Engineered mesenchymal stem cells and their uses

Abstract

The present invention provides engineered mesenchymal stem cells (MSCs), pharmaceutical compositions comprising the same, and uses thereof in the preparation of medicaments for treating cancer, wherein the MSCs comprise an introduced NQO1 protein coding sequence and / or an introduced hypoxia response element (HRE), and nucleic acids encoding one or more immune-stimulating cytokines.

Description

[Technical Field]

[0001] The present invention relates to engineered genetically modified mesenchymal stem cells (MSCs) and their use in expressing immunostimulatory cytokines for tumor immunotherapy. [Background technology]

[0002] Cancer is a serious disease that threatens human health due to abnormal proliferation or differentiation of cancer cells, resulting in dysfunction, uncontrolled cell growth, local tissue invasion, and metastasis. Genetic mutations allow cancer cells to express specific tumor neoantigens, which can be recognized and killed by immune T lymphocytes, forming the basis of tumor immunotherapy. However, tumors evolve through various mechanisms, including upregulation of inhibitory immune checkpoint molecules and recruitment of suppressive myeloid cells, creating an immunosuppressive microenvironment. After immunotherapy, a lack of T lymphocyte growth factors (e.g., IL-2 and IL-15) to maintain or reactivate stimulation can lead to T lymphocyte dysfunction and tumor recurrence. As an agonist molecule for T lymphocytes and NK cells, IL-2 was the first cytokine approved by the FDA and is used to treat metastatic melanoma and renal cancer. However, clinical application of IL-2 for cancer therapy has been hampered by its short half-life and severe dose-dependent toxic side effects. More importantly, IL-2 also expands immunosuppressive regulatory T cells (Tregs), limiting their antitumor efficacy. To overcome these issues, various strategies have been attempted to engineer IL-2 variants that selectively expand effector T cells but not Tregs. However, systemic administration of IL-2 variants tends to expand T cells and NK cells in lymphoid and non-tumor tissues, potentially resulting in severe toxicity. Therefore, how to specifically deliver cytokines, such as IL-2 or IL-2 variants, to the tumor microenvironment to effectively activate and expand lymphocytes while avoiding peripheral toxicity remains an important current challenge.

[0003] Within solid tumor tissues, stromal cells interact with tumor cells and immune cells to form a complex tumor microenvironment (TME). MSCs are an important source of tumor-associated stromal cells. In many cases, MSCs injected into animals exhibit tumor-homing ability. MSCs can promote tumor progression and suppress inflammation and immune responses. Meanwhile, due to their pleiotropic nature in tumors, MSCs have great potential as cell carriers for delivering or producing antitumor molecules. Several recent studies have reported strategies for using genetically modified MSCs to treat tumors. However, the use of MSCs in tumor therapy faces significant challenges: first, how to convert immunosuppressive MSCs into MSCs with antitumor capabilities; and second, how to specifically express antitumor factors within tumors by MSCs to avoid toxic side effects due to peripheral expression. Furthermore, the short in vivo survival time of transplanted MSCs also impacts their clinical use. Therefore, for clinical application, genetically modified MSCs must be able to improve their in vivo survival time and to regulatably generate tumor-therapeutic factors that activate anti-tumor immune responses locally in the tumor. Summary of the Invention

[0004] In order to solve the above-mentioned problems of the conventional technology, the present inventors precisely modified MSCs to obtain genetically modified MSCs that have improved tumor treatment ability, stronger in vivo survival ability, and the ability to specifically express target gene products in tumor tissue, thereby completing the present invention.

[0005] In a first aspect, the present invention provides engineered mesenchymal stem cells (MSCs) whose genome comprises an introduced NQO1 protein coding sequence. In one embodiment, the introduced NQO1 protein coding sequence enables the engineered MSCs to overexpress NQO1 protein. Overexpression of the NQO1 protein can extend the survival time of the engineered MSCs in tumor tissue. In one embodiment, the NQO1 protein coding sequence is introduced into the genome of an MSC by lentiviral vector transfection, adenoviral vector transfection, or mRNA transfection, resulting in the engineered MSCs. In one embodiment, the genome of the engineered MSCs further comprises introduced regulatory elements and a target gene, and expression of the target gene is regulated by the regulatory element.

[0006] In a second aspect, the present invention provides an engineered MSC, the genome of which comprises an introduced hypoxia response element (HRE) and optionally introduced regulatory elements and a target gene located downstream of the HRE, wherein expression of the target gene is regulated by the regulatory elements. The HRE can promote expression of a gene downstream of the regulatory element by activating a downstream regulatory element (e.g., a promoter) under a hypoxic environment in tumor tissue. In one embodiment, the HRE is introduced into the genome of the engineered MSC by lentiviral vector transfection, adenoviral vector transfection, or mRNA transfection. In one embodiment, the HRE, together with its downstream regulatory element (e.g., a promoter) and target gene, is introduced into the genome of the engineered MSC by lentiviral vector transfection, adenoviral vector transfection, or mRNA transfection. In one embodiment, the HRE and the downstream regulatory element and target gene are operably linked in the genome of the engineered MSC. In one embodiment, the sequence of the HRE is derived from any one of the Eno1, Epo, VEGF-A, PGK, Ldha, ALDA and GAPDH genes, preferably selected from SEQ ID No: 2 and SEQ ID No: 6-11, more preferably SEQ ID No: 2 derived from the Eno1 gene.

[0007] In a third aspect, the present invention provides an engineered MSC, the genome of which comprises (i) an introduced NQO1 protein coding sequence and (ii) an introduced hypoxia response element (HRE). In one embodiment, the genome of the engineered MSC further comprises an introduced regulatory element and a target gene downstream of the HRE, wherein expression of the target gene is regulated by the regulatory element. In one embodiment, the HRE and the downstream regulatory element and target gene are operably linked in the genome of the engineered MSC.

[0008] In any of the above embodiments, when the engineered MSCs further comprise a regulatory element and a target gene, the regulatory element may comprise a promoter.

[0009] In any of the above embodiments, when the engineered MSCs further comprise a regulatory element and a target gene, the target gene may encode an anti-tumor protein, such as an immunostimulatory cytokine or an anti-tumor cytokine. In one embodiment, the target gene encodes any one of IL-2, IL-1, IL-7, IL-21, IL-12, IL-15, and variants thereof. In one embodiment, the IL-1 is IL-1β. In one embodiment, the IL-2 variant is sumIL2.

[0010] The engineered MSCs of the present invention utilize MSCs as a carrier to specifically deliver immunostimulatory or antitumor cytokines to the tumor microenvironment, thereby improving the antitumor effect and avoiding the peripheral toxic side effects caused by systemic administration of the cytokines. Furthermore, no tumorigenesis-promoting activity was observed in the engineered MSCs of the present invention.

[0011] In a fourth aspect, the present invention further provides a pharmaceutical composition for treating cancer comprising the engineered MSC of any one of the first to third aspects, wherein the genome of the engineered MSC further comprises the introduced regulatory element and a target gene encoding an immunostimulatory cytokine or an anti-tumor cytokine. In one embodiment, the pharmaceutical composition further comprises an immune checkpoint inhibitor and / or adoptive T cells that specifically recognize tumor cells.

[0012] In a fifth aspect, the present invention further provides a method for treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the engineered MSC of any one of the first to third aspects, wherein the genome of the engineered MSC further comprises the introduced regulatory element and a target gene encoding an immunostimulatory cytokine or an anti-tumor cytokine. In one embodiment, the subject is human. In one embodiment, the method further comprises administering an immune checkpoint inhibitor to the subject. In one embodiment, the method further comprises administering adoptive T cell therapy (ACT) to the subject.

