Immunotherapy targeting chemokine and cytokine delivery by mesenchymal stem cells

Abstract

The present invention provides an immunotherapy that targets chemokine and cytokine delivery by mesenchymal stem cells. Specifically, the mesenchymal stem cells express an immune stimulatory factor selected from the group consisting of CCL3, CCL19, CCL21, XCL1, CXCL9, OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof. The mesenchymal stem cells can specifically attract and activate immune cells that kill tumor tissue at the tumor site. Furthermore, the mesenchymal stem cells and the chemokines and / or cytokines have a synergistic effect, resulting in highly efficient immunotherapeutic effects with few side effects, and significantly enhanced killing ability against tumor tissue, particularly colorectal cancer cells.

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention is in the field of biotechnology, and specifically, the present invention relates to immunotherapy that targets chemokine and cytokine delivery by mesenchymal stem cells.

[0002] [Background technology] The rapid development of immunotherapy has brought new hope to cancer. Chimeric antigen receptor T-cell immunotherapy (CAR-T) and immune checkpoint blockade are currently the two most advanced cancer immunotherapies. While CAR-T cell therapy has demonstrated some therapeutic efficacy in several types of cancer, it faces several bottlenecks, including low T cell delivery efficiency, short duration of action, limited immune cell numbers at the tumor site, and side effects associated with systemic administration. Antibodies targeting the T cell inhibitory receptors PD-1 or CTLA-4 can achieve highly significant antitumor effects. However, the therapeutic efficacy of antibodies is often limited by many factors, including low T cell infiltration and loss of activity in solid tumors. Furthermore, the systemic use of immunotherapeutic agents such as interferon-α, interleukin-2, or PD-1 antibodies can cause serious side effects. Therefore, there is an urgent need in the art for immunotherapies that can specifically target tumor cells.

[0003] [Summary of the Invention] [Problem to be solved by the invention] It is an object of the present invention to provide an immunotherapy that can specifically target tumor cells. Another object of the present invention is to provide immunotherapies that target chemokine and cytokine delivery by mesenchymal stem cells.

[0004] [Means for solving the problem] A first aspect of the present invention provides mesenchymal stem cells, wherein the mesenchymal stem cells express an immune stimulatory factor, the immune stimulatory factor being selected from the group consisting of CCL3, CCL19, CCL21, XCL1, CXCL9, OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof.

[0005] In another preferred example, the mesenchymal stem cells comprise an exogenous nucleic acid molecule, wherein the exogenous nucleic acid molecule comprises a nucleic acid sequence encoding an immune stimulatory factor, and the immune stimulatory factor is selected from the group consisting of CXCL9, OX40L, CCL3, CCL19, CCL21, XCL1, CXCL9, OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof.

[0006] In another preferred embodiment, the exogenous nucleic acid molecule further comprises a promoter or a promoter / enhancer combination, and the nucleic acid sequence encoding the immunostimulatory factor is operably linked to the promoter or promoter / enhancer combination.

[0007] In another preferred embodiment, the promoter is a constitutive promoter or an inducible promoter, preferably a constitutive promoter. In another preferred embodiment, the immune stimulator comprises at least one chemokine, and the chemokine comprises CCL3, CCL19, CCL21, XCL1, CXCL9, or a combination thereof.

[0008] In another preferred embodiment, the immune stimulator comprises at least one cytokine, and the cytokine comprises OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof.

[0009] In another preferred embodiment, the immune stimulator comprises at least one chemokine and at least one cytokine, wherein the chemokine comprises CCL3, CCL19, CCL21, XCL1, CXCL9, or a combination thereof, and the cytokine comprises OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof.

[0010] In another preferred embodiment, the immune stimulator is a combination of one or two of CCL3, CCL19, CCL21, and XCL1 with CD40L. In another preferred embodiment, the immune stimulatory factor is a combination of one or two of OX40L, 4-1BBL, and GITRL with CXCL9.

[0011] In another preferred embodiment, the immune stimulator is CXCL9 and / or OX40L. In another preferred example, the exogenous nucleic acid molecule comprises a first expression cassette and / or a second expression cassette, wherein the first expression cassette comprises a nucleic acid sequence encoding a chemokine, and the second expression cassette comprises a nucleic acid sequence encoding a cytokine, wherein the chemokine comprises CCL3, CCL19, CCL21, XCL1, CXCL9, or a combination thereof, and the cytokine comprises OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof.

[0012] In another preferred example, the exogenous nucleic acid molecule comprises a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a nucleic acid sequence encoding CXCL9 and the second expression cassette comprises a nucleic acid sequence encoding OX40L.

[0013] In another preferred embodiment, the first expression cassette and the second expression cassette are independent of each other or combined into one. In another preferred example, the first expression cassette and the second expression cassette each further comprise a promoter and / or a terminator.

[0014] In another preferred example, the first expression cassette and the second expression cassette are the same expression cassette, and the expression cassette comprises a promoter, a nucleic acid sequence encoding a chemokine, and a nucleic acid sequence encoding a cytokine.

[0015] In another preferred example, the first expression cassette and the second expression cassette are located on a carrier or integrated into the chromosomes of the mesenchymal stem cells. In another preferred embodiment, the first expression cassette and the second expression cassette are independent or linked.

[0016] In another preferred embodiment, the first and second expression cassettes are located on the same or different carriers. In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same carrier.

[0017] In another preferred embodiment, the carrier is selected from the group consisting of DNA, RNA, a plasmid, a lentiviral carrier, an adenoviral carrier, a retroviral carrier, a transposon, another gene transfer system, or a combination thereof.

[0018] In another preferred embodiment, the mesenchymal stem cells include adipose-derived mesenchymal stem cells, umbilical cord mesenchymal stem cells, or a combination thereof. In another preferred embodiment, the mesenchymal stem cells are isolated. In another preferred embodiment, the mesenchymal stem cells are autologous or allogeneic.

[0019] A second aspect of the present invention provides a method for preparing mesenchymal stem cells according to the first aspect of the present invention, comprising: (1) providing mesenchymal stem cells to be modified; and (2) obtaining the mesenchymal stem cells according to the first aspect of the present invention by introducing an exogenous nucleic acid comprising a nucleic acid sequence encoding an immunostimulatory factor into the mesenchymal stem cells to be modified, wherein the immune stimulatory factor is selected from the group consisting of CCL3, CCL19, CCL21, XCL1, CXCL9, OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof.

[0020] A third aspect of the present invention provides a formulation, said formulation comprising mesenchymal stem cells according to the first aspect of the present invention and a pharmaceutically acceptable carrier, diluent or excipient. In another preferred embodiment, the formulation is a liquid formulation.

