WO2022011651A1 - 间充质干细胞靶向运输趋化因子和细胞因子的免疫疗法 - Google Patents

间充质干细胞靶向运输趋化因子和细胞因子的免疫疗法 Download PDF

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WO2022011651A1
WO2022011651A1 PCT/CN2020/102473 CN2020102473W WO2022011651A1 WO 2022011651 A1 WO2022011651 A1 WO 2022011651A1 CN 2020102473 W CN2020102473 W CN 2020102473W WO 2022011651 A1 WO2022011651 A1 WO 2022011651A1
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mesenchymal stem
tumor
stem cells
cells
stem cell
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PCT/CN2020/102473
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French (fr)
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高维强
马斌
印盼
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上海交通大学
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Priority to PCT/CN2020/102473 priority Critical patent/WO2022011651A1/zh
Priority to US18/005,166 priority patent/US20230257713A1/en
Priority to KR1020237005492A priority patent/KR20230038776A/ko
Priority to EP20945214.3A priority patent/EP4183872A4/en
Priority to JP2023503083A priority patent/JP2023534055A/ja
Publication of WO2022011651A1 publication Critical patent/WO2022011651A1/zh

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    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
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    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention belongs to the field of biotechnology, in particular, the present invention relates to the immunotherapy of mesenchymal stem cells targeting chemokines and cytokines.
  • CAR-T chimeric antigen receptor T-cell immunotherapy
  • immune regulatory checkpoint blockade immune regulatory checkpoint blockade
  • CAR-T cell therapy has shown certain efficacy in some types of cancer, but it has also encountered inefficiency of T cells reaching the solid tumor site and short duration of action, the number of existing immune cells in the tumor site is small, and systemic medication brings side effects etc. bottlenecks.
  • the use of antibodies targeting the T cell inhibitory receptors PD-1 or CTLA-4 can produce very significant anti-tumor effects.
  • the therapeutic efficacy of antibodies is often limited by a number of factors, such as low T cell infiltration and loss of activity in solid tumors.
  • systemic use of immunotherapy drugs such as interferon alpha, interleukin 2 or PD-1 antibodies may cause serious side effects.
  • the purpose of the present invention is to provide an immunotherapy that can specifically target tumor cells.
  • Another object of the present invention is to provide an immunotherapy for mesenchymal stem cells to target and transport chemokines and cytokines.
  • a first aspect of the present invention provides a mesenchymal stem cell, the mesenchymal stem cell expresses 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 contain an exogenous nucleic acid molecule, and the exogenous nucleic acid molecule includes 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.
  • 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.
  • the exogenous nucleic acid molecule further comprises a promoter or a promoter/enhancer combination, and the nucleic acid sequence encoding an immunostimulatory factor is operably linked to the promoter or the promoter/enhancer combination.
  • the promoter is a constitutive promoter or an inducible promoter, preferably a constitutive promoter.
  • the immunostimulatory factor includes at least one chemokine, and the chemokine includes: CCL3, CCL19, CCL21, XCL1, CXCL9, or a combination thereof.
  • the immunostimulatory factor includes at least one cytokine, and the cytokine includes: OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof.
  • the immunostimulatory factor includes at least one chemokine and at least one cytokine, wherein the chemokine includes: CCL3, CCL19, CCL21, XCL1, CXCL9, or a combination thereof, wherein The cytokines include: OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof.
  • the immunostimulatory factor is one or both of CCL3, CCL19, CCL21, and XCL1 in combination with CD40L.
  • the immunostimulatory factor is one or both of OX40L, 4-1BBL, GITRL and CXCL9 in combination.
  • the immunostimulatory factor is CXCL9 and/or OX40L.
  • the exogenous nucleic acid molecule includes a first expression cassette and/or a second expression cassette, the first expression cassette contains a nucleic acid sequence encoding a chemokine, and the second expression cassette contains a cell encoding The nucleic acid sequence of a factor, 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.
  • the exogenous nucleic acid molecule includes a first expression cassette and a second expression cassette, the first expression cassette contains a nucleic acid sequence encoding CXCL9, and the second expression cassette contains a nucleic acid sequence encoding OX40L.
  • first expression cassette and the second expression cassette are independent of each other, or combined into one.
  • first expression cassette and the second expression cassette further comprise a promoter and/or a terminator, respectively.
  • 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.
  • the first expression cassette and the second expression cassette are located on a vector or integrated into the chromosome of the mesenchymal stem cell.
  • first expression cassette and the second expression cassette are independent or linked.
  • first expression cassette and the second expression cassette are located on the same or different vectors.
  • first expression cassette and the second expression cassette are located in the same vector.
  • the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, other gene transfer systems, or a combination thereof.
  • the mesenchymal stem cells include: adipose-derived mesenchymal stem cells, umbilical cord mesenchymal stem cells, or a combination thereof.
  • the mesenchymal stem cells are ex vivo.
  • the mesenchymal stem cells are autologous or allogeneic.
  • the second aspect of the present invention provides a method for preparing the mesenchymal stem cells described in the first aspect of the present invention, comprising the following steps:
  • the immunostimulatory factor is selected from the group consisting of CCL3, CCL19, CCL21, XCL1, CXCL9, OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof.
  • the third aspect of the present invention provides a preparation comprising the mesenchymal stem cells according to the first aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the preparation is a liquid preparation.
  • the dosage form of the preparation includes injection.
  • the concentration of mesenchymal stem cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml.
  • the fourth aspect of the present invention provides the use of the mesenchymal stem cells according to the first aspect of the present invention for preparing a medicament or preparation for preventing and/or treating cancer or tumor.
  • the tumor is selected from the group consisting of hematological tumors, solid tumors, or a combination thereof.
  • the tumor is a solid tumor.
  • 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 B-cell lymphoma (DLBCL), or a combination thereof.
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • DLBCL diffuse B-cell lymphoma
  • 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, colorectal 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 its combination.
  • gastric cancer gastric cancer peritoneal metastasis
  • liver cancer leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, Cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endo
  • the solid tumor is colorectal cancer.
  • the tumor includes recurrent and metastatic tumors.
  • the tumor includes an MHC-I-deficient tumor.
  • the fifth aspect of the present invention provides a medicine box, the medicine box contains
  • the anti-tumor immunotherapy agent is selected from the group consisting of antibodies, immune cells, or a combination thereof.
  • the immune cells are T cells or NK cells.
  • the anti-tumor immunotherapy agent is an immune checkpoint antibody.
  • the immune checkpoint antibody includes PD-1 antibody and/or CTLA-4 antibody.
  • the present invention also provides a method for treating a disease, comprising administering an appropriate amount of the cells of the first aspect of the present invention, or the preparation of the third aspect of the present invention, or the fifth aspect of the present invention to a subject in need of treatment. drug combination.
  • the disease is cancer or tumor, preferably solid tumor, more preferably colorectal cancer.
  • Figure 1 shows the identification of mouse adipose-derived mesenchymal stem cell phenotype and tumor-migrating properties.
  • A The expression of surface molecules of adipose-derived mesenchymal stem cells was detected by flow cytometry.
  • B Migration of MSC-GFP in vivo in a CT26 subcutaneous xenograft mouse model. The infiltration of MSC-GFP in tumor tissue was detected by immunofluorescence, green indicates MSC-GFP, blue (DAPI staining) indicates nuclei, scale bar: 20 ⁇ M.
  • FIG. 2 shows that overexpression of CXCL9 in tumor cells inhibits tumor growth in vivo.
  • FIG. 3 shows that overexpression of OX40L in tumor cells inhibits tumor growth in vivo.
  • A WB detected the overexpression of IL36 ⁇ and OX40L in CT26.
  • B Flow cytometry detection of OX40L overexpression in CT26.
  • C Cell proliferation assay of CT26-Vector, CT26-IL36 ⁇ and CT26-OX40L.
  • FIG. 4 shows the identification of CXCL9 and OX40L overexpression in mesenchymal stem cells.
  • A WB detection of CXCL9 overexpression in MSCs.
  • C WB detected the overexpression of OX40L in MSCs.
  • D The overexpression of OX40L in MSCs was detected by flow cytometry.
  • Figure 5 shows that mesenchymal stem cells overexpressing CXCL9 and OX40L inhibit the growth of subcutaneous xenografts.
  • A ELISA to detect the secretion of CXCL9.
  • B The expression of OX40L was detected by flow cytometry.
  • Figure 6 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.
  • B Representative images of colorectal tumors. Scale bar: 5mm.
  • D Immunofluorescence staining of CD8 and NK cells. Scale bar: 50 ⁇ m.
