lysis for subsequent identification.

[0136] 6. Clone identification

[0137] ①Cell crude lysis

[0138] 2 mL of 1M Tris-HCl (pH = 8.2) + 0.2 ml of Triton-x100 + 18.8 ml of H2O to make 20 ml of cell crude lysis solution, and then add proteinase K at 1:100. After collecting the cells, wash them once with DPBS and discard the supernatant, resuspend the cells with 50 μl of lysis solution, and then lyse them at 56°C overnight. Then heat them at 95°C for 10 min to inactivate the proteinase K.

[0139] ②PCR identification of single cell clones

[0140] Design primers spanning the upstream homologous arm region and the downstream homologous arm region for PCR amplification and identification.

[0141] The primer sequences used are shown in Table 1:

[0142] Table 1. Primer sequences

[0143] AAVSI, B2M each site were picked 48 iPSCs monoclonal to expand culture, using the primer shown in Table 2, cross upstream homologous arm region, cross downstream homologous arm region PCR amplification identification. As shown in Figure 3, wherein AAVS1 site has 2 clones PCR upstream and downstream positive, sequencing results are correct, named AAVS1-NKG2D-11 and AAVS1-NKG2D-146 respectively. As shown in Figure 4, B2M site has 5 clones upstream and downstream PCR identification positive, but there are 4 clones sequencing shows that the downstream deletion of 100 bp fragment, so the final positive clone of B2M site has one, named B2M-NKG2D-103. As shown in Figure 5, in the rDNA region targeting experiment, a total of 21 G418 resistant clones were picked, of which 12 were PCR positive, named rDNA-NKG2D-(1, 2, 4, 6, 7, 8, 9, 11, 16, 18, 19, 21), and part of the better clones were continued qPCR verification.

[0144] 7. Flow identification of NKG2D expression level of site-specific integration clone

[0145] In the rDNA region, the highest qPCR clone No. 18 was selected for flow identification, and the clones of the remaining two sites were all identified by flow. The flow staining steps were the same as the staining steps when sorting NKG2D positive cells.

[0146] As shown in Figure 6, the positive clones of rDNA region and the two positive clones of AAVS1 site cannot successfully express NKG2D. The positive clone of B2M site can stably express NKG2D. Therefore, when the NKG2D-CAR expressing gene is site-specifically integrated into the cell genome, not any site is suitable for the expression of NKG2D-CAR. For example, the rDNA region and the AAVS1 site are difficult to successfully and effectively express NKG2D-CAR, and the inventors of the present application have found that the B2M site is an effective site suitable for expressing NKG2D-CAR. Therefore, in subsequent experiments, the positive clone of B2M site was selected for the differentiation of iMSCs.

[0147] Example 4 Induction differentiation and cell expression of site-specific integration clone B2M-NKG2D-CAR-iMSCs

[0148] 1. Induction differentiation and cell culture of site-specific integration clone B2M-NKG2D-CAR-iMSCs

[0149] According to the STEMdiff TMMesenchymal Progenitor Kit (Catalog #05240) using the instruction manual, the integrated B2M-NKG2D-CAR-iPSCs clone B2M-NKG2D-103 was directionally differentiated into iMSCs, which was named B2M-NKG2D-CAR-iMSCs. After analysis, as shown in Figure 7, it had the typical characteristics of MSCs: including cell growth morphology, surface markers. B2M-NKG2D-CAR-iMSCs showed fibroblast-like morphology, and had a clear fingerprint spiral distribution trend when reaching confluence. The surface markers of B2M-NKG2D-CAR-iMSCs were flow cytometry identified, and the results showed that the cell surface markers were CD44+, CD73+, CD90+, CD105+, CD34- / CD45- / HLA-DR-, which was consistent with the characteristics of tissue-derived MSCs, and met the identification standard of ISCT (Figure 8).

[0150] 2. Flow cytometry identification of NKG2D expression level of B2M-NKG2D-CAR-iMSCs

[0151] Aspirate the culture medium of iMSCs, wash twice with DPBS, and resuspend with TrypLE TM Incubate the cells at room temperature for 3 min, resuspend with an appropriate amount of complete culture medium, and transfer to a 15 mL centrifuge tube. Centrifuge at 175 g for 5 min.