[0013] In a sixth aspect, the present invention further provides the use of the engineered MSCs of any one of the first to third aspects in the preparation of a medicament for treating cancer, wherein the genome of the engineered MSCs further comprises the introduced regulatory element and a target gene encoding an immunostimulatory cytokine or an anti-tumor cytokine. In one embodiment, the medicament further comprises a second anti-cancer agent, such as an immune checkpoint inhibitor, and / or adoptive T cells that specifically recognize tumor cells.

[0014] In each of the fourth to sixth aspects, the regulatory element may comprise a promoter. In one embodiment, the promoter is an SV40 promoter. In one embodiment, in the engineered MSC, the HRE, the regulatory element, and the target gene are operably linked. In one embodiment, the target gene encodes any one of IL-2, IL-1, IL-7, IL-21, IL-12, IL-15, and variants thereof. In one embodiment, the IL-1 is IL-1β. In one embodiment, the IL-2 variant is sumIL2. In one embodiment, the cancer may be selected from B-cell lymphoma, bronchial cancer, prostate cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral cancer, laryngeal cancer, salivary gland cancer, thymus cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, adipose cancer, testicular cancer, malignant fibrous histiocytoma, colorectal cancer, melanoma, gastric cancer, pancreatic cancer, lung cancer, liver cancer, kidney cancer, bile duct cancer, small intestine or appendix cancer, squamous cell carcinoma, breast cancer, and ovarian cancer.

[0015] The present invention further provides the use of the engineered mesenchymal stem cells of any one of the first to third aspects as a carrier for delivering a target gene. In one embodiment, the carrier is used to deliver the target gene to tumor tissue. In one embodiment, the target gene is a tumor therapeutic gene, preferably a gene encoding an immunostimulatory protein or an anti-tumor protein, more preferably an immunostimulatory cytokine or an anti-tumor cytokine, such as the specific cytokines mentioned above.

[0016] In other aspects, the present invention further provides use of an NQO1 protein for improving the survival time of mesenchymal stem cells in tumor tissue, use of a hypoxia response element (HRE) for enhancing expression of a target gene in mesenchymal stem cells under hypoxic conditions, and use of an NQO1 protein-coding sequence in combination with a hypoxia response element (HRE) for preparing a pharmaceutical composition comprising mesenchymal stem cells carrying a target gene. In one embodiment, the hypoxic environment is a tumor microenvironment. In one embodiment, the genome of the mesenchymal stem cell comprises (i) an introduced NQO1 protein-coding sequence, and / or (ii) the introduced hypoxia response element and an introduced regulatory element downstream of the hypoxia response element and the target gene, and expression of the target gene is regulated by the regulatory element. [Brief explanation of the drawings]

[0017] Hereinafter, a description will be given of an embodiment of the present invention with reference to the drawings, but neither the drawings nor the following embodiment of the present invention are intended to limit the scope of the present invention. [Figure 1] Figure 1 shows the survival of engineered MSCs expressing NQO1 in the tumor microenvironment. (a) Representative graph of flow cytometry analysis results. (b) Number of viable MSCs in tumor tissue per unit weight. [Figure 2] Figure 1 shows the effect of HRE-engineered MSCs on target gene expression. (a) Schematic diagram showing the mechanism of action of HRE. (b) IL-1β expression in NQO1 / HRE-engineered MSCs carrying the IL-1β gene under normoxic and hypoxic conditions. (c) SIL-2 expression in NQO1 / HRE-engineered MSCs carrying the sumIL-2 (SIL-2) gene under normoxic and hypoxic conditions. (d) Antitumor effects of non-engineered MSCs, NQO1-engineered MSCs, and NQO1 / HRE-engineered MSCs carrying the sumIL-2 (SIL-2) gene. [Figure 3]Figure 1 shows that NQO1 / HRE-engineered MSCs expressing SIL2 (MSC-NQO1-HRE-SIL2) specifically express SIL2 in tumor tissue. (a) SIL2 levels in tumors after intratumoral (it) or peritumoral (pt) injection of NQO1 / HRE-engineered MSCs expressing SIL2-Fc protein or SIL-2. (b) SIL2 levels in serum after intratumoral (it) or peritumoral (pt) injection of NQO1 / HRE-engineered MSCs expressing SIL2-Fc protein or SIL-2. (c) IFNγ levels in serum after intratumoral (it) or peritumoral (pt) injection of NQO1 / HRE-engineered MSCs expressing SIL2-Fc protein or SIL-2. (d) Body weight change of mice after intratumoral (it) or peritumoral (pt) injection of NQO1 / HRE-engineered MSCs expressing SIL2-Fc protein or SIL-2, except for intratumoral injection of phosphate buffered saline (PBS) as a control. [Figure 4] Figure 1 shows the effect of different doses of SIL2 protein and engineered MSCs expressing SIL2 (MSC-NQO1-HRE-SIL2) cells on tumor volume, except for the control injected with phosphate buffered saline (PBS). [Figure 5] Figure 1 shows the percentage of Ki67+ CD8+ T cells (a) and the ratio of CD8+ T cells to Treg cells (b and c) in tumors after injection of control (PBS), engineered MSCs without tumor-inhibitory cytokines (MSC-NQO1), engineered MSCs expressing wild-type IL-2 (wtIL2), and engineered MSCs expressing SIL2. Statistical significance: ns - not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. [Figure 6]The effects of engineered MSCs expressing SIL2 (MSC-NQO1-HRE-SIL2) and / or immune checkpoint inhibitors on tumor volume and survival in tumor-bearing mice are shown. Tumor volume (a) and survival time (b) of mice were measured after injection with control MSCs (without MSC-NQO1, HRE, and SIL2) (●), control MSCs plus immune checkpoint inhibitors (black squares), MSC-NQO1-HRE-SIL2 (▲), or MSC-NQO1-HRE-SIL2 plus immune checkpoint inhibitors (▼). Statistical significance: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. [Figure 7] Figure 1 shows the effect of engineered MSCs (MSC-NQO1-HRE-SIL2) expressing SIL2 locally on distant tumors. (a) Volume change of the right tumor, (b) volume change of the left tumor after injection of control MSCs (without MSC-NQO1, HRE, and SIL2) or MSC-NQO1-HRE-SIL2 into the right tumor of tumor-bearing mice. [Figure 8] The effect of engineered MSCs expressing SIL2 (MSC-NQO1-HRE-SIL2) in combination with adoptive cell therapy (ACT) on advanced tumors is shown. Figure 1 shows the change in tumor volume (a) and survival percentage (b) after treatment with control MSCs (MSC-NQO1), MSC-NQO1-HRE-SIL2, control MSCs plus two concentrations of ACT, and MSC-NQO1-HRE-SIL2 plus low-concentration ACT. Statistical significance: *** p<0.001; **** p<0.0001 (two-way ANOVA (a) and log-rank test (b)). [Figure 9]This shows that engineered MSCs expressing IL1β (MSC-NQO1-HRE-IL1β) specifically express IL1β in tumor tissues. (a) IL1β levels in tumors, organs, and serum after intratumoral injection of IL1β protein or MSC-NQO1-HRE-IL1β. (b) Inflammatory factor parameter levels in serum after intratumoral injection of PBS control, IL1β protein, or MSC-NQO1-HRE-IL1β. (c) Changes in body weight of mice after intratumoral injection of PBS control, IL1β protein, or MSC-NQO1-HRE-IL1β. (d) Changes in tumor volume of mice after intratumoral injection of PBS control, IL1β protein, or MSC-NQO1-HRE-IL1β. Statistical significance: ns - not significant; * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0018] Terms and Definitions In the present invention, the term "introduced NQO1 protein coding sequence" refers to a nucleic acid sequence encoding an NQO1 protein that is introduced into the genome of a target cell (e.g., an MSC) by genetic modification to distinguish it from the NQO1 gene naturally present in the genome of the cell. The NQO1 protein coding sequence may be, for example, but is not limited to, the coding sequence of human NQO1 protein (e.g., NCBI sequence NP_000894.1), the coding sequence of mouse NQO1 protein (e.g., SEQ ID No: 1), or the coding sequence of NQO1 protein of other mammals.