  • Figure 7 shows that PD-1 and CTLA-4 antibody combination therapy has no significant therapeutic efficacy on AOM/DSS-induced colorectal cancer.
  • A Schematic diagram of AOM/DSS treatment and antibody treatment protocol.
  • Figure 8 shows the efficacy of mesenchymal stem cells overexpressing CXCL9 and OX40L to promote PD-1 blockade therapy.
  • A Schematic diagram of mesenchymal stem cells (MSC-CXCL9+OX40L) (5X10 5 cells per injection) and PD-1 antibody blocking ( ⁇ PD-1) treatment;
  • B mesenchymal stem cells overexpressing CXCL9 and OX40L and PD
  • Figure 9 shows that mesenchymal stem cells overexpressing CXCL9 and OX40L inhibit the growth of MHC-I-deficient tumors with immune escape properties.
  • sgB2m B2m gene
  • H2Kb/H2Db MHC-I molecules
  • n 4 animals/group
  • B the transplanted tumor model of MHC-I-deficient MC38-sgB2m or control MC38-sgLacZ tumor cells overexpressing OVA was established in vivo.
  • the cells grew faster and were treated with a tumor vaccine (OVA257-264 polypeptide plus poly:IC adjuvant), and found that MC38-sgLacZ cells were significantly inhibited by the tumor vaccine, while the MHC-I-deficient MC38-sgB2m tumors were not resistant to vaccine treatment.
  • FIG 10 shows the therapeutic efficacy of mesenchymal stem cells (MSCs) overexpressing immune activators/cytokines or chemokines.
  • MSCs mesenchymal stem cells
  • Each mouse received 5 ⁇ 10 5 mesenchymal stem cells overexpressing one of the factors at a time, once every three days, for a total of six treatments.
  • mesenchymal stem cells overexpress chemokines CCL3, CCL19, CCL21, XCL1, CXCL9 and/or cytokines OX40L, 4-1BBL, GITRL, CD40L can specifically attract and activate immune cells that kill tumor tissue at the tumor site, and mesenchymal stem cells have a synergistic effect with chemokines and/or cytokines, resulting in more efficient immune efficacy with less side effects.
  • mesenchymal stem cells overexpress CXCL9 and OX40L, they have a synergistic effect and significantly enhance the killing ability of tumor tissues, especially colorectal cancer cells.
  • the inventors have completed the present invention.
  • immunotherapies such as cytokines, CAR-T cells or immune checkpoint blockade have produced good effects in some cancer patients, but they have also encountered many obstacles, such as the low efficiency of T cells reaching the tumor site, and the existing tumor sites.
  • the number of immune cells is low, and the side effects caused by systemic medication.
  • Mesenchymal stem cells can be obtained from various tissues in vivo. The inventors' studies in mouse models confirmed that adipose-derived mesenchymal stem cells can specifically migrate to tumor sites and are not enriched in other organs, so they can be used as ideal drug carriers.
  • the present invention utilizes adipose mesenchymal stem cells as carriers to overexpress chemokine CXCL9 and cytokine OX40L with immunoregulatory effects, and through active migration of mesenchymal stem cells to tumors, specifically attracts and activates the immune system that kills tumor tissues at tumor sites cells, and ultimately achieve more efficient immunotherapy efficacy with less side effects.
  • the term “about” means that the value may vary by no more than 1% from the recited value.
  • the expression “about 100” includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the terms "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “consisting essentially of,” or “consisting of.”
  • the term "administration" refers to the physical introduction of a product of the present invention into a subject using any of a variety of methods and delivery systems known to those of skill in the art, including intravenous, intramuscular, subcutaneous, peritoneal Intra, spinal or other parenteral routes of administration, for example by injection or infusion.
  • MSCs Mesenchymal Stem Cells
  • MSCs Mesenchymal stem cells
  • BM-MSCs bone marrow-derived MSCs
  • UB-MSCs umbilical cord blood-derived MSCs
  • UC-MSCs umbilical cord-derived MSCs
  • AT-MSCs adipose tissue-derived MSCs
  • adipose tissue or umbilical cord tissue MSCs can be a more ideal alternative because they contain more MSCs than bone marrow and umbilical cord blood, and the tissue is easier to obtain and collect.
  • AT-MSCs have minimal risk to the donor and no ethical concerns.
  • the present invention utilizes MSC to target and deliver chemokines CCL3, CCL21, XCL1, CXCL9 and cytokines OX40L, 4-1BBL, GITRL, CD40L to attract and activate effector T cells in the tumor microenvironment, NK cells and antigen-presenting cells, resulting in a more precise and sustained immune response to kill tumor cells.
  • chemokines CCL3, CCL21, XCL1, CXCL9 and cytokines OX40L, 4-1BBL, GITRL, CD40L to attract and activate effector T cells in the tumor microenvironment, NK cells and antigen-presenting cells, resulting in a more precise and sustained immune response to kill tumor cells.
  • adipose-derived mesenchymal stem cells were used as vectors to overexpress chemokines and cytokines to treat colorectal cancer in a mouse model.
  • an "expression cassette” or “expression cassette of the invention” includes a first expression cassette and/or a second expression cassette.
  • the first expression cassette contains a nucleic acid sequence of a chemokine;
  • the second expression cassette contains a nucleic acid sequence encoding a cytokine, wherein the chemokine includes: CCL3, CCL19, CCL21, XCL1, CXCL9, or a combination thereof ,
  • the cytokines include: OX40L, 4-1BBL, GITRL, CD40L, or a combination thereof.
  • the exogenous nucleic acid molecule comprises a first expression cassette containing a nucleic acid sequence encoding CXCL9 and a second expression cassette containing a nucleic acid sequence encoding OX40L.
  • the first expression cassette and the second expression cassette are independent of each other, or combined into one.
  • the first expression cassette and the second expression cassette further comprise a promoter and/or a terminator, respectively.
  • the first expression cassette and the second expression cassette are located on a vector or integrated into the chromosome of the mesenchymal stem cell.
  • the first expression cassette and the second expression cassette are located on the same or different vectors.
  • the first expression cassette and the second expression cassette are located in the same vector.
  • the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, other gene transfer systems, or a combination thereof.
  • the present invention also provides a vector containing the expression cassette of the present invention.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools to achieve long-term gene transfer because they allow long-term, stable integration of the transgene and its proliferation in daughter cells.
  • Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus because they can transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity.
  • an expression cassette or nucleic acid sequence of the invention to a promoter, and incorporating it into an expression vector.
  • the vector is suitable for replication and integration in eukaryotic cells.
  • Typical cloning vectors contain transcriptional and translational terminators, initial sequences and promoters that can be used to regulate the expression of the desired nucleic acid sequence.
  • the expression constructs of the present invention can also be used in nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, eg, US Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated by reference in their entirety.
  • the present invention provides gene therapy vectors.
  • the expression cassette or nucleic acid sequence can be cloned into many types of vectors.
  • 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.
  • vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Particular vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • expression vectors can be provided to cells in the form of viral vectors.
  • Viral vector techniques are well known in the art and are described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.
  • suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (eg, WO01/96584; WO01/29058; and U.S. Patent No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to subject cells in vivo or ex vivo.
  • Many retroviral systems are known in the art.
  • promoter elements can regulate the frequency of transcription initiation. Typically, these are located in a region of 30-110 bp upstream of the initiation site, although it has recently been shown that many promoters also contain functional elements downstream of the initiation site.
  • the spacing between promoter elements is often flexible so that promoter function is maintained when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before activity begins to decline.
  • individual elements appear to act cooperatively or independently to initiate transcription.
  • 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 ⁇ (EF-1 ⁇ ).
  • constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, the mouse breast cancer virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russell sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter , myosin promoter, heme promoter and creatine kinase promoter.
  • the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention.
  • an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is not desired.
  • inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
  • Expression vectors introduced into cells may also contain either or both selectable marker genes or reporter genes to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector.
  • the selectable marker can be carried on a single piece 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 and the like.
  • Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that 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 cells, the expression of the reporter gene is measured at an appropriate time.
  • Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Typically, constructs with a minimum of 5 flanking regions showing the highest levels of reporter gene expression are identified as promoters. Such promoter regions can be linked to reporter genes and used to assess the ability of an agent to modulate promoter-driven transcription.
  • an expression vector can be readily introduced into a host cell, eg, mammalian (eg, human T cells), bacterial, yeast, or insect cells, by any method known in the art.
  • a host cell eg, mammalian (eg, human T cells), bacterial, yeast, or insect cells
  • an expression vector can be transferred into a host cell by physical, chemical or biological means.