[0021] In another preferred embodiment, the dosage form of the preparation includes an injection. In another preferred embodiment, the concentration of mesenchymal stem cells in the preparation is 1×10 3 -1×10 8 cells / ml, preferably 1 x 10 4 -1×10 7 cells / ml.

[0022] A fourth aspect of the present invention provides the use of the mesenchymal stem cells according to the first aspect of the present invention for use in the preparation of a medicament or formulation for preventing and / or treating cancer or tumors.

[0023] In another preferred embodiment, the tumor is selected from the group consisting of a hematological tumor, a solid tumor, or a combination thereof. Preferably, the tumor is a solid tumor. In another preferred embodiment, the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0024] In another preferred embodiment, the solid tumor is selected from the group consisting of gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, squamous cell carcinoma of the lung, anal cancer, head and neck cancer, or a combination thereof.

[0025] In another preferred embodiment, the solid tumor is colorectal cancer. In another preferred embodiment, the tumor comprises a recurrent metastatic tumor. In another preferred embodiment, the tumor comprises an MHC-I deficient tumor.

[0026] A fifth aspect of the present invention provides a kit, said kit comprising: (1) a first container and mesenchymal stem cells according to the first aspect of the present invention contained in the first container; and (2) A second container and an anti-tumor immunotherapeutic agent contained in the second container.

[0027] In another preferred embodiment, the anti-tumor immunotherapeutic agent is selected from the group consisting of an antibody, an immune cell, or a combination thereof. In another preferred embodiment, the immune cells are T cells or NK cells.

[0028] In another preferred embodiment, the anti-tumor immunotherapeutic agent is an immune checkpoint antibody. In another preferred example, the immune checkpoint antibody comprises a PD-1 antibody and / or a CTLA-4 antibody.

[0029] The present invention provides a method of treating a disease comprising administering to a subject in need thereof an appropriate amount of cells according to the first aspect of the invention, or a formulation according to the third aspect of the invention, or a drug combination according to the fifth aspect of the invention. In another preferred embodiment, the disease is cancer or a tumor, preferably a solid tumor, more preferably colorectal cancer.

[0030] [Effects of the invention] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be repeated here due to space limitations. [Brief explanation of the drawings]

[0031] [Figure 1] Identification of the phenotype and tumor migration characteristics of mouse adipose-derived mesenchymal stem cells. A: Flow cytometry was used to detect the expression of surface molecules in adipose-derived mesenchymal stem cells. B: In vivo migration of MSC-GFP in a CT26 subcutaneously implanted tumor mouse model. Immunofluorescence was used to detect MSC-GFP infiltration in tumor tissue. Green indicates MSC-GFP, and blue (DAPI staining) indicates cell nuclei. Scale bar: 20 μM. C: Flow cytometry was used to detect the specific number of MSC-GFP cells throughout the entire CT26 subcutaneously implanted tumor. Data represent the mean ± SEM (n = 3). [Figure 2] Figure 1 shows that overexpression of CXCL9 in tumor cells inhibits tumor growth in vivo. A, Overexpression of CCL3 and CXCL9 in CT26 cells was detected by WB and qPCR. Data represent mean ± SEM (n = 3). B, Cell proliferation experiments of CT26-Vector, CT26-CCL3, and CT26-CXCL9. Data represent mean ± SEM (n = 3). C, Tumor growth curves of BALB / c mice subcutaneously injected with CT26-Vector, CT26-CCL3, or CT26-CXCL9 tumor cells were measured every 3 days. Data represent mean ± SEM (n = 4). D, Percentages of CD8+, CD4+ T, and NK cells within total cells. Data represent mean ± SEM (n = 4). [Figure 3]Figure 1 shows that overexpression of OX40L in tumor cells inhibits tumor growth in vivo. A, WB detection of IL36β and OX40L overexpression in CT26. B, Flow cytometry detection of OX40L overexpression in CT26. C, Cell proliferation experiment of CT26-Vector, CT26-IL36β, and CT26-OX40L. Data represent mean ± SEM (n = 3). D, Tumor growth curve of BALB / c mice subcutaneously injected with CT26-Vector, CT26-CCL3, or CT26-CXCL9 tumor cells. Tumor size was measured every 3 days. Four mice per group. Data represent mean ± SEM (n = 4). *p < 0.05, **p < 0.01. [Figure 4] Figure 1 shows the identification of CXCL9 and OX40L overexpression in mesenchymal stem cells. A, WB detection of CXCL9 overexpression in MSCs. B, ELISA detection of CXCL9 overexpression in MSCs. Data represent mean ± SEM (n=3). ***p<0.001. C, WB detection of OX40L overexpression in MSCs. D, Flow cytometry detection of OX40L overexpression in MSCs. [Figure 5] Figure 1 shows that mesenchymal stem cells overexpressing CXCL9 and OX40L inhibit the growth of subcutaneously implanted tumors. A, ELISA detection of CXCL9 secretion. B, Flow cytometry detection of OX40L expression. C, The size of CT26 subcutaneously implanted tumors at different time points is shown in the figure. Arrows indicate the corresponding time when mice were injected with PBS or MSCs. Data represent mean ± SEM (n = 5). D, Flow cytometry analysis of the proportion of immune cells in tumors after MSC treatment. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. [Figure 6]Figure 1 shows that mesenchymal stem cells overexpressing CXCL9 and OX40L inhibit AOM / DSS-induced colorectal cancer. A, Schematic diagram of AOM / DSS treatment and MSC treatment scheme. B, Representative images of colorectal tumors. Scale bar: 5 mm. C, Average tumor number and size. Data represent mean ± SEM (n=3-4). *p<0.05, **p<0.01, ***p<0.001, ns=not significant). D, Immunofluorescence staining of CD8 and NK cells. Scale bar: 50 μm. [Figure 7] This shows that combined treatment with antibodies against PD-1 and CTLA-4 has no significant therapeutic effect on AOM / DSS-induced colorectal cancer. A, Schematic diagram of AOM / DSS treatment and antibody treatment scheme. B, Average tumor number and size. Data represent mean ± SEM (n = 4). ns = not significant. [Figure 8] This shows that mesenchymal stem cells overexpressing CXCL9 and OX40L enhance the efficacy of PD-1 blockade therapy. A, Schematic diagram of the treatment of mesenchymal stem cells (MSC-CXCL9+OX40L) (5x105 cells per needle) and PD-1 antibody blockade (αPD-1). B, Combination treatment of mesenchymal stem cells overexpressing CXCL9 and OX40L with PD-1 antibody blockade has a significantly greater tumor inhibitory effect than either treatment alone (n=7-8 / group). **p<0.01. [Figure 9]We demonstrate that mesenchymal stem cells overexpressing CXCL9 and OX40L can inhibit the growth of MHC-I-deficient tumors with immune evasion properties. A, Flow cytometry analysis was performed on subcutaneously transplanted tumors of mouse colorectal cancer cell MC38 in vivo, confirming that MC38 tumor cells with B2m gene knockout (sgB2m) do not express MHC-I molecules (H2Kb / H2Db) compared to control sgLacZ tumor cells (n=4 per group). B, An in vivo transplantation tumor model of MHC-I-deficient MC38-sgB2m or control MC38-sgLacZ tumor cells overexpressing OVA was established. MC38-sgB2m tumor cells express MHC-I normally, whereas control MC38 tumor cells express MHC-I normally. We found that MC38-sgLacZ cells grew faster than MC38-sgLacZ cells. Treatment with a tumor vaccine (OVA257-264 polypeptide + poly:IC adjuvant) significantly inhibited the growth of MC38-sgLacZ cells. MHC-I-deficient MC38-sgB2m tumors did not respond to vaccine treatment (n=5 per group). Mesenchymal stem cells overexpressing C, CXCL9, and OX40L significantly inhibited the growth of MHC-I-deficient MC38-sgB2m tumors (n=7 per group). **p<0.01, ***p<0.001, ****p<0.0001. [Figure 10] The therapeutic effects of mesenchymal stem cells (MSCs) overexpressing immune activators / cytokines or chemokines are shown. Each mouse received 5 x 105 MSCs overexpressing one of these factors every time, once every three days for a total of six treatments. A. MSCs overexpressing GITRL, 4-1BBL, or CD40L had highly significant tumor-inhibitory effects, whereas MSCs overexpressing IL5 had no significant therapeutic effect (n = 5 mice / group). B. MSCs overexpressing CCL3, CCL19, CCL21, or XCL1 had significant tumor-inhibitory effects (n = 5 mice / group). **p < 0.05, **p < 0.01, ***p < 0.001. DETAILED DESCRIPTION OF THE INVENTION