[0019] In the present invention, the term "introduced hypoxia response element (HRE)" refers to a hypoxia response element (HRE) introduced into the genome of a target cell (e.g., MSC) by genetic modification, thereby distinguishing it from an HRE naturally present in the genome of the cell. The introduced HRE is typically operably linked downstream to a regulatory element and a target gene, so as to promote specific expression of the target gene under hypoxic conditions. The sequence of the HRE may be derived from a human or mouse, and may be derived from genes such as Eno1, Epo, VEGF-A, PGK, Ldha, ALDA, and GAPDH. The sequence may be, for example, SEQ ID No. 2 derived from Eno1.

[0020] In the present invention, the "mesenchymal stem cells (MSCs)" used for engineering may be, but are not limited to, mammalian-derived MSCs, such as human, rat, or mouse-derived MSCs, for example, MSCs derived from the bone marrow thereof.

[0021] In the present invention, "sumIL-2" or "SIL-2" may be used interchangeably and is a mutant form of IL-2 that contains F42A, L80F, R81D, L85V, I86V and I92F mutations based on wild-type IL-2.

[0022] Herein, NQO1 / HRE double-engineered MSCs expressing a target gene are referred to as "MSC-NQO1-HRE-(target gene)." For example, if the target gene is SIL-2, NQO1 / HRE-engineered MSCs expressing SIL-2 are referred to as "MSC-NQO1-HRE-SIL2." Similarly, for example, "MSC-NQO1" refers to MSCs engineered with NQO1 alone, without an HRE and a target gene regulated by the HRE; "MSC-NQO1-SIL2" refers to MSCs engineered with NQO1 but not an HRE, but containing an introduced SIL-2 gene; and "MSC-SIL2" refers to MSCs containing an introduced SIL2 gene but not engineered with NQO1 or an HRE.

[0023] Hereinafter, several embodiments of the present invention will be described, but these embodiments do not limit the scope of the present invention, and it will be apparent to those skilled in the art that the embodiments can be modified into equivalent embodiments that can be foreseen by those skilled in the art and combined with each other.

[0024] I. NQO1 Engineering MSC The present invention provides engineered mesenchymal stem cells (MSCs) whose genome contains an introduced NQO1 protein coding sequence. NQO1 stands for NAD(P)H:quinone oxidoreductase 1. In the present invention, NQO1 is overexpressed in MSCs to promote the survival of adoptively transferred therapeutic MSCs in tumor tissue. Surprisingly, the inventors found that engineered MSCs of the present invention overexpressing NQO1 have a significantly longer survival time in the tumor microenvironment compared to non-engineered MSCs (see Figure 1). This significantly increases the usefulness of MSCs as intratumoral cell carriers, and may be used, for example, to more sustainedly or stably deliver or produce antitumor molecules or other functional molecules into tumors. The NQO1 protein coding sequence in the engineered MSCs of the present invention may be any NQO1 protein coding sequence, such as, but not limited to, the coding sequence of human NQO1 or mouse NQO1 protein (e.g., SEQ ID No. 1).

[0025] Methods for introducing the NQO1 protein coding sequence into the MSC genome may include, but are not limited to, introducing a vector (e.g., a lentiviral vector or an adenoviral vector) containing the NQO1 protein coding sequence into the MSC by transfection, transduction, infection or other methods, or inserting the NQO1 protein coding sequence into the genome of the MSC using gene editing methods such as mRNA transfection or the CRISPR Cas9 system.

[0026] In one embodiment, the genome of the engineered MSC further comprises an introduced regulatory element and a target gene, the expression of which is regulated by the regulatory element, and the target gene may be any gene. In one embodiment, the target gene encodes an immunostimulatory cytokine or an anti-tumor cytokine. In one embodiment, the target gene encodes any one of T cell stimulatory factors, IL-2, IL-1, IL-7, IL-21, IL-12, IL-15, and variants thereof. In one embodiment, IL-1 is IL-1β. In one embodiment, the IL-2 variant is sumIL2 (SIL2). In one embodiment, when the engineered MSC is administered to a tumor, the target gene can express and produce an anti-tumor factor to exert a specific anti-tumor function, and the engineered MSC has a prolonged lifespan, thereby enhancing the anti-tumor effect of the anti-tumor factor.

[0027] II. HRE Engineering MSC The present invention also provides engineered MSCs whose genome includes an introduced hypoxia response element (HRE) and optionally introduced regulatory elements and target genes downstream of the HRE, whereby expression of the target gene is regulated by the regulatory elements. The inventors discovered that, under the hypoxic environment within tumor tissue, the HRE binds to HIF1α, which is cumulatively expressed due to oxygen deprivation, and then activates downstream regulatory elements (e.g., promoters), thereby significantly promoting the expression of genes downstream of the regulatory elements (see Figure 2). This significantly increases the ability and efficiency of MSCs as cell carriers to deliver or produce antitumor molecules or other functional molecules into tumors. In one embodiment, the regulatory element includes a promoter. In one embodiment, the promoter is an SV40 promoter. In one embodiment, the engineered MSCs include an operably linked HRE, the regulatory element, and the target gene.

[0028] The target gene may be any gene. In one embodiment, the target gene encodes an immunostimulatory cytokine or an anti-tumor cytokine. In one embodiment, the target gene encodes any one of IL-2, IL-1, IL-7, IL-21, IL-12, IL-15, and variants thereof. In one embodiment, the IL-1 is IL-1β. In one embodiment, the IL-2 variant is sumIL2 (SIL2), which has a superior anti-tumor effect to wild-type IL-2 (see Figure 5).

[0029] Methods for introducing the HRE, regulatory elements (e.g., promoters), and target genes to be expressed into the genome of MSCs may include, but are not limited to, introducing vectors (e.g., lentiviral vectors or adenoviral vectors) containing these nucleic acid sequences into MSCs by transfection, transduction, infection, or other methods, or inserting these nucleic acid sequences into the genome of MSCs using gene editing methods such as mRNA transfection or the CRISPR / Cas9 system. In one embodiment, the HRE, its downstream regulatory elements (e.g., promoters), and target genes are operably linked together in a lentiviral vector, and then the engineered MSCs are transfected with the lentiviral vector.