  • Biological methods for introducing polynucleotides into host cells include the use of DNA and RNA vectors.
  • Viral vectors especially retroviral vectors, have become the most widely used method of inserting genes into mammalian, eg, human cells.
  • Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, among others. See, eg, US Patent Nos. 5,350,674 and 5,585,362.
  • colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipids plastid.
  • lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and lipids plastid.
  • Exemplary colloidal systems for use as in vitro and in vivo delivery vehicles are liposomes (eg, artificial membrane vesicles).
  • exemplary delivery vehicles are liposomes.
  • lipid formulations is contemplated to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo).
  • nucleic acid can be associated with a lipid.
  • Nucleic acids associated with lipids can be encapsulated into the aqueous interior of liposomes, interspersed within the lipid bilayer of liposomes, attached via linker molecules associated with both liposomes and oligonucleotides to liposomes, entrapped in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, associated with lipids, contained in lipids as a suspension, contained in micelles or Complex with micelles, or otherwise associated with lipids.
  • the lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution.
  • Lipids are fatty substances, which can be naturally occurring or synthetic lipids.
  • lipids include lipid droplets, which occur naturally in the cytoplasm as well as in such compounds comprising long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols and aldehydes.
  • the present invention provides a method for preparing mesenchymal stem cells, comprising introducing a first expression cassette and/or a second expression cassette into the mesenchymal stem cells to be engineered, wherein the first expression cassette is used to express chemotaxis cytokines, and the second expression cassette is used to express cytokines, thereby obtaining the mesenchymal stem cells.
  • the present invention provides a mesenchymal stem cell according to the first aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the formulation is a liquid formulation.
  • the formulation is an injection.
  • the concentration of the mesenchymal stem cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, more preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml.
  • the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine ; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, etc.
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as glycine
  • chelating agents such as EDTA or glutathione
  • adjuvants eg, aluminum hydroxide
  • preservatives e.g, aluminum hydroxide
  • the present invention includes the therapeutic use of mesenchymal stem cells transduced with the vector containing the expression cassette of the present invention.
  • the mesenchymal stem cells of the present invention can actively migrate to the tumor site, and are not enriched in organs such as liver, spleen, kidney, etc., and have the specificity and safety as a drug carrier for tumor treatment. Sexual side effects provide an effective means.
  • the adipose-derived mesenchymal stem cell therapy system overexpressing chemokine CXCL9 and cytokine OX40L has the characteristics of specific targeting to tumor sites, and can attract and activate T cells and NK cells to achieve ideal anti-tumor efficacy.
  • the present invention provides a type of cell therapy comprising administering to a mammal the mesenchymal stem cells of the present invention.
  • the mesenchymal stem cells of the present invention are capable of replicating in vivo, resulting in long-term persistence that can lead to sustained tumor control.
  • Cancers that can be treated include tumors that are not vascularized or substantially not vascularized, as well as tumors that are vascularized. Cancers may include non-solid tumors (such as hematological tumors, eg, leukemias and lymphomas) or may include solid tumors. Cancer types to be treated with the mesenchymal stem cells of the invention include, but are not limited to, carcinomas, blastomas and sarcomas, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors such as sarcomas, carcinomas and melanomas . Also includes adult tumors/cancers and pediatric tumors/cancers.
  • Hematological cancers are cancers of the blood or bone marrow.
  • hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastoid, promyelocytic, myelomonocytic type) , monocytic and erythroleukemia), chronic leukemia (such as chronic myeloid (myeloid) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non- Hodgkin's lymphoma (painless and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.
  • acute leukemias such
  • Solid tumors are abnormal masses of tissue that typically do not contain cysts or areas of fluid. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell type that forms 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, and ovarian cancer.
  • the mesenchymal stem cells of the present invention can also be used as a type of vaccine for ex vivo immunization and/or in vivo therapy of mammals.
  • the mammal is a human.
  • cells are isolated from mammals (preferably human) and genetically modified (ie, transduced or transfected in vitro) with vectors containing the expression cassettes of the invention.
  • the mesenchymal stem cells of the present invention can be administered to a mammalian recipient to provide therapeutic benefit.
  • the mammalian recipient can be a human, and the mesenchymal stem cells of the invention can be autologous to the recipient.
  • the cells may be allogeneic, syngeneic or xenogeneic with respect to the recipient.
  • the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.
  • cells activated and expanded as described herein can be used to treat and prevent diseases that arise in individuals who are immune-compromised. Accordingly, the present invention provides a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of mesenchymal stem cells of the present invention.
  • the mesenchymal stem cells of the present invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as certain cytokines or cell populations.
  • a pharmaceutical composition or formulation of the present invention may include mesenchymal stem cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions of the present invention can be administered in a manner appropriate to the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease - although appropriate doses may be determined by clinical trials.
  • the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the patient (subject ) individual differences in age, weight, tumor size, degree of infection or metastasis, and condition. It may generally be noted that the pharmaceutical composition comprising the mesenchymal stem cells described herein may be administered at a dose of 10 4 to 10 9 cells/kg body weight, preferably 10 5 to 10 6 cells/kg body weight (including those within those ranges). all integer values) are administered. The mesenchymal stem cell composition can also be administered multiple times at these doses.
  • Cells can be administered by using infusion techniques well known in immunotherapy (see, eg, 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 treatment accordingly.
  • compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinal, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the T cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection.
  • the T cell composition of the present invention is preferably administered by i.v. injection.
  • Compositions of mesenchymal stem cells can be injected directly into tumors, lymph nodes, or sites of infection.
  • cells activated and expanded using the methods described herein, or other methods known in the art to expand mesenchymal stem cells to therapeutic levels are combined with any number of relevant therapeutic modalities (e.g. , before, at the same time, or after) administration to a patient in a form 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 natalizumab therapy for MS patients or elfazizumab therapy for psoriasis patients or other treatments for PML patients.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also Known as ARA-C) or natalizumab therapy for MS patients or elfazizumab therapy for psoriasis patients or other treatments for PML patients.
  • 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 immunotherapeutics.
  • the cellular composition of the present invention is administered in combination with (eg, before, concurrently or after) bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • the subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • the subject receives an infusion of expanded mesenchymal stem cells of the invention.
  • the expanded cells are administered before or after 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. Dosage ratios for human administration can be carried out according to art-accepted practice. Typically, 1 x 10 3 to 1 x 10 10 mesenchymal stem cells of the present invention may be administered to a patient, eg, by intravenous infusion, per treatment or per course of treatment.
  • the mesenchymal stem cells of the present invention can be obtained from various tissues in vivo, and are easy to be cultured, expanded and transformed by genetic engineering methods in vitro, and have low immunogenicity.
  • the method of the present invention does not depend on the presence of tumor-infiltrating lymphocytes, and is also suitable for the treatment of tumors with very low or resistant lymphocyte infiltration in clinical practice.
  • 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 and kill tumor tissue at the tumor site
  • the immune cells have more efficient and less side effects of immune efficacy.
  • the mesenchymal stem cells of the present invention overexpress CXCL9 and OX40L, there is a synergistic effect, and the killing ability of tumor tissue is significantly enhanced, especially colorectal cancer cells.
  • This method also has a killing effect on MHC-I-negative tumor cells that are resistant to traditional immunotherapy (such as CAR-T or PD-1/PD-L1 antibodies).
  • the efficacy of these immunotherapies can also be enhanced.
  • the combined therapy of the mesenchymal stem cells of the present invention and the PD-1 antibody has a more significant tumor suppressing effect than the single therapy of the two, and the two show a lower synergistic effect.
  • 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.
  • Antibodies used for flow cytometry were obtained 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 for immunofluorescence staining included anti-GFP (Abcam), anti-CD8a (BioLegend) and anti-NKp46 (CD335) (BioLegend).
  • Immune checkpoint blocking antibodies anti-PD-1 (clone RMP1-4) and anti-CTLA-4 (clone 9D9) were purchased from Bio X Cell Company. The two antibodies (anti-PD-1: 200 ⁇ g/mouse; anti-CTLA-4: 100 ⁇ g/mouse) were administered to mice by intraperitoneal injection.
  • AT-MSCs were isolated by digesting mouse subcutaneous adipose tissue with type I collagenase. Cells were cultured in ⁇ -MEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. The expression of cell surface marker proteins was identified by flow cytometry after the cells were adherently cultured for three passages.
  • the cDNA was cloned into a lentiviral vector.
  • Packaging and titer determination of lentiviruses were performed by Heyuan Biotechnology (Shanghai) Co., Ltd.
