CN111849905A

CN111849905A - Immunotherapy of mesenchymal stem cell targeted transport of chemokines and cytokines

Info

Publication number: CN111849905A
Application number: CN201910314298.6A
Authority: CN
Inventors: 高维强; 马斌; 印盼
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2019-04-18
Filing date: 2019-04-18
Publication date: 2020-10-30
Anticipated expiration: 2039-04-18
Also published as: CN115851605A; CN111849905B

Abstract

The invention provides an immunotherapy of mesenchymal stem cell targeted delivery of chemokines and cytokines. Specifically, the present invention provides a mesenchymal stem cell expressing an immunostimulatory 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 for killing tumor tissues at tumor parts, and the mesenchymal stem cells have a synergistic effect with chemotactic factors and/or cytokines, so that the mesenchymal stem cells have a more efficient and low-side-effect immune curative effect, and the killing capacity on the tumor tissues is remarkably enhanced, particularly colorectal cancer cells.

Description

Immunotherapy of mesenchymal stem cell targeted transport of chemokines and cytokines

Technical Field

The invention belongs to, in particular to an immunotherapy of mesenchymal stem cell targeted transportation of chemotactic factors and cytokines.

Background

The rapid development of immunotherapy brings new eosin to cancer, and in particular chimeric antigen receptor T cell immunotherapy (CAR-T) and immune regulation checkpoint blockade are two cancer immunotherapies that are currently on the forefront of comparison. CAR-T cell therapy has shown some efficacy in some types of cancer, but also suffers from the bottlenecks of low efficiency of T cells reaching solid tumor sites, short duration of action, low number of existing immune cells at tumor sites, side effects from systemic administration, etc. The use of antibodies targeting the T cell inhibitory receptors PD-1 or CTLA-4 can produce very significant anti-tumor effects. However, the therapeutic efficacy of antibodies is often limited by a number of factors, such as low infiltration of T cells and loss of activity in solid tumors. In addition, systemic use of immunotherapeutic drugs such as interferon alpha, interleukin 2, or PD-1 antibody, etc. may cause serious side effects.

There is therefore a pressing need in the art for an immunotherapy that specifically targets tumor cells.

Disclosure of Invention

The object of the present invention is to provide an immunotherapy specifically targeting tumor cells.

Another object of the present invention is to provide an immunotherapy for targeting and transporting chemokines and cytokines by mesenchymal stem cells.

In a first aspect of the present invention, there is provided a mesenchymal stem cell expressing an immunostimulatory factor selected from the group consisting of: CCL3, CCL19, CCL21, XCL1, CXCL9, OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof.

In another preferred embodiment, the mesenchymal stem cell contains an exogenous nucleic acid molecule comprising a nucleic acid sequence encoding an immunostimulatory factor selected from the group consisting of: CXCL9, OX40L, CCL3, CCL19, CCL21, XCL1, CXCL9, OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof.

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 the promoter/enhancer combination.

In another preferred embodiment, the promoter is a constitutive promoter or an inducible promoter, preferably a constitutive promoter.

In another preferred embodiment, the immunostimulatory factor comprises at least one chemokine, the chemokine comprising: CCL3, CCL19, CCL21, XCL1, CXCL9, or a combination thereof.

In another preferred embodiment, the immunostimulatory factor includes at least one cytokine including: OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof.

In another preferred embodiment, the immunostimulatory factor comprises at least one chemokine and at least one cytokine, wherein the chemokine comprises: CCL3, CCL19, CCL21, XCL1, CXCL9, or a combination thereof, the cytokine comprising: OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof.

In another preferred embodiment, the immunostimulatory factor is one or two of CCL3, CCL19, CCL21, XCL1 in combination with CD 40L.

In another preferred embodiment, the immunostimulatory factor is one or both of OX40L, 4-1BBL, GITRL in combination with CXCL 9.

In another preferred example, the immunostimulatory factor is CXCL9 and/or OX 40L.

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

In another preferred example, the exogenous nucleic acid molecule comprises a first expression cassette comprising a nucleic acid sequence encoding CXCL9 and a second expression cassette comprising a nucleic acid sequence encoding OX 40L.

In another preferred embodiment, the first expression cassette and the second expression cassette are each independent, or are combined into one.

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 the same expression cassette comprising a promoter, a nucleic acid sequence encoding a chemokine, and a nucleic acid sequence encoding a cytokine.

In another preferred embodiment, said first expression cassette and said second expression cassette are located on a vector or integrated in the chromosome of said mesenchymal stem cell.

In another preferred embodiment, the first expression cassette and the second expression cassette are independent or linked.

In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same or different vectors.

In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same vector.

In another preferred embodiment, the carrier is selected from the group consisting of: DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, transposons, other gene transfer systems, or combinations thereof.

In another preferred embodiment, the mesenchymal stem cell comprises: adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, or a combination thereof.

In another preferred embodiment, the mesenchymal stem cell is ex vivo.

In another preferred embodiment, the mesenchymal stem cells are autologous or allogeneic.