[0030] III. NQO1 / HRE Engineering MSC The present invention provides an engineered MSC, the genome of which comprises (i) an introduced NQO1 protein coding sequence, and (ii) an introduced hypoxia response element (HRE), as well as optionally a regulatory element and a target gene downstream of the HRE, wherein expression of the target gene is regulated by the regulatory element. In one embodiment, the regulatory element comprises a promoter. In one embodiment, the promoter is an SV40 promoter. In one embodiment, in the engineered MSC, the HRE, the regulatory element, and the target gene are operably linked. In one embodiment, the target gene encodes an immunostimulatory cytokine or an anti-tumor cytokine. In one embodiment, the target gene encodes any one of IL-2, IL-1, IL-7, IL-21, IL-12, IL-15, and variants thereof. In one embodiment, IL-1 is IL-1β. In one embodiment, the IL-2 variant is sumIL2.

[0031] The method for introducing NQO1, HRE, regulatory elements (e.g., promoters), and target genes to be expressed into the genome of MSCs is the same as described above. In one embodiment, the NQO1 protein-coding sequence, the HRE and its downstream regulatory elements (e.g., promoters), and target genes may all be constructed in a lentiviral vector, where the HRE, regulatory elements, and target genes are operably linked, but the NQO1 coding sequence is not operably linked thereto and may be regulated by an independent promoter. MSCs can then be transduced with the lentiviral vector.

[0032] The NQO1 / HRE-engineered MSCs of the present invention can use MSCs as a carrier to specifically deliver immunostimulatory or antitumor cytokines (e.g., IL-1, IL-2) to the tumor microenvironment, demonstrating superior antitumor effects compared to non-engineered MSCs expressing the cytokines and direct administration of the cytokines to the tumor (see Figures 2 and 4). Furthermore, the immunostimulatory or antitumor cytokines expressed by the engineered MSCs are substantially confined to the tumor, avoiding the peripheral toxic side effects caused by systemic administration of the cytokines (see Figure 3). However, their antitumor effects are not limited to the local tumor site where they are administered, but also suppress the growth of distant tumors (Figure 7). This is because T cells activated and expanded by them migrate to distant tumors and exert their antitumor effects. Furthermore, the inventors have found that the IL-2-expressing NQO1 / HRE-engineered MSCs of the present invention can also enhance the antitumor effect of immune checkpoint inhibitors (Figure 6), thereby showing promising potential for late-stage tumors resistant to immune checkpoint blockade (ICB) therapy, and can also expand antitumor adoptive T cells and enhance the therapeutic effect of adoptive cells on low-temperature tumors (Figure 8).

[0033] However, the immunostimulatory or antitumor cytokines delivered or produced by the NQO1 / HRE-engineered MSCs of the present invention as cell carriers are not limited to IL-2, and can be used to express any cytokine with immunostimulatory or antitumor effects, such as IL-1β. This is because NQO1 independently enhances the viability of MSCs in the tumor microenvironment, and the HRE's promoting effect on downstream regulatory elements and target gene expression depends on the hypoxic environment in the tumor microenvironment. In this study, we demonstrated that the target gene, IL-1β, can be expressed at a similarly significant level, is relatively localized within the tumor, has low peripheral toxicity, and exhibits superior tumor-suppressing effects compared to IL-1β protein injection alone (Figure 9).

[0034] IV. Pharmaceutical Compositions The present invention also provides a pharmaceutical composition for treating cancer, comprising an engineered MSC whose genome comprises (i) an introduced NQO1 protein coding sequence, and / or (ii) an introduced hypoxia response element (HRE), and a regulatory element and a target gene downstream of the HRE, wherein the target gene encodes an immunostimulatory cytokine or an anti-tumor cytokine.

[0035] In one embodiment, the pharmaceutical composition further comprises an immune checkpoint inhibitor, such as an anti-PD-L1 antibody, an anti-PD-1 antibody, and an anti-CTLA4 antibody. In one embodiment, the pharmaceutical composition further comprises adoptive T cells that specifically recognize tumor cells.

[0036] V. Methods for Treating Cancer The present invention also provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of engineered MSCs, wherein the genome of the engineered MSCs comprises (i) an introduced NQO1 protein coding sequence, and / or (ii) an introduced hypoxia response element (HRE), and a regulatory element and a target gene downstream of the HRE, wherein the target gene encodes an immunostimulatory cytokine or an anti-tumor cytokine.

[0037] In one embodiment, the method further comprises administering to the subject an immune checkpoint inhibitor, such as, for example, an anti-PD-L1 antibody, an anti-PD-1 antibody, and an anti-CTLA4 antibody. In one embodiment, the method further comprises administering to the subject adoptive T cell therapy (ACT), such as, for example, CAR-T cell therapy, TIL therapy, and TCR-T therapy. In one embodiment, the subject is human.

[0038] VI. Pharmaceutical Uses The present invention also provides the use of engineered MSCs in the preparation of a drug for treating cancer, wherein the genome of the engineered MSCs comprises (i) an introduced NQO1 protein coding sequence, and / or (ii) an introduced hypoxia response element (HRE), and a regulatory element and a target gene downstream of the HRE, wherein the target gene encodes an immunostimulatory cytokine or an anti-tumor cytokine.

[0039] In one embodiment, the medicament further comprises an immune checkpoint inhibitor, such as an anti-PD-L1 antibody, an anti-PD-1 antibody, and an anti-CTLA4 antibody. In one embodiment, the medicament further comprises adoptive T cells that specifically recognize tumor cells.

[0040] In the above-described embodiments of the pharmaceutical composition, the method for treating cancer, and the pharmaceutical use, the regulatory element may include a promoter. In one embodiment, the promoter is an SV40 promoter. In one embodiment, the HRE, the regulatory element, and the target gene are operably linked in the engineered MSC. In one embodiment, the target gene encodes any one of IL-2, IL-1, IL-7, IL-21, IL-12, IL-15, and variants thereof. In one embodiment, the IL-1 is IL-1β. In one embodiment, the IL-2 variant is sumIL2. In one embodiment, the cancer may be selected from B-cell lymphoma, bronchial cancer, prostate cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral cancer, laryngeal cancer, salivary gland cancer, thymus cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, adipose cancer, testicular cancer, malignant fibrous histiocytoma, colorectal cancer, melanoma, gastric cancer, pancreatic cancer, lung cancer, liver cancer, kidney cancer, bile duct cancer, small intestine or appendix cancer, squamous cell carcinoma, breast cancer, and ovarian cancer. [Example]

[0041] Example Hereinafter, the embodiments of the present invention will be described in more detail with reference to examples. However, it should be understood that these examples are for the purpose of specific explanation and do not limit the scope of the present invention in any way.

[0042] Materials and Methods 1. Mouse C57BL / 6J, BALB / c, and C57BL / 6J-Tg(TcraTcrb)1100Mjb / J (OT1 TCR transgenic) mice, 6–8 weeks old, were purchased from the Jackson Laboratory. Mice were housed in a specific-pathogen-free (SPF) environment, and all animal experiments were conducted in accordance with the Tsinghua University Laboratory Animal Care and Use Guidelines.

[0043] 2. Cell lines and reagent materials MC38 (mouse colon carcinoma cell lines), CT26 (mouse colon carcinoma cell lines), and B16F10 (mouse cutaneous melanoma cell lines) cell lines were purchased from the American Type Culture Collection (ATCC). MC38-OVA and B6-OVA cell lines were obtained by transfecting MC38 or B16F10 with a lentivirus expressing the chicken OVA protein gene. The cell line culture medium was DMEM medium (containing 10% heat-inactivated fetal bovine serum (FBS), 100 U / ml penicillin, and 100 μg / ml streptomycin), and the culture conditions were 37°C and 5% CO2. The anti-PD-L1 antibody (Atezolizumab) and anti-CTLA-4 antibody (9D9) were purchased from BioXCell.