[0032] Through extensive and thorough research and extensive screening, the present inventors have unexpectedly discovered that mesenchymal stem cells overexpressing the chemokines CCL3, CCL19, CCL21, XCL1, and CXCL9 and / or the cytokines OX40L, 4-1BBL, GITRL, and CD40L can specifically attract and activate immune cells that kill tumor tissue at the tumor site. Furthermore, mesenchymal stem cells and chemokines and / or cytokines have a synergistic effect, resulting in highly efficient immunotherapy with minimal side effects. In particular, overexpression of CXCL9 and OX40L in mesenchymal stem cells has a synergistic effect, significantly enhancing the killing ability of tumor tissue, particularly colorectal cancer cells. Based on this finding, the present inventors have completed the present invention.

[0033] In recent years, immunotherapies such as cytokines, CAR-T cells, and immune checkpoint blockade have shown favorable effects in some cancer patients. However, they also face many obstacles, such as low efficiency of T cells reaching tumor sites, low numbers of immune cells present at tumor sites, and side effects from systemic medication. Mesenchymal stem cells can be obtained from various tissues in vivo, are easy to culture and grow in vitro, are easily modified by genetic engineering techniques, and have low immunogenicity. Our research in mouse models confirmed that adipose-derived mesenchymal stem cells can specifically migrate to tumor sites and are not abundant in other organs, making them ideal drug carriers. This invention uses adipose-derived mesenchymal stem cells as carriers to overexpress the immunomodulatory chemokine CXCL9 and cytokine OX40L. Active migration of mesenchymal stem cells to tumors specifically attracts and activates immune cells that kill tumor tissue at the tumor site, ultimately achieving highly efficient immunotherapeutic effects with minimal side effects.

[0034] [Terminology] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0035] As used herein, when used in reference to a specifically recited numerical value, the term "about" means that the value can vary by no more than 1% from the recited value. For example, as used herein, the term "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0036] As used herein, the terms "containing" or "comprising" can refer to open, semi-closed, and closed systems. In other words, the terms also include "consisting essentially of" or "consisting of."

[0037] As used herein, the term "administration" refers to the physical introduction of any product of the present invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration such as injection or infusion.

[0038] [Mesenchymal stem cells (MSCs)] In recent years, mesenchymal stem cells (MSCs) have emerged as a potential cell carrier, possessing active migratory capacity against many different tumor types when administered systemically. They can be extracted from many different adult tissues, are easy to grow and culture, and can avoid immune rejection. At the same time, mesenchymal stem cells' tumor-tropic migratory properties and their ability to survive for long periods at target sites make them an important resource for cell therapy. Commonly used types of MSCs are bone marrow-derived MSCs (BM-MSCs), umbilical cord blood-derived MSCs (UCB-MSCs), umbilical cord-derived MSCs (UC-MSCs), and adipose tissue-derived MSCs (AT-MSCs). However, the isolation process for BM-MSCs and UCB-MSCs is highly complex and inefficient. Therefore, adipose tissue or umbilical cord tissue MSCs could be used as a more ideal alternative because they contain more MSCs than bone marrow and umbilical cord blood and are easier to obtain and collect. Furthermore, with regard to a source of autologous stem cells for personalized cell therapy, AT-MSCs pose minimal risk to the donor and are not a logical concern.

[0039] Through the tumor-homing ability of MSCs, the present invention uses MSCs to deliver chemokines CCL3, CCL21, XCL1, and CXCL9 and cytokines OX40L, 4-1BBL, GITRL, and CD40L to attract and activate effector T cells, NK cells, and antigen-presenting cells in the tumor microenvironment, thereby generating a more precise and sustained immune response to kill tumor cells. In the current study, adipose-derived mesenchymal stem cells were used as a carrier to overexpress chemokines and cytokines to treat colorectal cancer in a mouse model.

[0040] [Expression cassette] As used herein, an "expression cassette" or an "expression cassette of the invention" includes a first expression cassette and / or a second expression cassette, wherein the first expression cassette includes a nucleic acid sequence encoding a chemokine, and the second expression cassette includes a nucleic acid sequence encoding a cytokine, wherein the chemokine includes CCL3, CCL19, CCL21, XCL1, CXCL9, or a combination thereof, and the cytokine includes OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof.

[0041] In one embodiment, the exogenous nucleic acid molecule comprises a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a nucleic acid sequence encoding CXCL9 and the second expression cassette comprises a nucleic acid sequence encoding OX40L.