  • Mesenchymal stem cells were infected with lentivirus at a multiplicity of infection (MOI) of 60 in the presence of 6 ⁇ g/ml polybrene (Sigma-Aldrich).
  • the proliferation of tumor cells was measured using the CCK-8 kit (Dojindo Molecular Technologies) according to the procedure of the instructions. Absorbance was measured using a microplate reader (Tecan).
  • Cells were harvested and treated with cell lysis buffer (RIPA buffer + 1% protease inhibitors) (ThermoFisher Scientific) to prepare cell lysates. Protein concentrations in cell lysates were determined using the BCA kit (ThermoFisher Scientific). 15 to 30 micrograms of protein were loaded onto 5% to 15% SDS-PAGE protein gels (ThermoFisher Scientific) and then transferred to PVDF membranes (Millipore). Membranes were blocked with 5% nonfat milk prepared in TBST buffer and incubated overnight at 4°C with antibodies to Myc-tag, OX40L, CXCL9 and GAPDH.
  • cell lysis buffer (RIPA buffer + 1% protease inhibitors) (ThermoFisher Scientific)
  • Membranes were washed with TBS-T buffer and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Membranes were developed with enhanced chemiluminescence kit (Millipore) and exposed to membranes.
  • CT26 or MC38 (0.5 or 1 ⁇ 10 6 /mouse) cells were injected subcutaneously in the right lower back of 8- to 10-week-old BALB/c or C57BL/6 mice, respectively.
  • mice were randomly assigned to experimental groups.
  • Systemic administration was performed by tail vein injection of 250 ⁇ l PBS or 250 ⁇ l PBS suspension containing 5 ⁇ 10 5 AT-MSCs per animal.
  • the mice were monitored for tumor size and survival. Mice were sacrificed when the tumor volume reached 2 cm3 or when the tumor became ulcerated or the mice were dying.
  • adherent cells at passages 3 to 5 were isolated by digestion with 20 ⁇ M EDTA, then washed twice with PBS and stained with antibodies.
  • tumor-infiltrating immune cells For analysis of tumor-infiltrating immune cells, subcutaneously implanted tumors were dissected and transferred to RPMI medium, minced with scissors, and placed in 0.25 mg/ml Liberase TL (Roche) and 50 ⁇ g/ml DNase I (Sigma- Aldrich) serum-free RPMI medium, digested using gentleMAC Octo Dissociator (Milteniy Biotec) at 37°C, and dispersed through a 40 ⁇ m cell strainer (BD Biosciences). Single cells were further washed and stained with antibodies. Dead cells were excluded by staining with Zombie Fixable Viability Kit (BioLegend).
  • each mouse was injected intraperitoneally with 0.25 mg Brefeldin A (BFA) (Selleck) 4-6 hours before harvesting the samples. After surface staining in the presence of 5 ⁇ 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).
  • BFA Brefeldin A
  • Flow data were acquired 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.
  • mice were injected intraperitoneally with AOM (12.5 mg/kg body weight; Sigma-Aldrich) (14). After 1 week, mice were given drinking water containing 3% DSS (MP Biomedicals) for 7 days, followed by normal water for 2 weeks. DSS induction continued for two cycles and mice were sacrificed after five injections of MSCs via tail vein starting from the last week of the DSS induction cycle. Body weight was recorded during DSS treatment. Colons were removed from mice, rinsed with ice-cold PBS, opened longitudinally, fixed in 4% paraformaldehyde solution (Sigma-Aldrich) overnight at room temperature, and paraffin embedded. Before fixing, use digital calipers to take dimension measurements.
  • AOM 12.5 mg/kg body weight; Sigma-Aldrich
  • Tissue sections were blocked with 10% normal goat serum and then incubated with primary antibodies overnight at 4°C and secondary antibodies for 1 hour at room temperature. Slides were mounted in fade-resistant mounting medium with DAPI (ThermoFisher Scientific) and observed under a Nikon Eclipse Ti fluorescence microscope. Antibodies used for immunofluorescence were GFP antibody (Abeam), CD8a antibody (BioLegend) and NKp46 antibody (CD335) (BioLegend).
  • Example 1 Characteristics of adipose-derived mesenchymal stem cells migrating to tumors
  • Mesenchymal stem cells were extracted from mouse subcutaneous adipose, and detected by flow cytometry, these cells expressed specific mesenchymal stem cell marker molecules and did not express marker molecules of other cell types (Fig. 1A), confirming that the adipose used for the experiment was used. Purity of mesenchymal stem cells. Adipose-derived mesenchymal stem cells were transfected with lentivirus to express GFP, and then 5 ⁇ 10 5 cells were injected into tumor-bearing mice (CT26 intestinal cancer subcutaneous tumor) through the tail vein. After 7 days, the tumor and other organs were organized.
  • CT26 intestinal cancer subcutaneous tumor tumor-bearing mice
  • chemokines and cytokines with potential anti-tumor functions were selected, and the genes were cloned into lentiviral vectors to package lentiviruses carrying these genes.
  • the CT26 intestinal cancer cell lines transduced with these genes or blank control lentiviruses were subjected to subcutaneous transplantation tumor experiments to detect the antitumor efficacy of these molecules.
  • Fig. 2A After overexpression of the chemokines CCL3 and CXCL9 with potential antitumor efficacy in CT26 (Fig. 2A), neither cell proliferation was affected in vitro (Fig. 2B), while the growth of subcutaneous xenografts in vivo was significantly inhibited (Fig.
  • CXCL9 has the most significant anti-tumor effect. Detection of immune cell composition in tumors by flow cytometry revealed that CXCL9 indeed enhanced the infiltration of anti-tumor immune cells such as CD8, CD4 and NK (Fig. 2D).
  • a adipose-derived mesenchymal stem cell system overexpressing CXCL9 and OX40L was established by lentiviral infection.
  • the successful expression and secretion of CXCL9 were identified by western blotting and ELISA techniques (Figure 4A&B), and the successful expression of OX40L on the cell membrane was confirmed by western blotting and flow cytometry ( Figure 4C&D).
  • MSCs overexpressing CXCL9 and OX40L were established (Fig. 5A&B).
  • mice were injected with 5 ⁇ 10 5 mesenchymal stem cells or PBS each time through the tail vein, after four days between each time.
  • Fig. 5C mesenchymal stem cells harboring both CXCL9 and OX40L exhibited the strongest antitumor efficacy.
  • Flow cytometry analysis found that the proportion of lymphocytes, especially anti-tumor CD8 T cells and NK cells in the tumor was significantly increased (Fig. 5D), indicating that the therapy effectively activated the anti-tumor immune response.
  • AOM/DSS was used to induce an orthotopic colorectal cancer model caused by inflammation.
  • mice were treated for a total of 5 times for 4 weeks.
  • Treatment with PBS or 5 x 10 5 MSCs per injection via tail vein, CXCL9- and OX40L-loaded MSCs resulted in an extremely significant reduction in intestinal tumors in mice (Fig. 6B&C) .
  • Immunofluorescence staining demonstrated a marked increase in infiltration of anti-tumor CD8 T cells and NK cells (Fig. 6D), consistent with the results observed in the xenograft model.
  • mice treated with a combination of PD-1 antibody (200 ⁇ g/mouse) and CTLA-4 (100 ⁇ g/mouse) antibody (Fig. 7A)
  • Example 4 Mesenchymal stem cells overexpressing chemokine CXCL9 and cytokine OX40L improve the therapeutic efficacy of PD-1 blockade therapy
  • mice were injected a total of four times with mesenchymal stem cells (MSCs) overexpressing CXCL9 and OX40L, a PD-1 blocking antibody ( ⁇ PD-1), or both ( Figure 8A).
  • MSCs mesenchymal stem cells
  • ⁇ PD-1 PD-1 blocking antibody
  • each of MSC and ⁇ PD-1 alone can significantly inhibit tumor growth, and the combined treatment of MSC and ⁇ PD-1 inhibits tumor growth more effectively than either treatment alone, indicating that MSC therapy is effective for PD-1 blockade.
  • the therapy has a significant enhancement effect.
  • Example 5 Inhibitory effect of mesenchymal stem cells overexpressing chemokine CXCL9 and cytokine OX40L on MHC-I-deficient tumors
  • mice were injected through the tail vein a total of six times with MSCs expressing different cytokines or chemokines (5 ⁇ 10 5 cells/mouse/time) or PBS, which overexpressed IL15.
  • Mesenchymal stem cells did not show obvious anti-tumor efficacy, while mesenchymal stem cells overexpressing GITRL, 4-1BBL, and CD40L all showed highly effective anti-tumor effects (Fig. 10A).