[0152] After aspirating the supernatant, wash the cells once with DPBS, and centrifuge at 175 g for 5 min. After centrifugation, aspirate the supernatant, resuspend the cells with 100 μL of 5% FBS-DPBS, and add 1 μl of PE-Cy7-NKG2D flow cytometry antibody. Incubate at room temperature in the dark for 30 min. After incubation, add 2 volumes of 5% FBS-DPBS to terminate incubation, centrifuge at 175 g for 5 min, discard the supernatant, resuspend the cells with 150 μL of 5% FBS-DPBS per tube, and detect by flow cytometry within 1 h. The flow cytometry results showed that B2M-NKG2D-CAR-iMSCs successfully and stably expressed NKG2D (Figure 9).

[0153] Example 5. Enhanced tumor adhesion ability of B2M-NKG2D-CAR-iMSCs in vitro

[0154] Evaluation of the adhesion ability of B2M-NKG2D-CAR-iMSCs cells to tumor cells:

[0155] Tumor cell lines: Breast cancer cell line MCF7 cells, non-small cell lung cancer cell line A549 cells, and large cell lung cancer H460 were selected as detection objects of iMSC adhesion ability, all of which were solid tumor cell lines.

[0156] Specifically, in order to detect whether NKG2D can enhance the targeting and adhesion ability of iMSCs to solid tumor cells, the interaction experiment of fluorescently labeled cells was carried out by flow cytometry in this embodiment. The tumor cells were co-cultured with B6-iMSCs cells or B2M-NKG2D-CAR-iMSCs cells. Before co-culture, the tumor cells were labeled with CellTrace FarRed (Invitrogen, C34564), and the B6-iMSCs cells and B2M-NKG2D-CAR-iMSCs cells were labeled with CellTrace Violet (Invitrogen, C34557). The specific operation steps of the labeling method are as follows:

[0157] 1. First, the B6-iMSCs cells or B2M-NKG2D-CAR-iMSCs cells were digested into single cells by TrypLE Select, and inoculated into a 12-well plate at a concentration of 200,000 / mL / well, and the cells were shaken evenly using the cross shaking method. TM Select digestion, inoculate into a 12-well plate at a concentration of 200,000 / mL / well, and shake the cells evenly using the cross shaking method.

[0158] 2. After 12h, the MSC culture medium in the 12-well plate where the iMSCs were fully spread was discarded. The solid tumor cells MCF7, A549 and H460 cells digested with 0.25% trypsin were inoculated into the iMSCs plated on the plate at a concentration of 100,000 / mL / well (effector to target ratio 2:1), 200,000 / mL / well (effector to target ratio 1:1), and 400,000 / mL / well (effector to target ratio 1:2), and the cells were shaken evenly using the cross shaking method.

[0159] 3. Incubate at 37°C in a 5% CO2 cell incubator for 90min.

[0160] 4. Discard the culture medium in the 12-well plate, wash once with 1×DPBS. Add 1×DPBS to the 12-well plate again, and place it on a horizontal shaker and rotate at a speed of 130rpm / min at room temperature for 3min to remove weakly adherent solid tumor cells.

[0161] 5. Collect the cells by TrypLE Select digestion, and digest at room temperature for 30s. Ensure that the blowing strength and blowing frequency are consistent when collecting the cells. TM Select digestion, and digest at room temperature for 30s. Ensure that the blowing strength and blowing frequency are consistent when collecting the cells.

[0162] 6. Centrifuge at 350g for 5min at room temperature, discard the supernatant, and resuspend the cells with 200μL of DPBS for flow cytometry detection.

[0163] The results of flow detection are shown in Figure 10. The CellTrace Violet / Far Red double-positive cell population is considered as iMSCs / solid tumor cell aggregates, and the percentage thereof is calculated. The results show that the degree of interaction of B2M-NKG2D-CAR-iMSCs with the three tumor cells is significantly higher than that of B6-iMSCs. This result shows that NKG2D enhances the binding ability of iMSCs to solid tumor cells A549, MCF7, and H460, significantly improving the targeting and adhesion ability of iMSCs to solid tumor cells.