[0042] In another preferred embodiment, the first expression cassette and the second expression cassette are independent of each other or combined together. In another preferred embodiment, the first expression cassette and the second expression cassette each further comprise a promoter and / or a terminator. In another preferred embodiment, the first expression cassette and the second expression cassette are located on a carrier or integrated into the chromosomes of the mesenchymal stem cells. In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same or different carriers. In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same carrier. In another preferred embodiment, the carrier is selected from the group consisting of DNA, RNA, a plasmid, a lentiviral carrier, an adenoviral carrier, a retroviral carrier, a transposon, another gene transfer system, or a combination thereof.

[0043] [Carrier] The present invention further provides a carrier comprising the expression cassette of the present invention. Carriers derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of the transgene and propagation in daughter cells. Lentiviral carriers have the advantage over carriers derived from oncogenic retroviruses, such as murine leukemia viruses, that they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of being less immunogenic.

[0044] Briefly summarized, an expression cassette or nucleic acid sequence of the invention is typically operably linked to a promoter and incorporated into an expression vehicle suitable for replication and integration in eukaryotic cells. Typical cloning vehicles contain transcription and translation terminators, early sequences, and promoters that can be used to regulate expression of the desired nucleic acid sequence.

[0045] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy carrier.

[0046] The expression cassette or nucleic acid sequence can be cloned into many types of carriers. For example, the expression cassette or nucleic acid sequence can be cloned into a carrier, including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Particular carriers of interest include expression carriers, replication carriers, probe production carriers, and sequencing carriers.

[0047] Furthermore, expression carriers can be provided to cells in the form of viral carriers.Viral carrier technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology handbooks.Viruses that can be used as carriers include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.Suitable carriers usually contain a replication origin that functions in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0048] Many virus-based systems have been developed and used to introduce genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Using techniques known in the art, a selected gene can be inserted into a carrier and packaged into retroviral particles. The recombinant virus can be isolated and delivered to target cells in vivo or ex vivo. Many retroviral systems are known in the art.

[0049] Additional promoter elements, such as enhancers, can modulate the frequency of transcription initiation. Typically, these are located in the 30-110 bp region upstream of the start site, although recent studies have shown that many promoters also contain functional elements downstream of the start site. Spacing between promoter elements is often flexible to preserve promoter function when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, activity begins to decline when an additional 50 bp is added between promoter elements. Depending on the promoter, individual elements appear to act cooperatively or independently to initiate transcription.

[0050] An example of a suitable promoter is the early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can induce high-level expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked to the inducible promoter when expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0051] The expression carrier introduced into the cells can also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from a population of cells desired to be transfected or infected by the viral carrier. In other embodiments, the selectable marker can be carried on a single segment of DNA and used in co-transfection procedures. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo.

[0052] Reporter genes are used to identify potentially transfected cells and evaluate the functionality of regulatory sequences. Typically, reporter genes are genes that are absent or expressed in the recipient organism or tissue and encode a polypeptide, the expression of which is clearly indicated by some easily detectable property, such as enzymatic activity. After DNA is introduced into the recipient cells, reporter gene expression is measured at an appropriate time. Suitable reporter genes include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green oral protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are known and can be prepared using known techniques or obtained commercially. Typically, a construct with at least five flanking regions that exhibits the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be linked to reporter genes and used to evaluate the ability of reagents to modulate promoter-driven transcription.

[0053] Methods for introducing genes into cells and expressing genes in cells are known in the art.As for expression carriers, the carriers can be easily introduced into host cells, such as mammalian (e.g., human T cells), yeast or insect cells, by any method in the art.For example, the expression carriers can be introduced into host cells by physical, chemical or biological means.

[0054] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for preparing carriers and / or cells containing exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0055] Biological methods for introducing polynucleotides into host cells include the use of DNA and RNA carriers. Viral carriers, particularly retroviral carriers, have become the most widely used method for inserting genes into animal, e.g., human, cells. Other viral carriers may be derived from lentiviruses, poxviruses, herpes simplex viruses, adenoviruses, and adeno-associated viruses, among others. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0056] Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, oil-in-water emulsions, micelles, mixed micelles, and lipid-based systems including liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0057] When a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid can be associated with a lipid. The lipid-associated nucleic acid can be encapsulated in the aqueous interior of a liposome, dispersed in the lipid bilayer of a liposome, bound to a liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with a lipid, associated with a lipid, contained in a liquid as a suspension, contained or complexed in a micelle, or otherwise associated with a liquid. The lipid, lipid / DNA, or lipid / expression carrier associated with the composition is not limited to a particular structure in solution. For example, they can exist in a bilayer structure as micelles or have a "collapsed" structure. They can also simply be dispersed in a solution or form aggregates of heterogeneous size or shape. Lipids are fatty substances and can be natural or synthetic. For example, lipids include lipid droplets that occur naturally in the cytoplasm and compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0058] [Preparation method] The present invention provides a method for preparing mesenchymal stem cells, comprising the step of introducing a first expression cassette and / or a second expression cassette into the mesenchymal stem cells to be modified to obtain the mesenchymal stem cells, wherein the first expression cassette is used to express a chemokine and the second expression cassette is used to express a cytokine.

[0059] Generally, the process involves (1) transforming or transducing suitable host cells with a polynucleotide encoding an immunostimulatory factor of the invention, or with a recombinant expression vehicle containing the polynucleotide, and (2) culturing the host cells in a suitable medium.

[0060] [formulation] The present invention provides a method comprising the mesenchymal stem cells according to the first aspect of the present invention and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injectable formulation. Preferably, the concentration of the mesenchymal stem cells in the formulation is 1 x 10 3 -1×10 8 cells / ml, more preferably 1 x 10 4 -1×10 7 cells / ml.

[0061] In one embodiment, the formulation may contain a buffer such as neutral saline or sulfate-buffered saline, a hydrocarbon such as glucose, mannose, sucrose, or dextran, mannitol, a protein, a polypeptide or amino acid such as glycine, an antioxidant, a chelating agent such as EDTA or glutathione, an adjuvant (e.g., aluminum hydroxide), and a preservative. The formulation of the present invention is preferably formulated for intravenous administration.

[0062] [Therapeutic application] The present invention includes therapeutic applications carried out using mesenchymal stem cells transduced with a carrier containing the expression cassette of the present invention. The mesenchymal stem cells of the present invention can actively migrate to tumor sites and are not concentrated in organs such as the liver, spleen, or kidneys. They are specific and safe carriers of tumor therapeutic drugs, providing an effective means for locally activating tumor immune responses and avoiding systemic side effects. A therapeutic system using adipose-derived mesenchymal stem cells that overexpress the chemokine CXCL9 and cytokine OX40L has the ability to specifically target tumor sites and can achieve ideal antitumor effects by attracting and activating T cells and NK cells.