[0044] 3. Isolation, culture, and viral transfection of bone marrow-derived MSC cells Bone marrow cells were isolated from 6-8 week-old female C57BL / 6J or BALB / c mice, resuspended in Mesencult Expansion Media (Stemcell Technologies) containing 1 μM GW2580, seeded onto 10 cm cell culture dishes, and cultured under hypoxic conditions (1% oxygen concentration). After 24 hours, cells not adhering to the bottom of the culture dish were removed by fluid exchange. Fresh medium was replaced every 3 days, and when the cell density reached 60%, the cells were passaged at a 1:3 ratio. MSC cells were identified by flow cytometry to detect the expression of positive and negative surface markers.

[0045] Live MSC cells were transfected with lentivirus expressing the EGFP gene or EGFP + mouse NQO1 protein-encoding gene (SEQ ID No: 1), and EGFP-positive cells were sorted by flow cytometry to obtain MSC-EGFP or MSC-EGFP-NQO1 cells.

[0046] The hypoxia response element (HRE) (SEQ ID No. 2) and minimal SV40 promoter (SEQ ID No. 3) were obtained by PCR from the HIF-1 reporter p2.1 plasmid. The cloned sumIL2 coding sequence was ligated to the HRE-SV40 sequence, then incorporated into a lentiviral vector, packaged into lentivirus, transfected into MSCs, and selected to obtain MSC-NQO1-HRE-SIL2 or MSC-HRE-SIL2. Expression of SIL2 was detected by ELISA. Lentiviral vectors containing HREs derived from genes such as Epo, VEGF-A, PGK, Ldha, ALDA, or GAPDH were obtained in a similar manner.

[0047] 4. Construction and treatment of mouse tumor models MC38(1x10 6 ), CT26(5x10 5 ), MC38-OVA (1x10 6 ), B16-OVA(3x10 5 Mice were subcutaneously inoculated with SIL2 cells into the right dorsal region to generate tumors, and then randomly assigned to groups. Mice were treated with either 20 μg of SIL2 protein or 1×10 6 1 x 10 control MSCs (MSC-NQO1) or 1 x 10 6 When treating advanced tumors in combination with immune checkpoint blockade, C57BL / 6J mice were inoculated with MC38 tumors on day 14 (tumors approximately 150 mm). 3 ) and 100 μg of anti-PD-L1 + 100 μg of anti-CTLA4 were injected intraperitoneally, along with 1x10 6Control EMSCs or MSC-NQO1-HRE-SIL2 were injected into the peritumoral area of ​​C57BL / 6J mice. The injections were given twice, once every three days. In the case of combined cell adoptive therapy, 3x10 control EMSCs or MSC-NQO1-HRE-SIL2 were injected into C57BL / 6J mice. 5 On day 11, OT-1 T cells (1 x 10) were preactivated by OT-1 polypeptide. 6 or 2x10 5 ) was intravenously injected, and 1x10 control mice were injected around the tumor on days 11 and 14. 6 EMSCs or MSC-NQO1-HRE-SIL2 were injected. The length (a), width (b), and height (c) of the tumor were measured twice a week, and tumor volume was calculated as a*b*c / 2. The survival curve of tumor-bearing mice was recorded, and tumors were excluded if the tumor length, width, or height exceeded 2 cm or the tumor size reached 1500 mm. 3 When the weight loss exceeded 100 mg / kg / day or the weight loss of tumor-bearing mice exceeded 20%, the mice were culled.

[0048] 5. Tumor tissue digestion The tumor tissue was resuspended in digestion solution (RPMI medium containing 2% FBS, 1 mg / ml collagen IV, and 100 μg / ml Dnase I) and digested at 37°C for 45 min at 120 rpm. The tumor suspension was passed through a 70 μm cell filter to remove large debris or undigested tumor tissue chunks. The filtrate was washed twice with PBS containing 2 mM EDTA and then resuspended in FACS buffer.

[0049] 6. Flow Cytometry Analysis Anti-Fcg III / II receptor (clone 2.4G2) antibody was added to the tumor cell suspension and left on ice for 15 min to block nonspecific binding. Then, the corresponding fluorescent dye-coupled antibody was added and stained for 30 min at 4°C in the dark. Fixable viability dye (eFluor) TM 506 or the dye eFluor TM Dead cells were excluded by staining with 780. Foxp3 nuclear staining was performed using True-Nuclear TMThe procedure was carried out using a transcription factor buffer set (BioLegend) according to the manufacturer's instructions. Data were acquired using a CytoFLEX flow cytometer (Beckman Coulter) and analyzed using the software FlowJo (Tree Star).

[0050] 7. Preparation of tumor tissue homogenates CT26 tumor-bearing mice were treated with 20 μg of sumIL2 protein or 1×10 6 EMSC-sumIL2 was injected intratumorally. Five days later, tumor tissue was harvested, cut into small pieces, resuspended using a cell lysis kit (Bio-Rad Laboratories), and homogenized using a FastPrep-24 5G homogenizer. The homogenate was then centrifuged at 13,000 rpm for 20 minutes. The supernatant was then collected and stored at -80°C.

[0051] 8. Analysis of serum and tissue samples by cytometric bead array (CBA) and ELISA Cytokine levels in mouse serum and tumor tissue homogenates were measured according to the CBA Mouse Th1 / Th2 / Th17 Kit (BD Biosciences) manual. For ELISA, a 96-well microplate (Corning Costar) was used. 2 μg / mL (100 μL / well) capture antibody was added to the plate and allowed to adsorb overnight at 4°C. After washing the plate with PBS, blocking solution (PBS containing 0.05% TWEEN-20 and 5% skim milk) was added. Serum or tumor homogenate diluted in blocking solution was then added and incubated for 1.5 hours at 37°C. After washing the plate with PBST, alkaline phosphatase-conjugated goat anti-human IgG secondary antibody was added and incubated for 50 minutes at 37°C. After washing the plate with PBST, 100 μL of p-nitrophenyl phosphate was added for color development, and the readout was then acquired at 405 nm using a SPECTROstar Nano (BMG LABTECH).

[0052] 9. Preparation of SumIL2-Fc Protein The plasmid was constructed by linking a mutant form of IL-2, SumIL-2 (abbreviated as SIL2, containing F42A, L80F, R81D, L85V, I86V, and I92F mutations), to the N-terminus of hIgG1-derived Fc (containing L233A, L234A, and P329G mutations) (SEQ ID No: 4). The plasmid was transiently transfected into FreeStyle TM The resulting vector was transfected into 293-F cells. Six days after transfection, the supernatant was collected and the SIL2-Fc protein in the supernatant was purified using a protein A affinity chromatography column.

[0053] 10. Production of IL-1β-Fc Protein IL-1β-Fc protein (the coding sequence of which is SEQ ID No: 5) was produced in a similar manner to SumIL2-Fc protein.

[0054] 11. Data Analysis Tumor-bearing mice were randomly assigned to different groups according to tumor volume before treatment. Data were analyzed using GraphPad Prism statistical software and expressed as mean ± SEM. P values ​​were calculated for tumor curves by two-way ANOVA analysis, P values ​​for mouse survival curves were calculated by log-rank test, and P values ​​for other data were calculated by unpaired two-tailed t-test. p<0.05 indicates significant differences.