In a second aspect of the present invention, there is provided a method for preparing the mesenchymal stem cell of the first aspect of the present invention, comprising the steps of:

(1) providing a mesenchymal stem cell to be modified; and

(2) introducing an exogenous nucleic acid comprising a nucleic acid sequence encoding an immunostimulatory factor into the mesenchymal stem cell to be engineered, thereby obtaining a mesenchymal stem cell of the first aspect of the invention;

wherein the immunostimulatory factor is selected from the group consisting of: CCL3, CCL19, CCL21, XCL1, CXCL9, OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof.

In a third aspect of the invention, there is provided a formulation comprising mesenchymal stem cells according to the first aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

In another preferred embodiment, the formulation is a liquid formulation.

In another preferred embodiment, the formulation comprises an injection.

In another preferred embodiment, the mesenchymal stem cells are present in the preparation at a concentration of 1 × 10³-1×10⁸Individual cells/ml, preferably 1X 10⁴-1×10⁷Individual cells/ml.

In a fourth aspect of the present invention, there is provided a use of the mesenchymal stem cell according to the first aspect of the present invention for preparing a medicament or preparation for preventing and/or treating cancer or tumor.

In another preferred embodiment, the tumor is selected from the group consisting of: a hematologic 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 Lymphoblastic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

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

In another preferred example, the solid tumor is colorectal cancer.

In a fifth aspect of the invention, there is provided a pharmaceutical composition comprising

(1) A mesenchymal stem cell according to the first aspect of the invention; and

(2) an anti-tumor immunotherapeutic agent.

In another preferred embodiment, the anti-tumor immunotherapeutic agent is selected from the group consisting of: antibodies, immune cells, or a combination thereof.

In another preferred embodiment, the immune cell is a T cell or NK cell.

In another preferred embodiment, the anti-tumor immunotherapeutic agent is an immune checkpoint antibody.

In another preferred embodiment, the immune checkpoint antibody comprises a PD-1 antibody and/or a CTLA-4 antibody.

The invention also provides a method of treating a disease comprising administering to a subject in need thereof an amount of a cell according to the first aspect of the invention, or a formulation according to the third aspect of the invention, or a pharmaceutical 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.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Figure 1 shows the identification of mouse adipose mesenchymal stem cell phenotype and migration to tumor characteristics. A, detecting the expression of the surface molecules of the adipose-derived mesenchymal stem cells by a flow cytometry method. B, migration of MSC-GFP in vivo in CT26 subcutaneous transplantation tumor mouse model. Immunofluorescence detects MSC-GFP infiltration in tumor tissue, green for MSC-GFP, blue (DAPI staining) for nuclei, scale bar: 20 μ M. C, flow cytometry measures the specific amount of MSC-GFP in the whole CT26 subcutaneous transplanted tumor. Data represent mean ± SEM (n ═ 3).

Figure 2 shows that overexpression of CXCL9 in tumor cells inhibits tumor growth in vivo. A, WB and qPCR detected overexpression of CCL3 and CXCL9 in CT 26. Data represent mean ± SEM (n ═ 3). Cell proliferation experiments in B, CT26-Vector, CT26-CCL3 and CT26-CXCL 9. Data represent mean ± SEM (n ═ 3). C, tumor growth curves in mice injected subcutaneously with CT26-Vector, CT26-CCL3 or CT26-CXCL9 tumor cells BALB/C, tumor size was measured every 3 days. Data represent mean ± SEM (n ═ 4). D, percentage of total intracellular CD8+, CD4+ T and NK cells. Data represent mean ± SEM (n ═ 4).

FIG. 3 shows that overexpression of OX40L in tumor cells inhibited tumor growth in vivo. A, WB detected the overexpression of IL36 β and OX40L in CT 26. B, flow detection of OX40L overexpression in CT 26. Cell proliferation assays for C, CT26-Vector, CT26-IL36 β and CT26-OX 40L. Data represent mean ± SEM (n ═ 3). D, tumor growth curves in mice injected subcutaneously with CT26-Vector, CT26-CCL3 or CT26-CXCL9 tumor cells BALB/c, tumor size was measured every 3 days. Four mice were used per group. Data represent mean ± SEM (n ═ 4). P <0.05, p < 0.01.

Figure 4 shows the identification of CXCL9 over-expression in mesenchymal stem cells with OX 40L. A, WB detects overexpression of CXCL9 in MSC. B, ELISA detects overexpression of CXCL9 in MSCs. Data represent mean ± SEM (n ═ 3). P < 0.001. C, WB detected overexpression of OX40L in MSCs. D, flow cytometry detects OX40L overexpression in MSCs. E, MSC-OX40L coculture with spleen cells stimulated proliferation of spleen T cells.

Figure 5 shows that mesenchymal stem cells overexpressing CXCL9 and OX40L inhibit the growth of subcutaneous transplantable tumors. A, ELISA detects secretion of CXCL 9. B, flow cytometry to detect expression of OX 40L. C, shown as CT26 subcutaneous graft tumor sizes at different time points. Arrows indicate the time of the corresponding PBS or MSC injection to the mice. 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, ns not significant from 0.001.