  • overexpression of several other chemokines CCL3, CCL19, CCL21, and XCL1 showed significant antitumor efficacy (Fig. 10B).
  • cytokines and immune checkpoint blockers have shown significant efficacy in the clinical treatment of tumors, systemic use of these drugs will non-specifically activate the immune system and affect most organs.
  • mesenchymal stem cells were selected as drug carriers. The research results show that adipose-derived mesenchymal stem cells can actively migrate to the tumor site and are not enriched in the liver, spleen, kidney and other organs, which fully supports the specificity and safety of mesenchymal stem cells as a drug carrier for tumor therapy. Local activation of immune responses in tumors provides an effective means to avoid systemic side effects.
  • T cell and NK cell infiltration in tumors is a key determinant of the efficacy of immunotherapy in solid tumors.
  • Tumors can restrict the infiltration of lymphocytes by different mechanisms.
  • Tumors with more T cell infiltration often also highly express chemokines that can attract T cells, including CCL3, CCL4, and CXCL10.
  • the present invention unexpectedly found that the use of mesenchymal stem cells as a carrier to express CXCL9 and transport it to the tumor site to attract anti-tumor lymphocytes shows stronger T cell and NK cell attraction ability, and solves the problem that lymphocytes are difficult to enter solid tumors. problem.
  • loading OX40L in the mesenchymal stem cell system can also activate T cells and NK cells more efficiently.
  • OX40L Activating antibodies to the OX40 receptor have entered the clinical stage (ClinicalTrials.gov), but potential problems are also the side effects of systemic medication.
  • the ligand of OX40, OX40L is a membrane protein. When expressed in mesenchymal stem cells, it can be directed and transported to the tumor site without being secreted like secreted cytokines and transferred to other sites, reducing the diffusion caused by secretion. It can activate existing or newly migrated lymphocytes at the tumor site.
  • the present invention also unexpectedly found that adipose-derived mesenchymal stem cells overexpressing CXCL9 and OX40L exhibited extremely significant therapeutic effects in mouse subcutaneous transplanted tumors and inflammation-induced in situ colorectal cancer models.
  • the adipose-derived mesenchymal stem cell treatment system overexpressing chemokine CXCL9 and cytokine OX40L established in the present invention has the characteristics of specifically targeting tumor sites, and can attract and activate T cells and NK cells to achieve an ideal anti-tumor effect. tumor efficacy. In contrast to most immunotherapies, this therapy does not depend on the presence of tumor-infiltrating lymphocytes and is clinically suitable for the treatment of tumors with very low or resistant lymphocyte infiltration. Adipose or umbilical cord mesenchymal stem cells are easy to extract and culture, and can be easily applied to individualized treatment. Its low immunogenicity also makes allogeneic use feasible. Therefore, the established mesenchymal stem cell-based immunotherapy has extremely high clinical translation value.

Abstract

提供了一种间充质干细胞靶向运输趋化因子和细胞因子的免疫疗法。具体提供了一种间充质干细胞，所述间充质干细胞表达免疫刺激因子，所述免疫刺激因子选自下组：CCL3、CCL19、CCL21、XCL1、CXCL9、OX40L、4-1BBL、GITRL、CD40L、或其组合。上述间充质干细胞在肿瘤部位能够特异地吸引并激活杀伤肿瘤组织的免疫细胞，并且间充质干细胞与趋化因子和/或细胞因子具有协同作用，具有更高效且副作用低的免疫疗效，对肿瘤组织杀伤能力显著增强，尤其是结直肠癌细胞。

Description

间充质干细胞靶向运输趋化因子和细胞因子的免疫疗法 技术领域
本发明属于生物技术领域，具体地，本发明涉及间充质干细胞靶向运输趋化因子和细胞因子的免疫疗法。
背景技术
免疫疗法的飞速发展为癌症带来了新的曙光，特别是嵌合抗原受体T细胞免疫疗法(CAR-T)和免疫调节检查点阻断是目前比较前沿的两种癌症免疫疗法。CAR-T细胞疗法在一些类型的癌症中表现出一定的疗效，但是也遇到了T细胞到达实体瘤部位效率低下以及作用时间较短、肿瘤部位已有的免疫细胞数量少、全身性用药带来的副作用等的瓶颈。使用靶向T细胞抑制性受体PD-1或CTLA-4的抗体可以产生十分显著的抗肿瘤效果。然而，抗体的治疗效果通常受到许多因素的限制，例如实体瘤中T细胞的低浸润以及活性的丧失。此外全身性使用免疫治疗药物比如干扰素α、白介素2或者PD-1抗体等可能引起严重的副作用。
因此本领域迫切需要一种能特异性靶向肿瘤细胞的免疫疗法。
发明内容
本发明的目的是提供一种能特异性靶向肿瘤细胞的免疫疗法。
本发明的另一目的是提供一种间充质干细胞靶向运输趋化因子和细胞因子的免疫疗法。
本发明的第一方面，提供了一种间充质干细胞，所述间充质干细胞表达免疫刺激因子，所述免疫刺激因子选自下组：CCL3、CCL19、CCL21、XCL1、CXCL9、OX40L、4-1BBL、GITRL、CD40L、或其组合。