[0055] 12. Sequence Description SEQ ID No: 1: Coding nucleic acid sequence of mouse NQO1 protein SEQ ID No: 2 Sequence of the hypoxia response element (HRE) from the Eno1 gene SEQ ID No: 3 Minimal SV40 promoter sequence SEQ ID No: 4: Coding nucleic acid sequence of sumIL2-Fc SEQ ID No: 5: Coding nucleic acid sequence of IL1β-Fc SEQ ID No: 6 Sequence of the hypoxia response element (HRE) from the EPO gene SEQ ID No: 7 Sequence of the hypoxia response element (HRE) from the VEGF-A gene SEQ ID No: 8 Sequence of the hypoxia response element (HRE) from the PGK1 gene SEQ ID No: 9 Sequence of the hypoxia response element (HRE) from the LdhA gene SEQ ID No: 10 Sequence of the hypoxia response element (HRE) from the ALDA gene SEQ ID No:11 Sequence of the hypoxia response element (HRE) from the GAPDH gene.

[0056] Example 1. NQO1-engineered MSCs exhibit longer survival time within tumors. To enhance the survival of MSCs in tumor tissue in vivo, we transfected MSC cells with a lentiviral vector carrying EGFP and NQO1 gene sequences as described above to obtain NQO1-engineered MSCs ("MSC-EGFP-NQO1"). MSCs carrying EGFP alone served as a control ("MSC-EGFP"). To evaluate the survival time of NQO1-engineered MSCs in tumor tissue in vivo, 8-week-old female C57BL / 6J mice (n=5) were used, and 1x10 6 MC38 cells were subcutaneously inoculated. On day 9, 5x10 cells were injected intratumorally. 5 MSC-EGFP or MSC-EGFP-NQO1 were injected, and tumor tissues were collected 5 days after injection. CD45 expression in the tumor was analyzed by flow cytometry. - GFP + The number of MSCs was analyzed, and as shown in Figure 1(a) and (b), the number of MSC-EGFP-NQO1 cells present in the tumor was significantly higher than that of MSC-EGFP cells, indicating that NQO1-expressing MSCs are better adapted to the tumor microenvironment and have a longer survival time.

[0057] Example 2. HRE-engineered MSC-NQO1 specifically expresses anti-tumor factors in hypoxic tumor tissues. To specifically express antitumor factors in tumor tissues and avoid peripheral toxicity, we used a hypoxia-inducible promoter element (HRE) derived from the Eno1 gene, derived by PCR from the HIF-1 reporter P2.1 plasmid, containing a minimal SV40 promoter and a hypoxia-inducible promoter. MSC-NQO1 cells were transfected with a lentiviral vector containing the target gene (IL-1β or SIL-2) regulated by the HRE-SV40 activator, or a lentiviral vector lacking the HRE element but containing the target gene, and then cultured under normoxic (20%) or hypoxic (1%) conditions for 24 hours. The supernatant concentrations of the expressed antitumor factors IL-1β and SIL-2 (n = 4) were detected by ELISA. Under hypoxic culture conditions, the levels of IL-1β or SIL-2 expressed by MSC-NQO1-HRE-IL-1β or -SIL-2 (gray bars in Figure 2) were approximately 10-fold higher than those under normoxic culture conditions (Figures 2b and 2c). In contrast, the control MSC-NQO1-IL-1β or -SIL-2 (black bars in Figure 2) showed no significant changes in IL-1β or SIL-2 production under hypoxic or normoxic culture conditions, and both were significantly lower than the levels secreted by MSC-NQO1-HRE-IL-1β or -SIL-2 under hypoxic conditions. This indicates that HRE-engineered MSCs can specifically promote target gene expression under hypoxic conditions.

[0058] To detect the in vivo antitumor effect of engineered MSCs, 8-week-old female C57BL / 6J mice were cultured at 1x10 6 MC38 cells were inoculated subcutaneously. On days 9 and 12, 1x10 cells were injected intratumorally. 6Engineered MSC-NQO1-SIL2, engineered MSC-NQO1-HRE-SIL2, or MSC-SIL2 (i.e., MSCs lacking the NQO1 and HRE elements and carrying only the SIL-2 gene) was injected, and tumor volume was measured daily (n = 5 or 6). As shown in Figure 2d, MSC-NQO1-SIL2 had an improved antitumor effect compared to MSC-SIL2, indicating that the prolonged survival of NQO1-engineered MSCs can improve their antitumor ability. Meanwhile, MSC-NQO1-HRE-SIL2 had the strongest antitumor effect, indicating that its antitumor ability can be enhanced by the additional introduction of HRE.

[0059] Example 3. Engineered MSC-NQO1-HRE-SIL2 can express sumIL2 specifically in tumor tissues and avoid peripheral toxicity. As described above, we constructed a mutant form of IL-2, sumIL-2 (SIL-2). Compared to unmutated IL-2, sumIL-2 tends to activate effector CD8+ T cells and reduce the activation of suppressor Treg cells. It exhibits reduced binding to IL2Rα but enhanced binding to IL2Rβ. To extend its half-life, SumIL-2 was linked to the Fc portion of hIgG, which contains L233A, L234A, and P329G mutations to eliminate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) and to avoid the elimination effect on activated T cells. SumIL-2-Fc has superior antitumor activity compared to IL-2-Fc, but also exhibits increased toxicity.

[0060] In this example, a lentiviral vector carrying NQO1-HRE-sumIL2-Fc (expression of SumIL-2-Fc is regulated by HRE, whereas NQO1 is regulated by an HRE-independent element) was constructed, packaged into a virus, and transfected into MSC cells to obtain "MSC-NQO1-HRE-SIL2." To evaluate the effect of MSCs as a carrier in delivering sumIL-2 to tumor tissues, 5x10 cells were transfected into 8-week-old female BALB / c mice. 5CT26 tumor cells were inoculated subcutaneously. On day 9, 20 μg of SIL2-Fc protein or 1×10 6 MSC-NQO1-HRE-SIL2 were injected into each mouse, and tumor tissues and serum were collected 5 days after injection. The sumIL-2 levels in the tumor tissue homogenates and serum were detected by ELISA (n=5 or 6).

[0061] We found that the sumIL2 concentrations in tumor tissue after intratumoral injection of SIL2-Fc protein or MSC-NQO1-HRE-SIL2 were comparable and higher than those after peritumoral injection of SIL2-Fc protein (Figure 3a). Interestingly, the sumIL2 protein concentrations produced by peritumoral injection of MSC-NQO1-HRE-SIL2 cells were comparable to those after intratumoral injection, indicating that peritumoral injection of MSCs can home to tumor tissue and secrete sumIL2 protein. High levels of sumIL2 protein were detected in serum after intratumoral or peritumoral injection of sumIL2 protein. In contrast, only trace amounts of SIL2 protein were detected in serum after intratumoral or peritumoral injection of MSC-NQO1-HRE-SIL2 cells (Figure 3b). High levels of sumIL2 protein in serum are known to activate peripheral T cells and NK cells, producing cytokines and causing peripheral toxicity. Consistent with this, CBA detected significantly elevated IFNγ in the serum of mice injected intratumorally or peritumorally with sumIL2 protein, whereas mice injected intratumorally or peritumorally with MSC-NQO1-HRE-SIL2 had no detectable IFNγ protein (Fig. 3c). After intratumorally or peritumorally injection of sumIL2 protein, mouse body weight was significantly reduced, whereas the body weight of mice injected intratumorally or peritumorally with MSC-NQO1-HRE-SIL2 cells did not change significantly compared with the untreated control group (Fig. 3d).