Figure 6 shows that mesenchymal stem cells overexpressing CXCL9 and OX40L inhibit AOM/DSS-induced colorectal cancer. A, AOM/DSS treatment and MSC treatment protocol schematic. B, representative images of colorectal tumors. Scale bar: 5 mm. C, mean tumor number and size. Data represent mean ± SEM (n ═ 3-4). P <0.05, p <0.01, ns notsirnificant (no statistical significance). Immunofluorescence staining of D, CD8 and NK cells. Scale bar: 50 μm.

Figure 7 shows that PD-1 and CTLA-4 antibody combination therapy had no significant therapeutic efficacy on AOM/DSS-induced colorectal cancer. A, AOM/DSS treatment and antibody treatment protocol. B, mean tumor number and size. Data represent mean ± SEM (n ═ 4). ns-not significant.

Detailed Description

The inventor of the invention has extensively and deeply studied and screened a lot of times, and unexpectedly found that when the chemotactic factors CCL3, CCL19, CCL21, XCL1, CXCL9 and/or the cytokines OX40L, 4-1BBL, GITRL and CD40L are over-expressed by the mesenchymal stem cells, the mesenchymal stem cells can specifically attract and activate immune cells killing tumor tissues at tumor parts, and the mesenchymal stem cells have synergistic action with the chemotactic factors and/or the cytokines, so that the invention has more efficient and low-side-effect immune curative effect. Especially when the mesenchymal stem cells over-express CXCL9 and OX40L, the mesenchymal stem cells have mutual synergistic effect and obviously enhance the killing capacity to tumor tissues, especially colorectal cancer cells. On this basis, the inventors have completed the present invention.

In recent years immunotherapy such as cytokine, CAR-T cell or immune checkpoint blockade has been effective in some cancer patients, and has met with many obstacles such as inefficient T cell access to tumor sites, low numbers of existing immune cells at tumor sites, side effects from systemic administration, and the like. The mesenchymal stem cells can be obtained from various tissues in vivo, are easy to culture and amplify in vitro and are transformed by utilizing a genetic engineering method, and have lower immunogenicity. The research of the inventor in a mouse model proves that the adipose-derived mesenchymal stem cells can specifically migrate to a tumor part and are not enriched in other organs, so that the adipose-derived mesenchymal stem cells can be used as an ideal drug carrier. The invention uses adipose-derived mesenchymal stem cells as a carrier to over-express chemotactic factor CXCL9 and cytokine OX40L with immunoregulation effect, and specifically attracts, activates and kills immune cells of tumor tissues at tumor parts through the active migration of the mesenchymal stem cells to the tumor, thereby finally achieving the immunotherapy efficacy with higher efficiency and low side effect.

Description of the terms

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.

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

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

As used herein, the term "administering" refers to physically introducing a product of the 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, e.g., by injection or infusion.

Mesenchymal Stem Cell (MSC)

Mesenchymal Stem Cells (MSCs) have emerged in recent years as potential cell carriers in the field of view with active migration capability against many different tumor types when administered systemically. It can be extracted from many different adult tissues, is easy to expand and culture, and can avoid immunological rejection. Meanwhile, the characteristic of tropism migration of the mesenchymal stem cell tumor and the capability of long-term survival at a target site make the mesenchymal stem cell tumor an important resource for cell therapy. The 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 process of separating BM-MSC and UCB-MSC is very complicated and inefficient. Adipose tissue or umbilical cord tissue MSCs may therefore be a more desirable alternative because they contain more MSCs than bone marrow and cord blood, and the tissue is easier to obtain and collect. Furthermore, with respect to autologous stem cell sources for personalized cell therapy, AT-MSC has minimal risk to the donor and no ethical issues.

Through the tumor homing capability of MSCs, the present invention utilizes MSC-targeted delivery of chemokines CCL3, CCL21, XCL1, CXCL9, and cytokines OX40L, 4-1BBL, GITRL, 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 mesenchymal stem cells are used as vectors to overexpress chemokines and cytokines in a mouse model to treat colorectal cancer.

Expression cassette

As used herein, "expression cassette" or "expression cassette of the invention" includes a first expression cassette and/or a second expression cassette. The first expression cassette comprises a nucleic acid sequence of a chemokine; 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, the cytokine comprising: OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof.

In one embodiment, the exogenous nucleic acid molecule comprises a first expression cassette comprising a nucleic acid sequence encoding CXCL9 and a second expression cassette comprising a nucleic acid sequence encoding OX 40L.

In another preferred embodiment, the first expression cassette and the second expression cassette are each independent, or are combined into one. 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, said first expression cassette and said second expression cassette are located on a vector or integrated in the chromosome of said mesenchymal stem cell. In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same or different vectors. In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same vector. In another preferred embodiment, the carrier is selected from the group consisting of: DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, transposons, other gene transfer systems, or combinations thereof.

Carrier

The invention also provides a vector containing the expression cassette. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, since they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity.

Briefly summarized, the expression cassettes or nucleic acid sequences of the invention are typically incorporated into expression vectors by operably linking them to a promoter. The vector is suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that may be used to regulate the expression of the desired nucleic acid sequence.