[0164] Example 6 B2M-NKG2D-CAR-iMSCs enhance tumor targeting infiltration effect in vivo

[0165] 1. Establish a human mouse xenograft tumor model expressing positive NKG2D ligand

[0166] In this embodiment, a subcutaneous tumor model of human lung cancer cell line A549 cells in nude mice (BALB / c nude, purchased from Vivotan) was established, as shown in Figure 11: 5 million A549-Luc2 cells were subcutaneously injected into the axillary of the mice on day 0, and after 2 weeks of tumor formation, 1 million iMSCs were injected into the mice through the tail vein, only once. After 2 weeks of injection, the mice were sacrificed, and the tumor tissue and internal organs were collected. Live imaging was performed every 2 days after injection, and the body weight of the mice was recorded until the end of the experiment.

[0167] Specifically, first, 5 million 549 cells (purchased from Zhejiang Meisen Cell Technology Co., Ltd.) were transplanted subcutaneously into the skin of nude mice (recorded as Day 0), and then the progress of the A549 cell graft at the injection site was observed. When the tumor volume reached about 100 mm 3At the time, mice were divided into 3 groups, one of which was injected with 1 million B6-iMSC cells for treatment, recorded as the control group (B6-iMSCs); one group was injected with 100w B2M-NKG2D-CAR-iMSC cells for treatment, recorded as the B2M-NKG2D-CAR-iMSCs treatment group (B2M-NKG2D-CAR-iMSCs); one group was injected with DPBS, recorded as the blank group, and the injection method was all tail vein injection. Before being injected, both B6-iMSC cells and B2M-NKG2D-CAR-iMSC cells were stained and labeled with fluorescent dye DiR 750 (IVISense DiR 750, 125964, PerkinElmer), and the specific labeling method was performed according to the instructions. The maximum excitation wavelength and the maximum emission wavelength of DiR 750 were 748 nm and 780 nm, respectively, which were used in this embodiment to monitor the in vivo content of iMSCs and the preferential migration to the tumor site (i.e. targeting). The specific analysis method is: the average value of the signal around the mouse's head and genital area is taken as the background signal. The ratio of the signal at the tumor site to the background signal represents the tendency of MSC cells to the tumor.

[0168] 2. NKG2D enhances the residence of iMSCs at the tumor site

[0169] As shown in Figure 12, because MSCs are cleared by the body, and the background signal of the mouse itself is stronger than that at the tumor site, the tumor site / background signal will gradually decrease over time. However, compared with B6-iMSCs, B2M-NKG2D-CAR-iMSCs have stronger residence ability at the tumor site. On the 3rd day after injection of iMSCs, the B6-iMSC signal was basically consistent with the B2M-NKG2D-CAR-iMSC signal, but on the 13th day after injection of iMSCs, the B6-iMSC signal decreased significantly, almost consistent with the blank group, while the B2M-NKG2D-CAR-iMSCs had a more obvious signal enrichment, and the B2M-NKG2D-CAR-iMSC signal was improved by 57% compared with B6-iMSC. These results prove that compared with B6-iMSCs, B2M-NKG2D-CAR-iMSCs indeed have better residence ability at the tumor site.

[0170] At the end of the experiment, the mouse tumor was dissected for imaging, which was consistent with the above results. Compared with the iMSC group, the tumor injected with B2M-NKG2D-CAR-iMSCs had stronger signal retention, and the signal value was improved by 27% (Figure 13).

[0171] Finally, it is to be explained that the above embodiments are only used to illustrate the technical solutions of the present application but not to limit the present application. Although the present application 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 application can be modified or equivalently replaced without departing from the purpose and scope of the present application, and all of them should be covered in the scope of the claims of the present application.

Claims

1. An engineered mesenchymal stem cell, characterized in that... The engineered mesenchymal stem cells express a chimeric antigen receptor targeting NKG2DL or contain a polynucleotide encoding a chimeric antigen receptor targeting NKG2DL.

2. The engineered mesenchymal stem cell as described in claim 1, characterized in that... The engineered mesenchymal stem cells have a polynucleotide encoding a chimeric antigen receptor targeting NKG2DL specifically integrated into their genome, and the engineered mesenchymal stem cells express the chimeric antigen receptor.