[0062] These results indicate that intratumoral or peritumoral injection of MSC-NQO1-HRE-SIL2 cells can specifically secrete high concentrations of sumIL2 protein within tumor tissues, while producing very little SIL2 protein and associated toxicity in peripheral tissues.

[0063] Example 4. MSC-NQO1-HRE-SIL2 has a significant tumor-suppressing effect. The tumor-suppressive effect of MSC-NQO1-HRE-SIL2 was evaluated using a CT26 colorectal cancer mouse tumor model. Eight-week-old female BALB / c mice were treated with 5x10 5 CT26 tumor cells were subcutaneously inoculated. On days 9 and 12 after inoculation, SIL2-Fc protein (sumIL-2-Fc) was injected intratumorally at the doses shown in Figure 4, or 1 x 10 6 MSC-NQO1-HRE-SIL2 cells were injected around the tumor. Mice were observed twice a week, and tumor size was measured (n=5). The results are shown in Figure 4. 6 MSC-NQO1-HRE-SIL2 cells could significantly suppress tumor growth at a dose that did not cause peripheral toxicity, and the effect was slightly better than the therapeutic effect of 2.5 μg or 10 μg SIL2-Fc protein.

[0064] Example 5. Selective expansion of intratumoral CD8+ T cells by administration of MSC-NQO1-HRE-SIL2 To further analyze the modulation of antitumor immunity by administration of MSC-NQO1-HRE-SIL2, 5x10 5 CT26 tumor cells were subcutaneously inoculated. On days 9 and 12 after inoculation, 1x10 cells were injected around the tumor. 6MSC-NQO1, PBS control, or MSC-NQO1-HRE-SIL2 were injected into each mouse. Five days after the second injection, tumor tissues were isolated and digested into single-cell suspensions using a digestion solution containing 1 mg / ml collagenase IV and 100 μg / ml DNase I. The cell counts of each subset were then determined by flow cytometry. As shown in Figure 5(a) and 5(b), MSC-NQO1-HRE-SIL2 significantly expanded CD8+ T cells, and the CD8+ T / Treg cell ratio was significantly higher than that of the other groups.

[0065] To further analyze the ability of SIL2 to specifically expand CD8+ T cells, but not Tregs, compared with wtIL2, 5x10 5 CT26 tumor cells were subcutaneously inoculated. On days 9 and 12 after inoculation, 1x10 cells were injected into the tumor. 6 MSC-NQO1, MSC-NQO1-HRE-SIL2, or MSC-NQO1-HRE-wtIL2 (expressing wild-type IL-2) cells were injected into the tumor tissues. Five days after the second injection, tumor tissues were isolated and digested into single-cell suspensions using a digestion solution (containing 1 mg / ml collagenase IV and 100 μg / ml DNase I). The cell counts of each subset were then determined by flow cytometry. As shown in Figure 5c, the CD8+ T / Treg cell ratio in the MSC-NQO1-HRE-SIL2-treated group was significantly higher than in the other groups. Because wtIL2 not only expanded antitumor CD8+ T cells but also significantly expanded suppressive Treg cells within the tumor, the CD8+ T / Treg ratio in the MSC-NQO1-HRE-wtIL2-treated group did not differ significantly from that in the MSC-NQO1 group. These results indicate that MSC-NQO1-HRE-SIL2 can selectively expand CD8+ T cells within tumors and suppress tumor growth.

[0066] Example 6. MSC-NQO1-HRE-SIL2 enhances the antitumor effect of immune checkpoint inhibitors. Although immune checkpoint inhibitors have shown favorable tumor therapeutic effects in clinical trials, only a portion of patients respond to immune checkpoint blockade (ICB) therapy, and some patients who respond in the early stage become unresponsive in the later stage. To evaluate whether MSC-NQO1-HRE-SIL2 can improve the efficacy of immune checkpoint inhibitor therapy, 1x10 MSCs were injected into 8-week-old female C57BL / 6J mice. 6 MC38 cells were subcutaneously inoculated, and late-stage tumors were selected for treatment. On the 14th and 17th days of tumor growth, 100 μg of anti-PD-L1 + anti-CTLA4 antibody was intraperitoneally injected, or 1x10 6 MSC-NQO1-HRE-SIL2 cells were injected around the tumor, and tumor growth and survival curves of the mice were recorded (n=5).

[0067] As shown in Figure 6, the MSC-NQO1-HRE-SIL2 treatment group (▲) was able to partially suppress the growth of advanced tumors, but was unable to eliminate them. JPEG2025532695000001.jpg5113 showed a slightly better effect, but only suppressed tumor growth. In contrast, the immune checkpoint inhibitor + MSC-NQO1-HRE-SIL2 combination treatment group (▼) was able to more significantly suppress tumor growth (Figure 6a). Looking at the survival curves, the MSC-NQO1-HRE-SIL2 monotherapy group (▲) and the immune checkpoint inhibitor + control MSC treatment group showed significantly higher survival rates than the control MSC treatment group. JPEG2025532695000002.jpg5113 extended the survival of tumor-bearing mice to a maximum of approximately 50 days, while in the immune checkpoint inhibitor + MSC-NQO1-HRE-SIL2 combination treatment group (▼), 75% of the tumors were removed and the mice survived for more than 70 days.

[0068] These results suggest that MSC-NQO1-HRE-SIL2 not only suppresses tumor growth by itself, but also enhances the antitumor effects of immune checkpoint inhibitors, and is effective against advanced tumors that are resistant to immune checkpoint blockade (ICB) therapy.

[0069] Example 7. sumIL2 produced locally in tumors by MSC-NQO1-HRE-SIL2 can activate T cells and effectively suppress distant tumor growth. To evaluate whether local administration of MSC-NQO1-HRE-SIL2 could induce a systemic antitumor immune response and control distant tumors from the primary tumor, we used a mouse model inoculated with dual tumors subcutaneously. Eight-week-old female C57BL / 6J mice were inoculated with 1x10 6 MC38 tumor cells were inoculated subcutaneously on the right side, and 5x10 cells were inoculated on the second day. 5 MC38 tumor cells were inoculated subcutaneously on the left side. On days 9 and 12, 1x10 MC38 tumor cells were inoculated intratumorally on the right side. 6 MSC-NQO1-HRE-SIL2 cells or control MSCs were injected. The tumor sizes on the left and right sides were recorded simultaneously twice weekly (n=7-10).

[0070] As shown in Figure 7, compared with the control MSCs, MSC-NQO1-HRE-SIL2 significantly suppressed tumor growth not only at the right treatment site but also at the left distal tumor site, indicating that MSC-NQO1-HRE-SIL2 can activate antitumor immune responses locally in the tumor and suppress distal tumor growth.

[0071] Example 8. Administration of MSC-NQO1-HRE-SIL2 improves the antitumor effect of adoptive cell therapy (ACT) against advanced tumors. SIL2 is CD8 + Because it can effectively expand T cells, it is expected to be used in combination with adoptive T cell therapy to treat low-temperature tumors with little immune cell infiltration. B16 melanoma was selected as a low-temperature tumor model. Eight-week-old female C57BL / 6J mice were inoculated with 3x10 cells on day 0. 5 B16-OVA tumor cells were subcutaneously inoculated, and on day 9, OT-1 T cells preactivated with OT-1 polypeptide were intravenously injected at the numbers shown in Figure 8. 6Control MSCs (MSC-NQO1) or MSC-NQO1-HRE-SIL2 were injected around the tumor and combined with ACT as indicated in the figure. Tumor size and survival of mice were recorded twice weekly (n = 5-10).