[0063] In one embodiment, the present invention provides a cell therapy comprising administering to a mammalian subject the mesenchymal stem cells of the present invention. Unlike antibody therapies, the mesenchymal stem cells of the present invention are capable of replicating in vivo, providing long-term persistence leading to sustained tumor inhibition.

[0064] Treatable cancers include non-vascularized or substantially non-vascularized tumors, as well as vascularized tumors.Cancers can include non-solid tumors (e.g., hematological tumors, such as leukemia and lymphoma) or solid tumors.The types of cancers treated with the mesenchymal stem cells of the present invention include, but are not limited to, carcinomas, blastomas, sarcomas, certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors such as sarcomas, carcinomas, and melanomas.Also included are adult tumors / cancers and pediatric tumors / cancers.

[0065] Hematological cancer is cancer of the blood or bone marrow. Examples of hematological (or hematopoietic) cancer include leukemia, including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid cell leukemia, acute myeloid leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia de novo, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0066] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or areas of fluid. Solid tumors can be benign or malignant. Various types of solid tumors are named for the type of cells that form them (e.g., sarcoma, carcinoma, and lymphoma). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, and ovarian cancer.

[0067] The mesenchymal stem cells of the present invention can be used as a type of vaccine for ex vivo immunization and / or in vivo therapy of mammals. Preferably, the mammal is a human.

[0068] For ex vivo immunization, at least one of the following is performed in vitro before administering the cells to a mammal: i) expanding the cells, ii) introducing an expression cassette of the invention into the cells, and / or iii) cryopreserving the cells.

[0069] Ex vivo procedures are known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (i.e., transduced or transfected in vitro) with a carrier containing an expression cassette of the invention. The mesenchymal stem cells of the invention can be administered to a mammalian recipient to provide a therapeutic effect. The mammalian recipient can be human, and the mesenchymal stem cells of the invention can be autologous to the recipient. Optionally, the cells can be allogeneic, syngeneic, or xenogeneic with respect to the recipient.

[0070] In addition to the use of cell-based vaccines for ex vivo immunization, the present invention provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

[0071] Typically, the activated and expanded cells described herein are useful for treating and preventing diseases that occur in immunocompromised individuals. Accordingly, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the mesenchymal stem cells of the present invention.

[0072] The mesenchymal stem cells of the present invention can be administered alone or as a pharmaceutical composition in combination with other components such as a diluent and / or certain cytokines or cell populations. Briefly, the pharmaceutical compositions or formulations of the present invention can comprise a combination of the mesenchymal stem cells described herein with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0073] The pharmaceutical composition of the present invention can be administered in a manner appropriate for the disease to be treated (or prevented). The number and frequency of administration are determined by factors such as the patient's condition, the type and severity of the patient's disease, etc., and the appropriate dosage can be determined by clinical trials.

[0074] When an "immunologically effective amount," "antitumor effective amount," "tumor-inhibiting effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). Generally, the pharmaceutical compositions comprising mesenchymal stem cells described herein are administered in an amount of 10 4 ~10 9 Dose of cells / kg body weight, preferably 10 5 ~10 6 The mesenchymal stem cell composition can be administered at a dose of 1000 mg / kg body weight (including all integer values ​​within these ranges). The mesenchymal stem cell composition can also be administered multiple times at these doses. The cells can be administered using injection techniques well known in immunotherapy (e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment scheme for a specific patient can be easily determined by those skilled in the medical field by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0075] Administration of the composition to a subject can be by any convenient means, including spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to a patient via subcutaneous, intradermal, intratumoral, intranodal, intraspinal, intramuscular, intravenous (iv) injection, or intraperitoneal administration. In one embodiment, the T cell composition of the present invention is administered to a patient via intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered via iv injection. The mesenchymal stem cell composition can be injected directly into a tumor, lymph node, or site of infection.

[0076] In certain embodiments of the present invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding mesenchymal stem cells to therapeutic levels are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) any number of relevant forms of treatment, including, but not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or agents such as natalizumab treatment for MS patients, elfatizumab treatment for psoriasis patients, or other treatments for PML patients. In further embodiments, the mesenchymal stem cells of the present invention can be used in combination with chemotherapy, radiation therapy, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to a patient in conjunction with (e.g., before, simultaneously, or after) chemotherapeutic agents such as bone marrow transplantation, fludarabine, external beam radiation therapy (XRT), cyclophosphamide, and the like. For example, in one embodiment, a subject may receive standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In some embodiments, after transplant, the subject receives an infusion of the expanded mesenchymal stem cells of the present invention. In additional embodiments, the expanded cells are administered before or after surgery.

[0077] The dosages administered to patients for the above treatments vary depending on the exact nature of the condition being treated and the person being treated. The dosage ratio for administration to a human can be carried out according to accepted practices in the art. Typically, for each treatment or each course of treatment, 1 x 10 3 ~1×10 10 The mesenchymal stem cells of the present invention can be administered to a patient, for example, by intravenous infusion.

[0078] The technical scheme of the present invention has the following beneficial effects: 1. The mesenchymal stem cells of the present invention can be obtained from various tissues in vivo, are easy to culture and proliferate in vitro, and are easily transformed by genetic engineering techniques, and have low immunogenicity.

[0079] 2. Compared with most immunotherapies, the method of the present invention does not depend on the presence of tumor-infiltrating lymphocytes, and is suitable for treating tumors with very low or resistant lymphocyte infiltration in clinical settings.

[0080] 3. When the mesenchymal stem cells of the present invention overexpress one or more of CCL3, CCL19, CCL21, XCL1, CXCL9, OX40L, 4-1BBL, GITRL, and CD40L, they can specifically attract and activate immune cells that kill tumor tissue at the tumor site, resulting in highly efficient immunotherapeutic effects with few side effects.

[0081] 4. When the mesenchymal stem cells of the present invention overexpress CXCL9 and OX40L, they have a synergistic effect, significantly enhancing their ability to kill tumor tissues, particularly colorectal cancer cells. This method also has a killing effect on MHC-I-negative tumor cells that are resistant to conventional immunotherapy (e.g., CAR-T or PD-1 / PD-L1 antibodies).

[0082] 5. When the present invention is used in combination with other clinically used immunotherapies, such as CAR-T or PD-1 / PD-L1 antibodies, the effects of these immunotherapies can be enhanced. The combined treatment of the mesenchymal stem cells of the present invention and PD-1 antibodies has a more significant tumor-inhibiting effect than the monotherapy of either of them, and the two exhibit a synergistic effect.