3. The engineered mesenchymal stem cell as described in claim 2, characterized in that... The site of the targeted integration is the B2M locus.

4. The engineered mesenchymal stem cell as described in claim 1, characterized in that... The chimeric antigen receptor includes an NKG2DL binding domain and a signal transduction domain, wherein the NKG2DL binding domain includes the extracellular domain of NKG2D.

5. An engineered mesenchymal stem cell as described in claim 4, characterized in that... The NKG2D includes any naturally occurring form of NKG2D or a variant of NKG2D that retains its protein activity.

6. An engineered mesenchymal stem cell as described in claim 5, characterized in that... Compared to the naturally occurring form of NKG2D, the NKG2D variant exhibits at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity; or, compared to the naturally occurring form of NKG2D, the NKG2D variant has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the entire or partial sequence of NKG2D. Preferably, the NKG2D has the NCBI reference sequence number NP_031386.2, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the protein or its functional fragment with the NCBI sequence reference sequence number NP_031386.

2.

7. An engineered mesenchymal stem cell as described in claim 4, characterized in that... The signal transduction domain includes a transmembrane domain and an intracellular domain, and the intracellular domain includes a signal transduction chain with an immune receptor tyrosine activation motif (ITAM).

8. An engineered mesenchymal stem cell as described in claim 7, characterized in that... The intracellular domain includes at least one of CD3γ, CD3δ, CD3ε, CD3ζ, Igα (CD79a), Igβ (CD79b), and FcγRIII (CD16).

9. An engineered mesenchymal stem cell as described in claim 7, characterized in that... The intracellular domain further includes an intracellular portion of a co-stimulatory domain, the intracellular portion of which includes CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27, and / or DAP10.

10. An engineered mesenchymal stem cell as described in claim 7, characterized in that... The transmembrane domain includes at least one of the following: NKG2D transmembrane domain, CD8α transmembrane domain, CD28 transmembrane domain, IgG4 transmembrane domain, TNFR transmembrane domain, and TLR transmembrane domain.

11. An engineered mesenchymal stem cell as described in claim 4, characterized in that... The chimeric antigen receptor also includes a hinge region.

12. An engineered mesenchymal stem cell as described in claim 1, characterized in that... The mesenchymal stem cells are derived from adult cells or stem cells.

13. An engineered mesenchymal stem cell as described in claim 12, characterized in that... The mesenchymal stem cells are derived from pluripotent stem cells, and preferably, the pluripotent stem cells are selected from induced pluripotent stem cells.

14. An engineered mesenchymal stem cell as described in claim 12, characterized in that... The mesenchymal stem cells are derived from bone marrow, fat, muscle, heart, umbilical cord blood, or umbilical cord.

15. An engineered mesenchymal stem cell as described in claim 1, characterized in that... The engineered mesenchymal stem cells stably overexpress NKG2D.

16. The engineered mesenchymal stem cell as described in claim 1, characterized in that, Compared with wild-type mesenchymal stem cells, the engineered mesenchymal stem cells have enhanced tumor-targeting invasion ability, tumor survival ability, and tumor cell binding ability.

17. An engineered mesenchymal stem cell as described in claim 1, characterized in that, Compared with wild-type mesenchymal stem cells, the engineered mesenchymal stem cells have a tumor survival rate that is improved by at least 27%; preferably, the engineered mesenchymal stem cells have a tumor survival rate that is improved by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 57%, or at least 60%.

18. A composition, characterized in that, Contains engineered mesenchymal stem cells as described in any one of claims 1-17.

19. The composition according to claim 18, characterized in that, The composition includes one or more other therapeutic agents.

20. The composition according to claim 19, characterized in that, The other therapeutic agents include antitumor drugs. Preferably, the antitumor drug includes at least one of paclitaxel and its derivatives, docetaxel, camptothecin and its derivatives, etoposide, teniposide, doxorubicin hydrochloride, cyclophosphamide, actinomycin, bleomycin, fenofibrate, doxorubicin, epirubicin, mitomycin, methotrexate, 5-fluorouracil, carboplatin, carmustine, lomustine, cisplatin, vincristine, tamoxifen, piperatoxin, and benzethonol.