在另一优选例中，所述间充质干细胞含有外源核酸分子，所述外源核酸分子包括编码免疫刺激因子的核酸序列，所述免疫刺激因子选自下组：CXCL9、OX40L、CCL3、CCL19、CCL21、XCL1、CXCL9、OX40L、4-1BBL、GITRL、CD40L、或其组合。
在另一优选例中，所述外源核酸分子还包括启动子或启动子/增强子组合，所述编码免疫刺激因子的核酸序列可操作地连接于启动子或启动子/增强子组合。
在另一优选例中，所述启动子为组成型启动子或诱导型启动子，较佳地为组 成型启动子。
在另一优选例中，所述免疫刺激因子包括至少一种趋化因子，所述趋化因子包括：CCL3、CCL19、CCL21、XCL1、CXCL9、或其组合。
在另一优选例中，所述免疫刺激因子包括至少一种细胞因子，所述细胞因子包括：OX40L、4-1BBL、GITRL、CD40L、或其组合。
在另一优选例中，所述免疫刺激因子包括至少一种趋化因子和至少一种细胞因子，其中，所述趋化因子包括：CCL3、CCL19、CCL21、XCL1、CXCL9、或其组合，所述细胞因子包括：OX40L、4-1BBL、GITRL、CD40L、或其组合。
在另一优选例中，所述免疫刺激因子为CCL3、CCL19、CCL21、XCL1之中的一个或两个和CD40L组合。
在另一优选例中，所述免疫刺激因子为OX40L、4-1BBL、GITRL之中的一个或两个和CXCL9组合。
在另一优选例中，所述免疫刺激因子为CXCL9和/或OX40L。
在另一优选例中，所述外源核酸分子包括第一表达盒和/或第二表达盒，所述第一表达盒含有编码趋化因子的核酸序列，所述第二表达盒含有编码细胞因子的核酸序列，其中，所述趋化因子包括：CCL3、CCL19、CCL21、XCL1、CXCL9、或其组合，所述细胞因子包括：OX40L、4-1BBL、GITRL、CD40L、或其组合。
在另一优选例中，所述外源核酸分子包括第一表达盒和第二表达盒，所述第一表达盒含有编码CXCL9的核酸序列，所述第二表达盒含有编码OX40L的核酸序列。
在另一优选例中，所述第一表达盒和第二表达盒为各自独立的、或合二为一的。
在另一优选例中，所述第一表达盒和第二表达盒分别还包含启动子和/或终止子。
在另一优选例中，所述第一表达盒和第二表达盒为同一表达盒，所述表达盒包含启动子、编码趋化因子的核酸序列和编码细胞因子的核酸序列。
在另一优选例中，所述的第一表达盒和第二表达盒位于载体上或整合在所述间充质干细胞的染色体中。
在另一优选例中，所述的第一表达盒和第二表达盒是独立的或相连的。
在另一优选例中，所述的第一表达盒和第二表达盒位于相同或不同的载体上。
在另一优选例中，所述的第一表达盒和第二表达盒位于同一载体。
在另一优选例中，所述的载体选自下组：DNA、RNA、质粒、慢病毒载体、腺病毒载体、逆转录病毒载体、转座子、其他基因转移系统、或其组合。
在另一优选例中，所述间充质干细胞包括：脂肪间充质干细胞、脐带间充质干细胞、或其组合。
在另一优选例中，所述间充质干细胞是离体的。
在另一优选例中，所述间充质干细胞是自体或异体的。
本发明的第二方面，提供了一种制备本发明第一方面所述的间充质干细胞的方法，包括以下步骤：
(1)提供一待改造的间充质干细胞；和
(2)将含有编码免疫刺激因子的核酸序列的外源核酸导入到所述待改造的间充质干细胞中，从而获得本发明第一方面所述的间充质干细胞；
其中，所述免疫刺激因子选自下组：CCL3、CCL19、CCL21、XCL1、CXCL9、OX40L、4-1BBL、GITRL、CD40L、或其组合。
本发明的第三方面，提供了一种制剂，所述制剂含有本发明第一方面所述的间充质干细胞，以及药学上可接受的载体、稀释剂或赋形剂。
在另一优选例中，所述制剂为液态制剂。
在另一优选例中，所述制剂的剂型包括注射剂。
在另一优选例中，所述制剂中间充质干细胞的浓度为1×10 3-1×10 8个细胞/ml，较佳地1×10 4-1×10 7个细胞/ml。
本发明的第四方面，提供了如本发明第一方面所述的间充质干细胞的用途，用于制备预防和/或治疗癌症或肿瘤的药物或制剂。
在另一优选例中，所述肿瘤选自下组：血液肿瘤、实体瘤、或其组合。优选地，所述肿瘤为实体瘤。
在另一优选例中，所述血液肿瘤选自下组：急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、或其组合。
在另一优选例中，所述实体瘤选自下组：胃癌、胃癌腹膜转移、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、宫颈癌、卵巢癌、淋巴癌、鼻咽癌、肾上腺肿瘤、膀胱肿瘤、非小细胞肺癌(NSCLC)、脑胶质瘤、子宫内膜癌、肺鳞癌、肛门癌、头颈部肿瘤、或其组合。
在另一优选例中，所述实体瘤为结直肠癌。
在另一优选例中，所述肿瘤包括复发性、转移性肿瘤。
在另一优选例中，所述肿瘤包括MHC-I缺失的肿瘤。
本发明的第五方面，提供了一种药盒，所述药盒含有
(1)第一容器，以及装于该第一容器中的本发明第一方面所述的间充质干细胞；和
(2)第二容器，以及装于该第二容器中的抗肿瘤免疫治疗剂。
在另一优选例中，所述抗肿瘤免疫治疗剂选自下组：抗体、免疫细胞、或其组合。
在另一优选例中，所述免疫细胞为T细胞或NK细胞。
在另一优选例中，所述抗肿瘤免疫治疗剂为免疫检查点抗体。
在另一优选例中，所述免疫检查点抗体包括PD-1抗体和/或CTLA-4抗体。
本发明还提供了一种治疗疾病的方法，包括给需要治疗的对象施用适量的本发明第一方面所述的细胞、或本发明第三方面所述的制剂、或本发明第五方面所述的药物组合。
在另一优选例中，所述疾病为癌症或肿瘤，较佳地为实体瘤，更佳地为结直肠癌。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了小鼠脂肪间充质干细胞表型与向肿瘤迁移特性的鉴定。A,流式细胞法检测脂肪间充质干细胞表面分子的表达。B,体内MSC-GFP在CT26皮下移植瘤小鼠模型中的迁移。免疫荧光检测肿瘤组织中MSC-GFP的浸润，绿色表示MSC-GFP，蓝色(DAPI染色)指示细胞核，比例尺：20μM。C,流式细胞法检测整个CT26皮下移植瘤中MSC-GFP的具体数量。数据代表平均值±SEM(n＝3)。
图2显示了肿瘤细胞中过表达CXCL9抑制肿瘤体内生长。A,WB和qPCR检测CT26中CCL3和CXCL9的过表达。数据代表平均值±SEM(n＝3)。B,CT26-Vector，CT26-CCL3和CT26-CXCL9的细胞增殖实验。数据代表平均值±SEM(n＝3)。C,皮 下注射CT26-Vector、CT26-CCL3或CT26-CXCL9肿瘤细胞BALB/c小鼠中的肿瘤生长曲线，每3天测量肿瘤大小。数据代表平均值±SEM(n＝4)。D,总细胞内CD8+，CD4+T和NK细胞的百分比。数据代表平均值±SEM(n＝4)。
图3显示了肿瘤细胞中过表达OX40L抑制肿瘤体内生长。A,WB检测CT26中IL36β和OX40L的过表达。B,流式检测CT26中OX40L的过表达。C,CT26-Vector，CT26-IL36β和CT26-OX40L的细胞增殖实验。数据代表平均值±SEM(n＝3)。D,皮下注射CT26-Vector、CT26-CCL3或CT26-CXCL9肿瘤细胞BALB/c小鼠中的肿瘤生长曲线，每3天测量肿瘤大小。每组四只只小鼠。数据代表平均值±SEM(n＝4)。*p<0.05，**p<0.01。
图4显示了CXCL9与OX40L在间充质干细胞中过表达的鉴定。A,WB检测MSC中CXCL9的过表达。B,ELISA检测MSC中CXCL9的过表达。数据代表平均值±SEM(n＝3)。***p<0.001。C,WB检测MSC中OX40L的过表达。D,流式细胞法检测MSC中OX40L的过表达。
图5显示了过表达CXCL9与OX40L的间充质干细胞抑制皮下移植瘤的生长。A,ELISA检测CXCL9的分泌。B,流式细胞法检测OX40L的表达。C,图中显示为不同时间点的CT26皮下移植瘤大小。箭头指示为相应的给小鼠注射的PBS或MSC的时间。数据代表平均值±SEM(n＝5)。D,流式细胞法分析MSC治疗后肿瘤中免疫细胞的比例。*p<0.05，**p<0.01，***p<0.001，ns＝not significant(无统计学显著性)。
图6显示了过表达CXCL9与OX40L的间充质干细胞抑制AOM/DSS诱导的结直肠癌。A,AOM/DSS处理以及MSC治疗方案示意图。B,结直肠肿瘤的代表性图像。比例尺：5mm。C,平均肿瘤数量和大小。数据代表平均值±SEM(n＝3-4)。*p<0.05，**p<0.01，***p<0.001，ns＝not significant(无统计学显著性)。D,CD8和NK细胞的免疫荧光染色。比例尺：50μm。
图7显示了PD-1和CTLA-4抗体联合治疗对AOM/DSS诱导的结直肠癌没有显著的治疗功效。A,AOM/DSS处理以及抗体治疗方案示意图。B，平均肿瘤数量和大小。数据代表平均值±SEM(n＝4)。ns＝not significant(无统计学显著性)。
图8显示了过表达CXCL9和OX40L的间充质干细胞促进PD-1阻断疗法的功效。A，间充质干细胞(MSC-CXCL9+OX40L)(每针5X10 5个细胞)与PD-1抗体阻断(αPD-1)治疗示意图；B，过表达CXCL9和OX40L的间充质干细胞与PD-1抗体阻断联合治疗比两者的单独治疗有更显著的肿瘤抑制作用(n＝7-8只/组)。**p<0.01。