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

The expression cassette or nucleic acid sequence can be cloned into many types of vectors. For example, the expression cassette or nucleic acid sequence can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to the cell in the form of a viral vector. Viral vector 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 manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors comprise an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject cells in vivo or ex vivo. Many retroviral systems are known in the art.

Additional promoter elements, such as enhancers, may regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50bp apart, and activity begins to decline. Depending on the promoter, it appears that the individual elements may function cooperatively or independently to initiate transcription.

An example of a suitable promoter is the early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1 α (EF-1 α). However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40(SV40) early promoter, the mouse mammary cancer virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr (Epstein-Barr) virus immediate early promoter, the 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. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch that is capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

The expression vector introduced into the cells may also contain either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in a host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Typically, the reporter gene is the following: which is not present in or expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzymatic activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is assayed at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al, 2000FEBS Letters479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, the construct with the minimum of 5 flanking regions that showed the highest level of reporter gene expression was identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate the ability of an agent to modulate promoter-driven transcription.

Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vector can be readily introduced into a host cell, e.g., a mammalian (e.g., human T cell), bacterial, yeast, or insect cell, by any method known in the art. For example, the expression vector may be transferred into a host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or 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).

Biological methods for introducing polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. patent nos. 5,350,674 and 5,585,362.

Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).

In the case of non-viral delivery systems, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for use to introduce nucleic acids into host cells (ex vivo or in vivo). In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with the lipid may be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution comprising the lipid, mixed with the lipid, associated with the lipid, contained as a suspension in the lipid, contained in or complexed with a micelle, or otherwise associated with the lipid. The lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution. For example, they may be present in bilayer structures, either as micelles or with a "collapsed" structure. They may also simply be dispersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include fatty droplets that occur naturally in the cytoplasm as well as such compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Preparation method

The invention provides a method for preparing a mesenchymal stem cell, which comprises introducing a first expression cassette and/or a second expression cassette into the mesenchymal stem cell to be modified, wherein the first expression cassette is used for expressing chemotactic factors, and the second expression cassette is used for expressing cytokines, thereby obtaining the mesenchymal stem cell.

Generally comprising the steps of: (1) transforming or transducing a suitable host cell with a polynucleotide encoding an immunostimulatory factor of the invention, or with a recombinant expression vector comprising the polynucleotide; (2) host cells cultured in a suitable medium.

Preparation

The invention provides a pharmaceutical composition comprising the mesenchymal stem cell of the first aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the concentration of the mesenchymal stem cells in the preparation is 1 x 10³-1×10⁸Individual cells/ml, more preferably 1X 10⁴-1×10⁷Individual cells/ml.

In one embodiment, the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The formulations of the present invention are preferably formulated for intravenous administration.

Therapeutic applications

The invention includes therapeutic applications of mesenchymal stem cells transduced with a vector comprising an expression cassette of the invention. The mesenchymal stem cells can actively migrate to the tumor part, are not enriched in liver, spleen, kidney and other organs, have specificity and safety as tumor treatment drug carriers, and provide an effective means for locally activating immune reaction in the tumor to avoid systemic side effects. The adipose-derived mesenchymal stem cell therapy system over-expressing the chemotactic factor CXCL9 and the cytokine OX40L has the characteristic of specifically targeting a tumor part, and can attract and activate T cells and NK cells so as to achieve ideal anti-tumor efficacy.

In one embodiment, the invention provides a class of cell therapy comprising administering to a mammal the mesenchymal stem cells of the invention. Unlike antibody therapy, mesenchymal stem cells of the invention are capable of replication in vivo, resulting in long-term persistence that can lead to sustained tumor control.

Treatable cancers include tumors that are not vascularized or have not substantially vascularized, as well as vascularized tumors. The cancer may comprise a non-solid tumor (such as a hematological tumor, e.g., leukemia and lymphoma) or may comprise a solid tumor. The types of cancer treated with the mesenchymal stem cells of the invention include, but are not limited to, carcinomas, blastomas and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, such as sarcomas, carcinomas and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or hematological) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, granulo-monocytic, monocytic and erythrocytic leukemias), chronic leukemias (such as chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, hodgkin's disease, non-hodgkin's lymphoma (indolent and higher forms), multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

A solid tumor is an abnormal mass of tissue that generally does not contain cysts or fluid regions. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer.

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

For ex vivo immunization, at least one of the following occurs in vitro prior to administration of the cells into a mammal: i) expanding the cells, ii) introducing the expression cassette of the invention into the cells, and/or iii) cryopreserving the cells.

Ex vivo procedures are well 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 vector comprising an expression cassette of the invention. The mesenchymal stem cells of the invention may be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human, and the mesenchymal stem cells of the invention may be autologous with respect to the recipient. Alternatively, the cells may be allogeneic, syngeneic (syngeneic), or xenogeneic with respect to the recipient.

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

Generally, cells activated and expanded as described herein are useful for the treatment and prevention of diseases arising in immunocompromised individuals. Accordingly, the present invention provides a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the mesenchymal stem cells of the present invention.