21. The composition according to claim 19, characterized in that, The one or more other therapeutic agents are administered in combination with the engineered mesenchymal stem cells; Preferably, the combined administration includes administration in any order or at any time interval, such that two or more therapeutic agents exert their biological activity simultaneously; Preferably, the combined administration produces a synergistic therapeutic effect.

22. The composition according to claim 18, characterized in that, The composition also includes pharmaceutically acceptable excipients; Preferably, the excipients include at least one of the following: buffer solution, antioxidant, preservative, protein, hydrophilic polymer, amino acid, sugar, chelating agent, surfactant, metal complex, nonionic surfactant, liposome, albumin microsphere, polyester, micelle, and sustained-release matrix.

23. The method for preparing engineered mesenchymal stem cells according to any one of claims 1-17, characterized in that, This includes introducing the nucleic acid encoding the chimeric antigen receptor into mesenchymal stem cells to obtain engineered mesenchymal stem cells.

24. The preparation method according to claim 23, characterized in that, This includes introducing the aforementioned nucleic acid encoding the chimeric antigen receptor into induced pluripotent stem cells and inducing differentiation to obtain engineered mesenchymal stem cells.

25. The preparation method according to claim 23, characterized in that, This includes using a gene editing system to site-directedly integrate a nucleic acid encoding a chimeric antigen receptor into the genome of mesenchymal stem cells; the site-directed integration gene locus is the B2M locus. Preferably, the gene editing system is selected from the Cre-lox system, Zinc Finger Nucleases (ZFNs), CRISPR-Cas or Transcription Activator-Like Effector Nucleases (TALENs), preferably CRISPR-Cas or TALENs; more preferably the CRISPR / Cas system; Preferably, the CRISPR / Cas system is selected from CRISPR-Cas9, CRISPR-Cas12a(Cpf1), CRISPR-Cas13, CRISPR-Cas14, CRISPR-CasX, and CRISPR-CasY, and more preferably CRISPR-Cas9; Preferably, a targeting vector containing nucleic acid encoding a chimeric antigen receptor or a CRISPER / Cas system is introduced into mesenchymal stem cells, and the nucleic acid encoding the chimeric antigen receptor is site-specifically integrated into the B2M locus through homologous recombination.

26. The preparation method according to claim 23, characterized in that, The introduction method is selected from: transformation, transfection, heat shock, electroporation, transduction, and microinjection; Preferably, the method of introduction is a non-viral method.

27. The use of the engineered mesenchymal stem cells according to any one of claims 1-17 and the composition according to any one of claims 18-22 in the preparation of diagnostic, preventive and therapeutic antitumor drugs; Preferably, the tumor includes at least one of ovarian cancer, uterine cancer, lung cancer, Merkel cell carcinoma, skin cancer, breast cancer, malignant soft tumor, neuroendocrine tumor, brain tumor, pharyngeal cancer, laryngeal cancer, thyroid cancer, esophageal cancer, stomach cancer, colon cancer, liver cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, and bone tumor, and is preferably selected from breast cancer, uterine cancer, ovarian cancer, lung cancer, Merkel cell carcinoma, neuroendocrine tumor, and brain tumor; Preferably, the tumor is selected from tumors expressing NKG2DL molecules; more preferably, the tumor highly expresses NKG2DL molecules. Preferably, the antitumor drug is administered via intravenous, intramuscular, intraperitoneal, cerebrospinal, subcutaneous, intramedullary, intrathecal, oral, local, or inhalation routes.

28. The use of the engineered mesenchymal stem cells according to any one of claims 1-17 or the chimeric antigen receptor targeting NKG2DL according to claims 1-17 in the preparation of a drug that improves the tumor invasiveness of mesenchymal stem cells or the survival of mesenchymal stem cells at tumor sites.

29. A method for improving the tumor infiltration capacity or survival capacity of mesenchymal stem cells at tumor sites, comprising causing the mesenchymal stem cells to express a chimeric antigen receptor targeting NKG2DL.

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