[0083] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are used only to illustrate the present invention and do not limit the scope of the present invention. In the following examples, experimental methods without specific conditions are generally in accordance with conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions suggested by manufacturers. Unless otherwise specified, percentages and parts are calculated by weight.

[0084] [material and method] [Cell line] CT26 cells are colon adenocarcinoma cells derived from BALB / c mice, and MC38 is a colon adenocarcinoma cell line derived from C57BL / 6 mice. CT26 and MC38 cells were cultured in RPMI 1640 and DMEM containing 10% fetal bovine serum and 1% penicillin / streptomycin, respectively.

[0085] [antibody] Antibodies used for flow cytometry analysis were from BD Biosciences, BioLegend, or eBioscience. Antibodies used for Western blot analysis included anti-CXCL9 (Abcam), anti-Myc-tag (Cell Signaling Technology), anti-OX40L (Abcam), and anti-GAPDH (Abcam). Antibodies used for immunofluorescence staining included anti-GFP (Abcam), anti-CD8a (BioLegend), and anti-NKp46 (CD335) (BioLegend).

[0086] Immune checkpoint blocking antibodies anti-PD-1 (clone RMP1-4) and anti-CTLA-4 (clone 9D9) were purchased from Bio X Cell. These two antibodies (anti-PD-1: 200 μg / mouse, anti-CTLA-4: 100 μg / mouse) were administered intraperitoneally to mice.

[0087] [Isolation, culture, and identification of mesenchymal stem cells from mouse adipose tissue] AT-MSCs were isolated from mouse subcutaneous adipose tissue using collagenase type I. The cells were cultured in α-MEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. After three passages of adherent cell culture, the expression of cell surface marker proteins was identified using flow cytometry.

[0088] Lentivirus production and transduction The cDNA was cloned into a lentiviral carrier. Lentiviral packaging and titration were completed by OBiO Technology (Shanghai) Co., Ltd. Mesenchymal stem cells were infected with the lentivirus at a multiplicity of infection (MOI) of 60 in the presence of 6 μg / ml polybrene (Sigma-Aldrich).

[0089] [Tumor cell proliferation experiment] Tumor cell proliferation is measured using a CCK-8 kit (Dojindo Molecular Technologies) according to the manual procedure. Absorbance is measured using a microplate reader (Tecan).

[0090] [Western Blotting] Cells were harvested and treated with cell lysis buffer (RIPA buffer + 1% protease inhibitor) (ThermoFisher Scientific) to prepare cell lysates. Protein concentration in the cell lysates was measured using a BCA kit (ThermoFisher Scientific). 15–30 μg of protein was loaded onto a 5%–15% SDS-PAGE protein gel (ThermoFisher Scientific) and then transferred to a PVDF membrane (Millipore). The membrane was blocked with 5% skim milk in TBST buffer and incubated overnight at 4°C with antibodies against Myc tag, OX40L, CXCL9, and GAPDH. The membrane was washed with TBS-T buffer and incubated for 1 hour at room temperature with horseradish peroxidase-coupled secondary antibodies. The membrane was developed and exposed to light using an enhanced chemiluminescence kit (Millipore).

[0091] [Enzyme-linked immunosorbent assay (ELISA)] The supernatant of lentivirally transduced AT-MSCs was collected and stored in a refrigerator at -80°C until assay. CXCL9 secretion was tested using an ELISA kit from Abcam according to the manufacturer's instructions.

[0092] [Subcutaneous tumor transplantation model in mice] Inject CT26 or MC38 (0.5 or 1 × 10) into the right lower back of 8- to 10-week-old BALB / c or C57BL / 6 mice, respectively. 6 Mice were subcutaneously injected with 250 μl of PBS or 5 × 10 cells per mouse. When the tumor reached a maximum diameter of 0.5–0.7 cm, the mice were randomly assigned to experimental groups. Each animal received 250 μl of PBS or 5 × 10 cells per mouse. 5 Systemic administration was performed by injecting 250 μl of AT-MSCs in PBS via the tail vein. Tumor size was measured once every 3 days using a caliper and calculated as V = L × W. 2 Tumor volume was calculated using the formula: L / 2, where L and W are the long and short diameters of the tumor, respectively. Mice were monitored for tumor size and survival. When tumor volume was 2 cm 3Mice are sacrificed when the tumor reaches 0.5 mm Hg, or when the tumor ulcerates, or when the mice become moribund.

[0093] [Flow cytometry] To identify AT-MSCs, adherent cells isolated at passages 3 to 5 were digested with 20 µM EDTA, then washed twice with PBS, and stained with antibodies.

[0094] To analyze tumor-infiltrating immune cells, subcutaneously implanted tumors were dissected into RPMI medium, minced with scissors, and placed in serum-free RPMI medium containing 0.25 mg / ml Liberase TL (Roche) and 50 μg / ml DNase I (Sigma-Aldrich). The cells were digested at 37°C using a gentleMAC Octo Dissociator (Milteniy Biotec) and dispersed through a 40 μm cell strainer (BD Biosciences). Single cells were further washed and stained with antibodies. Dead cells were excluded by staining with the Zombie Fixable Viability Kit (BioLegend). For intracellular cytokine selection, each mouse was intraperitoneally injected with 0.25 mg of brefeldin A (BFA) (Selleck) 4–6 h before sample collection. Surface staining was performed in the presence of 5 μg / ml BFA, followed by intracellular staining using the Intracellular Fixation and Permeabilization Buffer Set (eBioscience). After surface staining, nuclear staining is performed using Foxp3 transcription factor staining buffer (eBioscience).

[0095] Flow data are acquired on a BD LSRFortessa cell analyzer (BD Biosciences) and analyzed using FlowJo software. All antibodies used for flow cytometry are purchased from BD Biosciences, BioLegend, or eBioscience.

[0096] [AOM / DSS-induced mouse colorectal cancer model] BALB / c mice were intraperitoneally injected with AOM (12.5 mg / kg body weight, Sigma-Aldrich) (14). One week later, the mice were given drinking water containing 3% DSS (MP Biomedicals) for 7 days, followed by regular water for 2 weeks. DSS induction was continued for two cycles, and starting from the last week of the DSS induction cycle, MSCs were injected five times via the tail vein before the mice were sacrificed. Body weights were recorded during DSS treatment. The colons were removed from the mice, rinsed with ice-cold PBS, opened longitudinally, fixed overnight in 4% paraformaldehyde solution (Sigma-Aldrich) at room temperature, and embedded in paraffin. Before fixation, the size was measured using a digital caliper.