The mesenchymal stem cells of the invention may be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as some cytokines or cell populations. Briefly, a pharmaceutical composition or formulation of the invention may comprise mesenchymal stem cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

The pharmaceutical compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease-although the appropriate dosage may be determined by clinical trials.

When referring to an "immunologically effective amount", "an anti-tumor effective amount", "a tumor-inhibiting effective amount", or a "therapeutic amount", the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, extent of infection or metastasis, and individual differences in the condition of the patient (subject). It can be generally pointed out that: a pharmaceutical composition comprising mesenchymal stem cells described herein may be in the range of 10⁴To 10⁹Dosage of individual cells/kg body weight, preferably 10⁵To 10 ⁶Doses of individual cells per kg body weight (including all integer values within those ranges) are administered. The mesenchymal stem cell composition may also be administered multiple times at these doses. Cells can be administered by using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al, New Eng.J.of Med.319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the subject composition may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by i.v. injection. The composition of mesenchymal stem cells may be injected directly into the tumor, lymph node or infection site.

In certain embodiments of the 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., prior to, concurrently with, or subsequent to) any number of relevant treatment modalities, including but not limited to treatment with: such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or efavirenz therapy for psoriasis patients or other therapy for PML patients. In further embodiments, the mesenchymal stem cells of the present invention may be used in combination with: chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In a further embodiment, the cell composition of the invention is administered to the patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) bone marrow transplantation with a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, following transplantation, the subject receives an infusion of the expanded mesenchymal stem cells of the invention. In an additional embodiment, the expanded cells are administered pre-or post-surgery.

The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The proportion of doses administered to a human can be effected in accordance with accepted practice in the art. Typically, 1X 10 may be administered per treatment or per course of treatment ³1 to 10¹⁰Mesenchymal stem cells of the invention, e.g. by quiescenceThe pulse back transfusion mode is applied to the patient.

The technical scheme of the invention has the following beneficial effects:

1. the mesenchymal stem cells can be obtained from various tissues in vivo, are easy to culture and amplify in vitro and are transformed by utilizing a genetic engineering method, and have lower immunogenicity.

2. In contrast to most immunotherapy approaches, the method of the invention is independent of the presence of tumor-infiltrating lymphocytes, and is also clinically useful for the treatment of tumors that have very low or are resistant to lymphocyte infiltration.

3. When the mesenchymal stem cells of the invention over-express one or more of CCL3, CCL19, CCL21, XCL1, CXCL9, OX40L, 4-1BBL, GITRL and CD40L, the mesenchymal stem cells can specifically attract and activate immune cells killing tumor tissues at tumor sites, and have more efficient and low-side-effect immune curative effects.

4. When the mesenchymal stem cells overexpress CXCL9 and OX40L, the mesenchymal stem cells have mutual synergistic effect, and obviously enhance the killing capacity to tumor tissues, especially colorectal cancer cells. This method also has a killing effect on MHC-I negative tumor cells resistant to traditional immunotherapy (such as CAR-T or PD-1/PD-L1 antibodies).

5. The present invention may also enhance the efficacy of other clinically used immunotherapies, such as CAR-T or PD-1/PD-L1 antibodies, when used in combination with these immunotherapies.

The invention is further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring Harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Materials and methods

Cell lines

CT26 cells were colon adenocarcinoma cells derived from BALB/c mice, purchased from Shanghai Life sciences institute. CT26 cells were cultured in RPMI 1640 containing 10% fetal bovine serum and 1% penicillin/streptomycin.

Antibodies

The antibodies used for flow cytometry were from the company BD Biosciences, BioLegent or eBioscience. Antibodies for western blot analysis include anti-CCL3(R & D Systems), anti-CXCL9(Abcam), anti-Myc-tag (cell Signaling technology), anti-OX40L (Abcam) and anti-GAPDH (Abcam). Immunofluorescent-stained antibodies include anti-GFP (Abcam), anti-CD8a (BioLegend) and anti-NKp46(CD335) (BioLegend).

Immune checkpoint blockade antibodies anti-PD-1(clone RMP1-4) and anti-CTLA-4(clone9D9) were purchased from Bio X Cell Co. These two antibodies (anti-PD-1: 200. mu.g/mouse; anti-CTLA-4: 100. mu.g/mouse) were injected intraperitoneally into mice.

Isolation, culture and identification of mesenchymal stem cells from mouse adipose tissue

AT-MSC was isolated by digesting subcutaneous adipose tissue of mice with collagenase type I. Cells were cultured in α -MEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. After the cells are cultured for three generations in an adherent way, the expression of the cell surface marker protein is identified by using a flow cytometry method.

Lentiviral production and transduction

The cDNA was cloned into a lentiviral vector. Lentivirus packaging and titer determination was performed by Heyuan Biotechnology (Shanghai) Inc. Mesenchymal stem cells were infected with lentivirus with a multiplicity of infection (MOI) of 60 in the presence of 6. mu.g/ml polybrene (Sigma-Aldrich).

Tumor cell proliferation assay

Proliferation of tumor cells was determined using the CCK-8 kit (Dojindo Molecular Technologies) according to the procedures of the instructions. The absorbance was measured using a microplate reader (Tecan).