[0072] As shown in Figures 8a and 8b, the results showed that MSC-NQO1-HRE-SIL2 (▼) or ACT (2x10 5 ) plus control MSCs (▲) only partially suppressed the growth of B16-OVA tumor cells, and tumor-bearing mice eventually died. In contrast, MSC-NQO1-HRE-SIL2 plus ACT (2 x 10 5 In the combined treatment group (◆) with ACT (2x10), tumors were significantly suppressed or eliminated, and survival of mice was prolonged. 6 ) The therapeutic effect was almost equivalent to that of JPEG2025532695000003.jpg5113. These results indicate that MSC-NQO1-HRE-SIL2 can expand anti-tumor adoptive T cells and improve the therapeutic effect of adoptive cells against low-temperature tumors.

[0073] Example 9. Engineered MSC-NQO1-HRE-IL1β can express IL1β specifically in tumor tissue and avoid peripheral toxicity. A lentiviral vector carrying NQO1-HRE-IL1β-Fc (IL1β-Fc expression is regulated by the HRE element) was constructed in the same manner as described in Example 3, packaged into a virus, and transfected into MSC cells. The transfected cells were designated "MSC-NQO1-HRE-IL1β." The IL1β expression level and antitumor effect in tumor tissue of the MSCs were measured in the same manner as described in Example 3.

[0074] As shown in Figure 9a, after intratumoral injection of IL1β protein or MSC-NQO1-HRE-IL1β, MSC-NQO1-HRE-IL1β expressed IL1β in the tumor, but the expression in serum and other organs was significantly lower than that observed with direct injection of IL1β protein. Furthermore, after MSC-NQO1-HRE-IL1β injection, serum levels of inflammatory factors IL-6 and MCP1, and liver damage indicators ALT and AST were not significantly different from those in the control group (intratumoral PBS injection group), whereas after IL1β protein injection, these inflammatory factors were significantly elevated (Figure 9b). This indicates that while IL1β protein administration causes toxic side effects in peripheral tissues, MSC-NQO1-HRE-IL1β specifically expresses IL1β protein in the tumor without causing peripheral tissue toxicity.

[0075] Furthermore, in terms of tumor-suppressing effects, the effects of MSC-NQO1-HRE-IL1β were significantly superior to those of IL-1β protein in both tumor volume and body weight in tumor-bearing mice (FIGS. 9b and 9c).

[0076] Although the technical concept and specific embodiments of the present invention have been described above, it should be understood that the above specific embodiments do not limit the scope of the present invention in any way. Those skilled in the art can make various modifications and / or changes to the invention shown in the specific embodiments without departing from the spirit of the present invention, and it will be understood that the modified and / or changed embodiments also fall within the scope of the present invention. Therefore, the embodiments of the present invention are merely illustrative and not limiting.

Claims

1. Engineered mesenchymal stem cells (MSCs) whose genome contains the introduced NQO1 protein coding sequence.

2. 2. The engineered mesenchymal stem cell of claim 1, wherein the genome further comprises an introduced regulatory element and a target gene, wherein expression of the target gene is regulated by the regulatory element.

3. Engineered mesenchymal stem cells (MSCs) whose genome contains an introduced hypoxia response element (HRE).

4. Engineered mesenchymal stem cells (MSCs) whose genome contains an introduced NQO1 protein coding sequence and an introduced hypoxia response element (HRE).

5. 5. The engineered mesenchymal stem cell of claim 3 or 4, wherein the genome further comprises an introduced regulatory element and a target gene located downstream of the HRE, and expression of the target gene is regulated by the regulatory element.

6. The engineered mesenchymal stem cell of claim 5 , wherein the HRE and the downstream regulatory element and target gene are operably linked.

7. 10. The engineered mesenchymal stem cell of claim 2 or 5, wherein the regulatory element comprises a promoter.

8. The engineered mesenchymal stem cell of claim 2 or 5, wherein the target gene encodes an anti-tumor protein, preferably an immunostimulatory cytokine or an anti-tumor cytokine.

9. 6. The engineered mesenchymal stem cell of claim 2 or 5, wherein the target gene encodes any one of IL-2, IL-1, IL-7, IL-21, IL-12, IL-15 and variants thereof, preferably sumIL-2 or IL-1β.

10. 6. The engineered mesenchymal stem cell of claim 1, wherein the introduction is carried out by lentiviral vector transfection, adenoviral vector transfection, or mRNA transfection.

11. The engineered mesenchymal stem cell of claim 1 or 4, wherein the NQO1 protein coding sequence is SEQ ID No:

1.

12. The engineered mesenchymal stem cell of claim 3 or 4, wherein the sequence of the HRE is derived from any one of Eno1, Epo, VEGF-A, PGK, Ldha, ALDA and GAPDH genes.

13. 5. The engineered mesenchymal stem cell of claim 3 or 4, wherein the sequence of the HRE is selected from SEQ ID No: 2 and SEQ ID No: 6-11, preferably SEQ ID No:

2.

14. A pharmaceutical composition for treating cancer, comprising the engineered mesenchymal stem cells of claim 8 or 9.

15. The pharmaceutical composition of claim 14, further comprising a second anti-cancer agent, such as an immune checkpoint inhibitor, and / or adoptive T cells that recognize cancer cells.

16. 10. Use of the engineered mesenchymal stem cells of claim 8 or 9 in the preparation of a medicament for treating cancer.

17. 17. The pharmaceutical composition of claim 14 or the use of claim 16, wherein the cancer is selected from B-cell lymphoma, bronchial cancer, prostate cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral cancer, laryngeal cancer, salivary gland cancer, thymus cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, adipose cancer, testicular cancer, malignant fibrous histiocytoma, colorectal cancer, melanoma, gastric cancer, pancreatic cancer, lung cancer, liver cancer, kidney cancer, bile duct cancer, small intestine or appendix cancer, squamous cell carcinoma, breast cancer and ovarian cancer.

18. Use of NQO1 protein to extend the survival period of mesenchymal stem cells in tumor tissue.

19. Use of hypoxia response elements (HRE) to enhance expression of target genes in mesenchymal stem cells under hypoxic conditions.

20. 20. The use according to claim 19, wherein the hypoxic environment is the intratumoral microenvironment.

21. The use described in claim 19, wherein the genome of the mesenchymal stem cell comprises the introduced hypoxia response element, and an introduced regulatory element and the target gene downstream thereof, and the expression of the target gene is regulated by the regulatory element.

22. Use of a combination of an NQO1 protein coding sequence and a hypoxia response element (HRE) for preparing a pharmaceutical composition comprising mesenchymal stem cells carrying a target gene.

23. The use described in claim 22, wherein the genome of the mesenchymal stem cell comprises the introduced NQO1 protein coding sequence, the introduced hypoxia response element, and an introduced regulatory element and the target gene downstream of the hypoxia response element, and the expression of the target gene is regulated by the regulatory element.

24. 14. Use of the engineered mesenchymal stem cells of any one of claims 1 to 13 as a carrier for delivering a target gene.

25. The carrier is used to deliver the target gene to tumor tissue; and 25. The use according to claim 24, wherein the target gene is a tumor therapeutic gene, preferably a gene encoding an anti-tumor protein, more preferably a gene encoding an immunostimulatory cytokine or an anti-tumor cytokine.

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