图9显示了过表达CXCL9和OX40L的间充质干细胞可抑制MHC-I缺失的具有免 疫逃逸特性的肿瘤的生长。A，对体内小鼠结直肠癌细胞MC38皮下移植瘤进行流式细胞分析，确认敲除B2m基因的MC38肿瘤细胞(sgB2m)与对照sgLacZ相比不表达MHC-I分子(H2Kb/H2Db)(n＝4只/组)；B，体内建立过表达OVA的MHC-I缺失的MC38-sgB2m或对照MC38-sgLacZ肿瘤细胞的移植瘤模型，发现MC38-sgB2m比MHC-I正常表达的对照MC38-sgLacZ细胞生长得更快，利用肿瘤疫苗(OVA257-264多肽加poly:IC佐剂)进行治疗，发现MC38-sgLacZ细胞被肿瘤疫苗显著抑制，而MHC-I缺失的MC38-sgB2m的肿瘤对疫苗治疗无响应(n＝5只/组)；C，过表达CXCL9和OX40L的间充质干细胞可显著抑制MHC-I缺失的MC38-sgB2m肿瘤的生长(n＝7只/组)。**p<0.01，***p<0.001，****p<0.0001。
图10显示了过表达免疫激活因子/细胞因子或者趋化因子的间充质干细胞(MSC)的治疗功效。每只小鼠每次接受5X10 5个过表达其中一个因子的间充质干细胞，每三天一次治疗，一共六次治疗。A，过表达GITRL、4-1BBL或CD40L的间充质干细胞具有非常显著的肿瘤抑制作用，而过表达IL5的间充质干细胞没有明显的治疗效果(n＝5只/组)；B，过表达CCL3、CCL19、CCL21或XCL1的间充质干细胞具有显著的肿瘤抑制作用(n＝5只/组)。**p<0.05，**p<0.01，***p<0.001。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，首次意外地发现间充质干细胞过表达趋化因子CCL3、CCL19、CCL21、XCL1、CXCL9和/或细胞因子OX40L、4-1BBL、GITRL、CD40L时，在肿瘤部位能够特异地吸引并激活杀伤肿瘤组织的免疫细胞，并且间充质干细胞与趋化因子和/或细胞因子具有协同作用，具有更高效且副作用低的免疫疗效。尤其是间充质干细胞过表达CXCL9和OX40L时，具有相互协同作用，对肿瘤组织杀伤能力显著增强，尤其是结直肠癌细胞。在此基础上，发明人完成了本发明。
近年来免疫疗法诸如细胞因子、CAR-T细胞或者免疫检查点阻断等，在一些癌症患者中产生良好的功效，也遇到了许多障碍，比如T细胞到达肿瘤部位的效率低、肿瘤部位已有的免疫细胞数量少、全身性用药带来的副作用等。间充质干细胞在体内可从多种组织中获取，在体外易于培养扩增和利用基因工程的方法改造，免疫原性较低。本发明人在小鼠模型中的研究证实脂肪间充质干细胞可特异地向肿瘤部位迁移，并不在其他脏器中富集，因此可作为理想的药物载体。本发明利用脂肪间充质干细胞为载体过表达具有免疫调节作用的趋化因子CXCL9和细胞因子OX40L，通 过间充质干细胞向肿瘤的主动迁移，在肿瘤部位特异地吸引并激活杀伤肿瘤组织的免疫细胞，最终达到更高效且副作用低的免疫治疗功效。
术语说明
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。
如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
如本文所用，术语“给予”是指使用本领域技术人员已知的各种方法和递送系统中的任一种将本发明的产品物理引入受试者，包括静脉内，肌内，皮下，腹膜内，脊髓或其它肠胃外给药途径，例如通过注射或输注。
间充质干细胞(MSC)
近些年来间充质干细胞(MSC)作为有潜力的细胞载体出现在人们的视野中，其在全身给药时具有针对许多不同肿瘤类型的主动迁移能力。它可以从许多不同的成体组织中提取，易于扩增培养，能避免免疫排斥。同时间充质干细胞肿瘤向性迁移的特性以及在靶向地点长期存活的能力，使其成为细胞治疗的一个重要资源。常用类型的MSC是骨髓来源的MSC(BM-MSC)，脐带血来源的MSC(UCB-MSC)、脐带来源的MSC(UC-MSC)和脂肪组织来源的MSC(AT-MSC)。然而，BM-MSC和UCB-MSC的分离过程十分复杂且低效。因此脂肪组织或脐带组织MSC可以作为更理想的替代方案，因为它们相比骨髓和脐带血含有更多的MSC，组织更易于获取和收集。此外，关于用于个体化细胞疗法的自体干细胞来源，AT-MSC对供体具有最小的风险并且没有伦理问题。
通过MSCs的肿瘤归巢能力，本发明利用MSC靶向递送趋化因子CCL3、CCL21、XCL1、CXCL9和细胞因子OX40L、4-1BBL、GITRL、CD40L来吸引和激活肿瘤微环境中的效应T细胞、NK细胞和抗原呈递细胞，从而产生更精准和持续的免疫应答来杀伤肿瘤细胞。在目前的研究中，在小鼠模型中以脂肪间充质干细胞为载体来过表达趋化因子和细胞因子来治疗结直肠癌。
表达盒
如本文所用，“表达盒”或“本发明表达盒”包括第一表达盒和/或第二表达盒。所述第一表达盒含有趋化因子的核酸序列；所述第二表达盒含有编码细胞因子的核酸序列，其中，所述趋化因子包括：CCL3、CCL19、CCL21、XCL1、CXCL9、或其组合，所述细胞因子包括：OX40L、4-1BBL、GITRL、CD40L、或其组合。
在一个实施方式中，所述外源核酸分子包括第一表达盒和第二表达盒，所述第一表达盒含有编码CXCL9的核酸序列，所述第二表达盒含有编码OX40L的核酸序列。
在另一优选例中，所述第一表达盒和第二表达盒为各自独立的、或合二为一的。在另一优选例中，所述第一表达盒和第二表达盒分别还包含启动子和/或终止子。在另一优选例中，所述的第一表达盒和第二表达盒位于载体上或整合在所述间充质干细胞的染色体中。在另一优选例中，所述的第一表达盒和第二表达盒位于相同或不同的载体上。在另一优选例中，所述的第一表达盒和第二表达盒位于同一载体。在另一优选例中，所述的载体选自下组：DNA、RNA、质粒、慢病毒载体、腺病毒载体、逆转录病毒载体、转座子、其他基因转移系统、或其组合。
载体
本发明还提供了含有本发明表达盒的载体。源于逆转录病毒诸如慢病毒的载体是实现长期基因转移的合适工具，因为它们允许转基因长期、稳定的整合并且其在子细胞中增殖。慢病毒载体具有超过源自致癌逆转录病毒诸如鼠科白血病病毒的载体的优点，因为它们可转导非增殖的细胞，诸如肝细胞。它们也具有低免疫原性的优点。
简单概括，通常通过可操作地连接本发明的表达盒或核酸序列至启动子，并将其并入表达载体。该载体适合于复制和整合真核细胞。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、初始序列和启动子。
本发明的表达构建体也可利用标准的基因传递方案，用于核酸免疫和基因疗法。基因传递的方法在本领域中是已知的。见例如美国专利号5,399,346、5,580,859、5,589,466，在此通过引用全文并入。在另一个实施方式中，本发明提供了基因疗法载体。
所述表达盒或核酸序列可被克隆入许多类型的载体。例如，该表达盒或核酸序可被克隆入如此载体，其包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。特定的感兴趣载体包括表达载体、复制载体、探针产生载体和测序载体。
进一步地，表达载体可以以病毒载体形式提供给细胞。病毒载体技术在本领域 中是公知的并在例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)和其他病毒学和分子生物学手册中进行了描述。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记(例如，WO01/96584；WO01/29058；和美国专利号6,326,193)。
已经开发许多基于病毒的系统，用于将基因转移入哺乳动物细胞。例如，逆转录病毒提供了用于基因传递系统的方便的平台。可利用在本领域中已知的技术将选择的基因插入载体并包装入逆转录病毒颗粒。该重组病毒可随后被分离和传递至体内或离体的对象细胞。许多逆转录病毒系统在本领域中是已知的。
额外的启动子元件，例如增强子，可以调节转录开始的频率。通常地，这些位于起始位点上游的30-110bp区域中，尽管最近已经显示许多启动子也包含起始位点下游的功能元件。启动子元件之间的间隔经常是柔性的，以便当元件相对于另一个被倒置或移动时，保持启动子功能。在胸苷激酶(tk)启动子中，启动子元件之间的间隔可被增加隔开50bp，活性才开始下降。取决于启动子，表现出单个元件可合作或独立地起作用，以起动转录。
合适的启动子的一个例子为早期巨细胞病毒(CMV)启动子序列。该启动子序列为能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。然而，也可使用其他组成型启动子序列，包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、艾伯斯坦-巴尔(Epstein-Barr)病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地，本发明不应被限于组成型启动子的应用。诱导型启动子也被考虑为本发明的一部分。诱导型启动子的使用提供了分子开关，其能够当这样的表达是期望的时，打开可操作地连接诱导型启动子的多核苷酸序列的表达，或当表达是不期望的时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