Western Blotting (Western Blotting)

Cells were harvested and treated with cell lysis buffer (RIPA buffer + 1% protease inhibitor) (ThermoFisherScientific) to prepare cell lysates. Protein concentration in cell lysates was determined using BCA kit (ThermoFisher Scientific). 15 to 30. mu.g of protein were loaded onto a 5% to 15% SDS-PAGE protein gel (ThermoFisher Scientific) and then transferred onto a PVDF membrane (Millipore). Membranes were blocked with 5% skim milk in TBST buffer and incubated overnight at 4 ℃ with antibodies to Myc tag, OX40L, CXCL9 and GAPDH. The membranes were washed with TBS-T buffer and incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. The membranes were developed with an enhanced chemiluminescence kit (Millipore) and exposed to the membranes.

Enzyme-linked immunosorbent assay (ELISA)

Supernatants of lentivirus transduced AT-MSCs were collected and stored in a freezer AT-80 ℃ until measured. Secretion of CXCL9 was tested using an ELISA kit from Abcam according to the instructions.

T cell proliferation assay

T lymphocytes were isolated from mouse spleens using the Pan T cell isolation kit II (Miltenyi Biotec). Immediately after isolation, T cells were activated and expanded using a T cell activation/expansion kit (Miltenyi Biotec) containing CD3 and CD28 antibodies and an additional 20ng/ml of recombinant mouse IL-2(R & D Systems). After 48 hours, the cells were harvested and washed thoroughly.

To examine the effect of cytokine-overexpressing mAT-MSCs on T cell proliferation, lentivirus-transduced mAT-MSCs (2.5X 10)⁴) Seeded on 24-well plates and incubated at 37 ℃ for 4 hours and washed 5 times with medium. Then 1X 10 labeled with CFSE (ThermoFisher Scientific)⁵Activated T cells and mAT-MSC (2.5X 10)⁴) Co-cultured in complete medium of RPMI 1640. After 96 hours, proliferation of T cells was detected by flow cytometry analysis of CFSE fluorescence intensity.

Mouse subcutaneous transplantation tumor model

Separately, CT26(0.5 or 1X 10) was injected subcutaneously in the right lower back of BALB/c mice aged 8 to 10 weeks ⁶Mice) cells. When the maximum tumor diameter reached 0.5 to 0.7cm, mice were randomly assigned to the experimental groups. The tail of each animal was injected with 250. mu.l PBS or 5X 10 PBS⁵AT-MSC 250 u l PBS suspension for systemic administration. Tumor size was measured every three days with a vernier caliper and using the formulaTumor volume was calculated: v ═ lxw²And/2, wherein L and W are the major and minor diameters of the tumor, respectively. Mice were monitored for tumor size and survival. When the tumor volume reaches 2cm³Or the tumor becomes ulcerated or the mouse is dying.

Flow analysis

To identify AT-MSCs, adherent cells from passage 3 to 5 were isolated using 20 μ M EDTA digestion, then washed twice with PBS and stained with antibody.

To analyze tumor-infiltrating immune cells, subcutaneously implanted tumors were dissected and transferred to RPMI medium, minced with scissors, placed in serum-free RPMI medium containing 0.25mg/ml Liberase TL (Roche) and 50 μ g/ml DNase I (Sigma-Aldrich), digested at 37 ℃ using gentleMAC OctoDissociator (Milteniy biotech), and dispersed through a 40 μm cell filter (BD Biosciences). The single cells were further washed and stained with antibody. Dead cells were excluded by staining with the Zombie Fixable visualization Kit (BioLegend). For intracellular staining of cytokines, each mouse was injected intraperitoneally with 0.25mg of brefeldin a (bfa) (selleck) 4-6 hours prior to harvesting the samples. After surface staining in the presence of 5. mu.g/ml BFA, intracellular staining was performed with an intracellular fixation and permeabilization buffer set (eBioscience). Surface staining was followed by nuclear staining with Foxp3 transcription factor staining buffer (eBioscience).

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

Model for inducing colorectal cancer of mice by AOM/DSS

BALB/c mice were injected intraperitoneally with AOM (12.5mg/kg body weight; Sigma-Aldrich) (14). After 1 week, the mice were given drinking water containing 3% dss (mp biomedicals) for 7 days, followed by 2 weeks of normal water. DSS induction was continued for two cycles, and mice were sacrificed after five MSC injections via tail vein starting from the last week of the DSS induction cycle. Body weights were recorded during DSS treatment. The colon was removed from the mice, rinsed with ice PBS, opened longitudinally, fixed overnight in 4% paraformaldehyde solution (Sigma-Aldrich) at room temperature, and paraffin embedded. Before fixation, dimensional measurements were made using digital calipers.

Immunofluorescence

Tissue sections were blocked with 10% normal sheep serum and then incubated with primary antibody overnight at 4 ℃ and secondary antibody for 1 hour at room temperature. Slides were mounted in a fade resistant mounting medium with dapi (thermofisher scientific) and viewed under a Nikon Eclipse Ti fluorescence microscope. Antibodies used for immunofluorescence were GFP antibody (Abcam), CD8a antibody (BioLegend) and NKp46 antibody (CD335) (BioLegend).