[0097] [Immunofluorescence] Tissue sections were blocked with 10% normal goat serum, then incubated with primary antibodies overnight at 4°C and secondary antibodies for 1 hour at room temperature. Slides were mounted in antifade mounts with DAPI (ThermoFisher Scientific) and observed under a Nikon Eclipse Ti fluorescence microscope. The antibodies used for immunofluorescence were GFP antibody (Abcam), CD8a antibody (BioLegend), and NKp46 (CD335) antibody (BioLegend).

[0098] [statistics] All results are expressed as mean ± SEM. Differences were assessed by Student's t-test, or, when comparing means of two groups of abnormalities, through two-way ANOVA followed by Bonferroni's multiple comparison test. Data were analyzed using Prism software (GraphPad). Statistical significance was set at a level of p<0.05.

[0099] [Research approval] All animal procedures are approved by the Animal Care and Use Committee of Shanghai Jiao Tong University.

[0100] Example 1. Migration characteristics of adipose-derived mesenchymal stem cells to tumors Mesenchymal stem cells were extracted from mouse subcutaneous fat and detected by flow cytometry. These cells expressed specific mesenchymal stem cell marker molecules, but did not express marker molecules for other cell types (Figure 1A), confirming the purity of the adipose-derived mesenchymal stem cells used in the experiment. Adipose-derived mesenchymal stem cells were transfected with lentivirus to express GFP, and 5 × 10 5 Cells were injected via the tail vein into tumor-bearing mice (CT26 colon cancer subcutaneous tumors). Seven days later, immunofluorescence staining of tumor and other organ tissue sections revealed that GFP-positive mesenchymal stem cells were present only in the tumor, but not in other organs such as the liver, spleen, or kidney (Figure 1B). Flow cytometry detected GFP-positive mesenchymal stem cells within the tumor, and a certain number of cells were detectable even 14 days after cell injection (Figure 1C). These results demonstrate that adipose-derived mesenchymal stem cells can specifically migrate to tumor sites and persist for long periods, supporting their potential as carriers of therapeutic molecules.

[0101] Example 2. Antitumor properties of CXCL9 and OX40L To explore immune-activating therapeutic molecules with higher efficacy, we selected chemokines and cytokines with potential antitumor functions, cloned their genes into lentiviral carriers, and packaged lentivirus carrying these genes. CT26 colon cancer cell lines transduced with these genes or blank control lentivirus were then subjected to subcutaneous tumor transplantation experiments to detect the antitumor effects of these molecules. After overexpressing the potential antitumor chemokines CCL3 and CXCL9 in CT26 (Figure 2A), cell proliferation in vitro was unaffected (Figure 2B), but subcutaneous tumor growth in vivo was significantly inhibited (Figure 2C), indicating that these chemokines may inhibit tumor growth through the immune system in vivo. Here, the antitumor effect of CXCL9 was most significant. Flow cytometry analysis of the immune cell composition within tumors demonstrated that CXCL9 can indeed increase the infiltration of antitumor immune cells, such as CD8, CD4, and NK cells (Figure 2D).

[0102] In CT26 cells overexpressing two immune-activating cytokines, IL36β and OX40L (Figure 3A and B), overexpression of these two cytokines had no effect on tumor cell proliferation in vitro (Figure 3C), but had a significant inhibitory effect on the growth of subcutaneously transplanted tumors (Figure 3D), suggesting that antitumor effects may be achieved via the immune system in vivo. Here, OX40L exhibited potent antitumor effects.

[0103] Example 3: Antitumor effect of mesenchymal stem cells overexpressing the chemokine CXCL9 and cytokine OX40L Adipose-derived mesenchymal stem cell systems overexpressing CXCL9 and OX40L were established by lentiviral infection. Successful expression and secretion of CXCL9 was confirmed by Western blot and ELISA techniques (Figure 4A and B), and successful expression of OX40L on the cell membrane was confirmed by Western blot and flow cytometry (Figure 4C and D). Subsequently, MSCs overexpressing CXCL9 and OX40L simultaneously (Figure 5A and B) were established in a CT26 subcutaneously implanted tumor model. 5 × 10 cells were injected into mice via the tail vein each time. 5 After three treatments of mesenchymal stem cells or PBS at 4-day intervals, mesenchymal stem cells bearing both CXCL9 and OX40L showed the strongest antitumor effect (Figure 5C).Flow cytometry analysis revealed a significant increase in the proportion of intratumor lymphocytes, particularly antitumor CD8 T cells and NK cells (Figure 5D), demonstrating that this treatment effectively stimulated antitumor immune responses.

[0104] To further explore the efficacy of the mesenchymal stem cell immunotherapy system, an inflammation-driven in situ intestinal cancer model was induced by AOM / DSS. Starting from the last week of the third DSS treatment cycle, mice were treated five times over a four-week period (Figure 6A). PBS or 5 × 10 5Injection of mesenchymal stem cells bearing CXCL9 and OX40L significantly reduced intestinal tumors in mice (Figure 6B and C). Immunofluorescence staining showed a significant increase in the infiltration of anti-tumor CD8 T cells and NK cells (Figure 6D), which is consistent with the results observed in the transplantation tumor model.

[0105] In comparison, in an AOM / DSS-induced in situ colon cancer model, treatment of mice with a combination of PD-1 (200 μg / mouse) and CTLA-4 (100 μg / mouse) antibodies (Figure 7A) did not significantly alter tumor growth (Figure 7B).

[0106] These results demonstrate that adipose-derived mesenchymal stem cells overexpressing CXCL9 and OX40L demonstrated highly significant therapeutic effects in both subcutaneously transplanted tumors in mice and in an inflammation-induced in situ colon cancer model. Mesenchymal stem cells expressing both CXCL9 and OX40L simultaneously exhibited a synergistic effect, demonstrating a more pronounced antitumor effect than mesenchymal stem cells expressing either CXCL9 or OX40L alone. Furthermore, the efficacy of mesenchymal stem cell therapy in an inflammation-induced in situ colon cancer model was significantly superior to that of immune checkpoint blockade therapy.

[0107] Example 4. Mesenchymal stem cells overexpressing the chemokine CXCL9 and cytokine OX40L improve the therapeutic effect of PD-1 blockade therapy. In a CT26 subcutaneous tumor-implanted mouse model, mice were injected a total of four times with mesenchymal stem cells (MSCs) overexpressing CXCL9 and OX40L, a PD-1 blocking antibody (αPD-1), or both (Figure 8A).

[0108] The results, as shown in Figure 8B, showed that while MSCs and αPD-1 alone could significantly inhibit tumor growth, the combined treatment of MSCs and αPD-1 inhibited tumor growth more effectively than either treatment alone, indicating that MSC therapy has a clear enhancing effect on PD-1 blockade therapy.