Statistics of

All results are expressed as mean ± SEM. Differences were assessed by Student's t-test or, when comparing two or more sets of averages, by two-way ANOVA followed by Bonferroni multiple comparison test. Data analysis was performed using Prism software (GraphPad). Statistical significance was set at a level of P < 0.05.

Study approval

All animal procedures were approved by the Shanghai university of transportation animal Care and use Committee.

Example 1 characteristics of adipose-derived mesenchymal stem cells migrating to tumor

Mesenchymal stem cells are extracted from subcutaneous fat of a mouse, and the cells express specific mesenchymal stem cell marker molecules and do not express marker molecules of other cell types through flow cytometry detection (figure 1A), so that the purity of the adipose mesenchymal stem cells for experiments is verified. Adipose-derived mesenchymal stem cells were transfected with lentivirus to express GFP, and 5X 10 cells were transfected⁵After 7 days, immunofluorescence staining of tissue sections of the tumor and other organs was performed on the cells injected into tumor-bearing mice (CT26 intestinal cancer subcutaneous tumor) through tail vein, and the results showed that GFP positive mesenchymal stem cells only reside in the tumor and are not found in other organs such as liver, spleen and kidney (FIG. 1B). The flow cytometry method is used for detecting GFP positive mesenchymal stem cells in the tumor, and a certain number of cells can still be detected 14 days after cell injection (figure 1C). These results all demonstrate that adipose-derived mesenchymal stem cells can specifically migrate to the tumor site And the longer-lasting nature of the molecule, supporting its potential as a carrier for therapeutic molecules.

Example 2 anti-tumor Properties of CXCL9 and OX40L

In order to search for immune activation type therapeutic molecules with higher efficacy, chemotactic factors and cytokines with potential anti-tumor functions are selected, genes are cloned into a lentiviral vector to package lentiviruses carrying the genes. These gene-loaded or placebo lentivirus-transduced CT26 intestinal cancer cell lines were tested for their anti-tumor efficacy in a subcutaneous tumor transplantation experiment. After the chemokines CCL3 and CXCL9 with potential anti-tumor efficacy were overexpressed in CT26 (fig. 2A), the in vitro proliferation of cells was not affected (fig. 2B), while the growth of subcutaneous transplanted tumors in vivo was significantly inhibited (fig. 2C), suggesting that these chemokines may have the effect of the immune system in vivo to inhibit tumor growth. Among them, CXCL9 has the most significant antitumor effect. Detection of immune cell composition in tumors by flow cytometry revealed that CXCL9 indeed increased infiltration of anti-tumor immune cells such as CD8, CD4, and NK (fig. 2D).

In CT26 cells overexpressing two immune-activating cytokines IL36 β and OX40L (fig. 3A & B), it was also found that overexpression of these two cytokines had no effect on the proliferation of tumor cells in vitro (fig. 3C) and that the growth of subcutaneous transplantable tumors was significantly inhibited (fig. 3D), indicating that it may be an antitumor effect via the immune system in vivo. Of these, OX40L showed strong antitumor efficacy.

Example 3 anti-tumor efficacy of mesenchymal Stem cells overexpressing the chemokine CXCL9 and the cytokine OX40L

Adipose-derived mesenchymal stem cell systems overexpressing CXCL9 and OX40L were established using lentiviral infection. Successful expression and secretion of CXCL9 was identified by western blotting and ELISA techniques (fig. 4A)&B) Successful expression of OX40L on cell membranes was confirmed by Western blotting and flow cytometry (FIG. 4C)&D) In that respect Mesenchymal stem cells overexpressing OX40L were also able to stimulate T cell proliferation after co-culture with T cells (fig. 4E), demonstrating their biological activity. After thatMSCs were established that overexpress both CXCL9 and OX40L (FIG. 5A)&B) In the model of subcutaneous transplantation tumor of CT26, mice were injected 5X 10 times each time through the tail vein⁵Mesenchymal stem cells, or PBS, were treated three times at four day intervals, while the mesenchymal stem cells carrying CXCL9 and OX40L showed the strongest antitumor efficacy (fig. 5C). Flow cytometry analysis found a significant increase in the proportion of lymphocytes in the tumor, particularly anti-tumor CD8T cells and NK cells (fig. 5D), indicating that the therapy effectively activated an anti-tumor immune response.

To further explore the efficacy of the mesenchymal stem cell immunotherapy system, AOM/DSS was used to induce an in situ intestinal cancer model caused by inflammation, and mice were treated for 5 total 4 weeks starting at the last week of the third DSS treatment cycle (fig. 6A), each time by tail vein injection of PBS or 5 × 10 ⁵The mesenchymal stem cells, which were loaded with CXCL9 and OX40L, provided a very significant reduction in intestinal tumors in mice (fig. 6B)&C) In that respect Immunofluorescent staining demonstrated a significant increase in infiltration of anti-tumor CD 8T cells and NK cells (fig. 6D), consistent with the results observed in the transplanted tumor model.