[0109] Example 5: Inhibitory effect of mesenchymal stem cells overexpressing the chemokine CXCL9 and cytokine OX40L on MHC-I-deficient tumors We successfully established an MHC-I-deficient tumor model by knocking out the B2m gene (sgB2m) in MC38 using CRISPR technology (Figure 9A). Based on this, overexpression of the OVA gene induces tumor cell-specific expression of OVA antigen. Compared to the control sgLacZ, MHC-I-deficient MC38-sgBm grows faster and is insensitive to OVA polypeptide vaccine treatment (Figure 9B). Mesenchymal stem cells overexpressing CXCL9 and OX40L can still significantly inhibit the growth of MHC-I-deficient MC38-sgBm tumors (Figure 9C). This indicates that mesenchymal stem cell treatment has a significant advantage over tumor vaccines in the case of MHC-I-deficient tumors.

[0110] Example 6: Antitumor effects of mesenchymal stem cells overexpressing other chemokines or cytokines In the CT26 transplanted tumor model, mesenchymal stem cells (5 × 10 ) expressing different cytokines or chemokines were cultured. 5 Mice were injected with IL15 (individually / mouse / next injection) or PBS via the tail vein. While mesenchymal stem cells overexpressing IL15 did not show any significant antitumor effects, mesenchymal stem cells overexpressing GITRL, 4-1BBL, and CD40L all exhibited highly effective antitumor effects (Figure 10A). Similarly, overexpression of several other chemokines, including CCL3, CCL19, CCL21, and XCL1, all exhibited significant antitumor effects (Figure 10B).

[0111] [Discussion] Immunotherapy has revolutionized cancer treatment. While some cytokines and immune checkpoint blockers have demonstrated significant therapeutic effects in clinical tumor treatment, the systemic use of these drugs nonspecifically activates the immune system and affects most organs. To address the side effects of systemic administration, mesenchymal stem cells have been selected as a drug carrier. Research has shown that adipose-derived mesenchymal stem cells can actively migrate to tumor sites and are not abundant in organs such as the liver, spleen, and kidneys. This fully supports the specificity and safety of mesenchymal stem cells as a tumor therapeutic drug carrier, providing an effective means of locally activating tumor immune responses and avoiding systemic side effects.

[0112] Tumor infiltration by T cells and NK cells is an important determinant of the efficacy of immunotherapy in solid tumors. Tumors can limit lymphocyte infiltration through various mechanisms. Tumors with higher T cell infiltration typically also express high levels of chemokines that can attract T cells, including CCL3, CCL4, and CXCL10. Surprisingly, the present invention discovered that expressing CXCL9 in mesenchymal stem cells as a carrier and delivering it to tumor sites to attract anti-tumor lymphocytes exhibits stronger T cell and NK cell attraction, solving the problem of lymphocytes' limited entry into solid tumors. Furthermore, delivery of OX40L to mesenchymal stem cell lines can also more efficiently activate T cells and NK cells. OX40 receptor-activating antibodies are currently in clinical trials (ClinicalTrials.gov), but potential side effects of systemic drugs remain a potential problem. The OX40 ligand, OX40L, is a membrane protein whose expression in mesenchymal stem cells allows targeted delivery to tumor sites, without being secreted and metastasized to other sites like secreted cytokines, and can reduce secreted nucleic acids and activate existing or newly migrated lymphocytes at the tumor site. The present inventors also unexpectedly discovered that adipose-derived mesenchymal stem cells overexpressing CXCL9 and OX40L exhibited highly significant therapeutic effects in both subcutaneously transplanted tumors in mice and in an inflammation-induced in situ colon cancer model.

[0113] In general, the adipose-derived mesenchymal stem cell therapeutic system established by the present invention, which overexpresses the chemokine CXCL9 and cytokine OX40L, has the ability to specifically target tumor sites and exerts ideal antitumor effects by attracting and activating T cells and NK cells. Compared to most immunotherapies, this therapy does not depend on the presence of tumor-infiltrating lymphocytes and is suitable for treating tumors with very low or resistant lymphocyte infiltration in clinical settings. Adipose or umbilical cord mesenchymal stem cells are easy to extract and culture, facilitating their application in personalized therapy. Their low immunogenicity also makes allogeneic use feasible. Therefore, the established immunotherapy based on mesenchymal stem cells has significant clinical translational value.

[0114] All documents mentioned in this application are incorporated by reference in this application as if each document were incorporated by reference individually. Furthermore, after reading the above teachings of the present invention, those skilled in the art will be able to make various changes or modifications to the present invention, and these equivalents will also fall within the scope defined by the appended claims of this application.

Claims

1. 1. Mesenchymal stem cells for use in the treatment of colorectal cancer, comprising: The mesenchymal stem cells express the following immune stimulatory factors: i) CXCL9 and ii) one or two of OX40L, 4-1BBL, and GITRL wherein the mesenchymal stem cells express GITRL, Here, the mesenchymal stem cells are characterized in that they contain an exogenous nucleic acid molecule containing a nucleic acid sequence encoding the immunostimulatory factor.

2. i) CXCL9 and ii) one of OX40L, 4-1BBL, and GITRL Expressing wherein the mesenchymal stem cells express GITRL. Mesenchymal stem cells for use according to claim 1.

3. The mesenchymal stem cells are characterized in that they include adipose-derived mesenchymal stem cells, umbilical cord mesenchymal stem cells, or a combination thereof. Mesenchymal stem cells for use according to claim 1.

4. 1. A formulation for use in the treatment of colon cancer, comprising: The formulation comprises mesenchymal stem cells and a pharmaceutically acceptable carrier, diluent, or excipient; The mesenchymal stem cells express the following immune stimulatory factors: i) CXCL9 and ii) one or two of OX40L, 4-1BBL, and GITRL The preparation, wherein the mesenchymal stem cells express GITRL.

5. The mesenchymal stem cells are i) CXCL9 and ii) one of OX40L, 4-1BBL, and GITRL Expressing wherein the mesenchymal stem cells express GITRL. A formulation for use according to claim 4.

6. 1. A kit for use in treating colon cancer, comprising: The kit comprises: (1) A first container and mesenchymal stem cells contained in the first container, wherein the mesenchymal stem cells express the following immunostimulatory factors: i) CXCL9 and ii) one or two of OX40L, 4-1BBL, and GITRL Here, the mesenchymal stem cells are mesenchymal stem cells that express GITRL. , and (2) A kit comprising a second container and an anti-tumor immunotherapeutic agent contained in the second container.

7. The antitumor immunotherapeutic agent is characterized in that it is an immune checkpoint antibody. A kit for use according to claim 6.

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