The results all show that the adipose-derived mesenchymal stem cells over-expressing CXCL9 and OX40L show extremely remarkable treatment effects in mouse subcutaneous transplanted tumor and inflammation-induced intestinal cancer models in situ. Meanwhile, the mesenchymal stem cells carrying CXCL9 and OX40L express CXCL9 and OX40L, the two have synergistic effect, and the anti-tumor effect is obviously better than that of the mesenchymal stem cells independently expressing CXCL9 or OX 40L.

Discussion of the related Art

Immunotherapy revolutionized the treatment of cancer. Although some cytokines and immune checkpoint blockers, etc., show significant efficacy in clinical treatment of tumors, systemic use of these drugs can non-specifically activate the immune system and affect most organs. In order to solve the side effect caused by systemic administration, mesenchymal stem cells are selected as a drug carrier. Research results show that the adipose-derived mesenchymal stem cells can actively migrate to tumor parts, are not enriched in liver, spleen, kidney and other organs, fully support the specificity and safety of the mesenchymal stem cells as tumor treatment drug carriers, and provide an effective means for locally activating immune reaction in tumors and avoiding systemic side effects.

Infiltration of T cells and NK cells in tumors is a key determinant of the efficacy of immunotherapy for solid tumors. Tumors can limit lymphocyte invasion by different mechanisms. Tumors with high T cell infiltration often express high levels of chemokines capable of attracting T cells, including CCL3, CCL4, CXCL10, and the like. The invention unexpectedly discovers that the mesenchymal stem cells are used as a vector to express CXCL9 and are transported to a tumor part to attract anti-tumor lymphocytes, so that stronger attraction capacity of T cells and NK cells is shown, and the problem that the lymphocytes are difficult to enter solid tumors is solved. In addition, the loaded OX40L in the mesenchymal stem cell system can also activate T cells and NK cells more efficiently. Activating antibodies to the OX40 receptor have entered clinical stage (clinical trials. gov), but potential problems are also side effects from systemic administration. The ligand OX40L of OX40 is a membrane protein, and the expression in mesenchymal stem cells can be directionally transported to a tumor site, and can not be secreted like secretory cytokines to transfer to other sites, so that the diffusion caused by secretion is reduced, and existing or newly migrated lymphocytes can be activated at the tumor site. The invention also unexpectedly discovers that adipose-derived mesenchymal stem cells over-expressing CXCL9 and OX40L show extremely remarkable treatment effects in mouse subcutaneous transplanted tumors and inflammation-induced intestinal cancer models in situ.

In summary, the adipose-derived mesenchymal stem cell therapy system over-expressing the chemokines CXCL9 and the cytokine OX40L established by the invention has the characteristic of specifically targeting a tumor part, and can attract and activate T cells and NK cells so as to achieve ideal anti-tumor efficacy. In contrast to most immunotherapy, this therapy is independent of the presence of tumor-infiltrating lymphocytes, and is also clinically applicable for the treatment of tumors with very low or resistant lymphocyte infiltration. The adipose or umbilical cord mesenchymal stem cells are easy to extract and culture and are easy to apply to individual treatment. Its low immunogenicity also makes the use of xenobiotics feasible. Therefore, the established mesenchymal stem cell-based immunotherapy has extremely high clinical transformation value.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A mesenchymal stem cell expressing an immunostimulatory factor selected from the group consisting of: CCL3, CCL19, CCL21, XCL1, CXCL9, OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof.

2. The mesenchymal stem cell of claim 1, wherein the immunostimulatory factor comprises at least one chemokine, wherein the chemokine comprises: CCL3, CCL19, CCL21, XCL1, CXCL9, or a combination thereof; and/or

The immunostimulatory factor includes at least one cytokine, the cytokine including: OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof.

3. The mesenchymal stem cell of claim 1, wherein the immunostimulatory factor is a combination of one or both of CCL3, CCL19, CCL21, XCL1 and CD 40L; or

The immunostimulant is one or two of OX40L, 4-1BBL and GITRL combined with CXCL 9.

4. The mesenchymal stem cell of claim 1, wherein the mesenchymal stem cell comprises: adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, or a combination thereof.

5. A method of preparing the mesenchymal stem cell of claim 1, comprising the steps of:

(1) providing a mesenchymal stem cell to be modified; and

(2) introducing an exogenous nucleic acid comprising a nucleic acid sequence encoding an immunostimulatory factor into the mesenchymal stem cell to be engineered, thereby obtaining the mesenchymal stem cell of claim 1;

6. A formulation comprising the mesenchymal stem cell of claim 1, and a pharmaceutically acceptable carrier, diluent or excipient.

7. Use of mesenchymal stem cells according to claim 1, for the preparation of a medicament or formulation for the prevention and/or treatment of cancer or tumour.

8. Use according to claim 7, wherein in another preferred embodiment the tumour is a solid tumour, preferably colorectal cancer.

9. A pharmaceutical composition comprising

(1) The mesenchymal stem cell of claim 1; and

(2) an anti-tumor immunotherapeutic agent.

10. The pharmaceutical combination of claim 9, wherein the anti-tumor immunotherapeutic agent is an immune checkpoint antibody.