被引入细胞的表达载体也可包含可选择的标记基因或报道基因中的任一个或两者，以便于从通过病毒载体寻求被转染或感染的细胞群中鉴定和选择表达细胞。在其他方面，可选择的标记可被携带在单独一段DNA上并用于共转染程序。可选择的标记和报道基因两者的侧翼都可具有适当的调节序列，以便能够在宿主细胞中表 达。有用的可选择标记包括例如抗生素抗性基因，诸如neo等等。
报道基因用于鉴定潜在转染的细胞并用于评价调节序列的功能性。通常地，报道基因为以下基因：其不存在于受体有机体或组织或由受体有机体或组织进行表达，并且其编码多肽，该多肽的表达由一些可容易检测的性质例如酶活性清楚表示。在DNA已经被引入受体细胞后，报道基因的表达在合适的时间下进行测定。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色萤光蛋白基因的基因(例如，Ui-Tei等，2000FEBS Letters479:79-82)。合适的表达系统是公知的并可利用已知技术制备或从商业上获得。通常，显示最高水平的报道基因表达的具有最少5个侧翼区的构建体被鉴定为启动子。这样的启动子区可被连接至报道基因并用于评价试剂调节启动子-驱动转录的能力。
将基因引入细胞和将基因表达入细胞的方法在本领域中是已知的。在表达载体的内容中，载体可通过在本领域中的任何方法容易地引入宿主细胞，例如，哺乳动物(如人T细胞)、细菌、酵母或昆虫细胞。例如，表达载体可通过物理、化学或生物学手段转移入宿主细胞。
将多核苷酸引入宿主细胞的物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔等等。生产包括载体和/或外源核酸的细胞的方法在本领域中是公知的。见例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)。
将多核苷酸引入宿主细胞的生物学方法包括使用DNA和RNA载体。病毒载体，特别是逆转录病毒载体，已经成为最广泛使用的将基因插入哺乳动物例如人细胞的方法。其他病毒载体可源自慢病毒、痘病毒、单纯疱疹病毒I、腺病毒和腺伴随病毒等等。见例如美国专利号5,350,674和5,585,362。
将多核苷酸引入宿主细胞的化学手段包括胶体分散系统，诸如大分子复合物、纳米胶囊、微球、珠；和基于脂质的系统，包括水包油乳剂、胶束、混合胶束和脂质体。用作体外和体内传递工具(delivery vehicle)的示例性胶体系统为脂质体(例如，人造膜囊)。
在使用非病毒传递系统的情况下，示例性传递工具为脂质体。考虑使用脂质制剂，以将核酸引入宿主细胞(体外、离体(ex vivo)或体内)。在另一方面，该核酸可与脂质相关联。与脂质相关联的核酸可被封装入脂质体的水性内部中，散布在脂质体的脂双层内，经与脂质体和寡核苷酸两者都相关联的连接分子附接至脂质体，陷入脂质体，与脂质体复合，分散在包含脂质的溶液中，与脂质混合，与脂质联合， 作为悬浮液包含在脂质中，包含在胶束中或与胶束复合，或以其他方式与脂质相关联。与组合物相关联的脂质、脂质/DNA或脂质/表达载体不限于溶液中的任何具体结构。例如，它们可存在于双分子层结构中，作为胶束或具有“坍缩的(collapsed)”结构。它们也可简单地被散布在溶液中，可能形成大小或形状不均一的聚集体。脂质为脂肪物质，其可为天然发生或合成的脂质。例如，脂质包括脂肪小滴，其天然发生在细胞质以及包含长链脂肪族烃和它们的衍生物诸如脂肪酸、醇类、胺类、氨基醇类和醛类的该类化合物中。
制备方法
本发明提供了一种制备间充质干细胞的方法，包括将第一表达盒和/或第二表达盒导入到所述待改造的间充质干细胞，其中所述第一表达盒用于表达趋化因子，所述第二表达盒用于表达细胞因子，从而获得所述的间充质干细胞。
一般包括以下步骤：(1)用编码本发明免疫刺激因子的多核苷酸，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；(2)在合适的培养基中培养的宿主细胞。
制剂
本发明提供了一种含有本发明第一方面所述的间充质干细胞，以及药学上可接受的载体、稀释剂或赋形剂。在一个实施方式中，所述制剂为液态制剂。优选地，所述制剂为注射剂。优选地，所述制剂中所述间充质干细胞的浓度为1×10 3-1×10 8个细胞/ml，更优地1×10 4-1×10 7个细胞/ml。
在一个实施方式中，所述制剂可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的制剂优选配制用于静脉内施用。
治疗性应用
本发明包括含本发明表达盒的载体转导的间充质干细胞进行的治疗性应用。本发明间充质干细胞可主动向肿瘤部位迁移，并且不在肝、脾、肾等脏器中富集，具有作为肿瘤治疗药物载体的特异性和安全性，为在肿瘤中局部激活免疫反应避免全身性副作用提供了有效的手段。过表达趋化因子CXCL9和细胞因子OX40L的脂肪间充质干细胞治疗体系具有特异靶向肿瘤部位的特性，能够吸引并激活T细胞和NK 细胞从而达到理想的抗肿瘤功效。
在一个实施方式中，本发明提供一类细胞疗法，包括给哺乳动物施用本发明的间充质干细胞。不像抗体疗法，本发明间充质干细胞能够体内复制，产生可导致持续肿瘤控制的长期持久性。
可治疗的癌症包括没有被血管化或基本上还没有被血管化的肿瘤，以及血管化的肿瘤。癌症可包括非实体瘤(诸如血液学肿瘤，例如白血病和淋巴瘤)或可包括实体瘤。用本发明的间充质干细胞治疗的癌症类型包括但不限于癌、胚细胞瘤和肉瘤，和某些白血病或淋巴恶性肿瘤、良性和恶性肿瘤、和恶性瘤，例如肉瘤、癌和黑素瘤。也包括成人肿瘤/癌症和儿童肿瘤/癌症。
血液学癌症为血液或骨髓的癌症。血液学(或血原性)癌症的例子包括白血病，包括急性白血病(诸如急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病)、慢性白血病(诸如慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤、霍奇金氏疾病、非霍奇金氏淋巴瘤(无痛和高等级形式)、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、重链疾病、骨髓增生异常综合征、多毛细胞白血病和脊髓发育不良。
实体瘤为通常不包含囊肿或液体区的组织的异常肿块。实体瘤可为良性或恶性的。不同类型的实体瘤以形成它们的细胞类型命名(诸如肉瘤、癌和淋巴瘤)。实体瘤诸如肉瘤和癌的例子包括纤维肉瘤、粘液肉瘤、脂肪肉瘤间皮瘤、淋巴恶性肿瘤、胰腺癌卵巢癌。
本发明的间充质干细胞也可用作对哺乳动物离体免疫和/或体内疗法的疫苗类型。优选地，哺乳动物为人。
对于离体免疫，以下中的至少一项在将细胞施用进入哺乳动物前在体外发生：i)扩展细胞，ii)将本发明表达盒引入细胞，和/或iii)冷冻保存细胞。
离体程序在本领域中是公知的，并在以下更完全地进行讨论。简单地说，细胞从哺乳动物(优选人)中分离并用含本发明表达盒的载体进行基因修饰(即，体外转导或转染)。本发明间充质干细胞可被施用给哺乳动物接受者，以提供治疗益处。哺乳动物接受者可为人，和本发明间充质干细胞可相对于接受者为自体的。可选地，细胞可相对于接受者为同种异基因的、同基因的(syngeneic)或异种的。
除了就离体免疫而言使用基于细胞的疫苗之外，本发明也提供了体内免疫以引起针对患者中抗原的免疫应答的组合物和方法。
通常地，如本文所述活化和扩展的细胞可用于治疗和预防无免疫应答的个体中 产生的疾病。因此，本发明提供了治疗癌症的方法，其包括施用给需要其的对象治疗有效量的本发明间充质干细胞。
本发明的间充质干细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分诸如一些细胞因子或细胞群结合施用。简单地说，本发明的药物组合物或制剂可包括如本文所述的间充质干细胞，与一种或多种药学或生理学上可接受载体、稀释剂或赋形剂组合。
本发明的药物组合物可以以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由这样的因素确定，如患者的病症、和患者疾病的类型和严重度——尽管适当的剂量可由临床试验确定。
当指出“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量”或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。可通常指出：包括本文描述的间充质干细胞的药物组合物可以以10 4至10 9个细胞/kg体重的剂量，优选10 5至10 6个细胞/kg体重的剂量(包括那些范围内的所有整数值)施用。间充质干细胞组合物也可以以这些剂量多次施用。细胞可通过使用免疫疗法中公知的注入技术(见例如Rosenberg等，NewEng.J.of Med.319:1676，1988)施用。对于具体患者的最佳剂量和治疗方案可通过监测患者的疾病迹象并因此调节治疗由医学领域技术人员容易地确定。
对象组合物的施用可以以任何方便的方式进行，包括通过喷雾法、注射、吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内(i.v.)注射或腹膜内施用给患者。在一个实施方式中，本发明的T细胞组合物通过皮内或皮下注射被施用给患者。在另一个实施方式中，本发明的T细胞组合物优选通过i.v.注射施用。间充质干细胞的组合物可被直接注入肿瘤，淋巴结或感染位置。
在本发明的某些实施方式中，利用本文描述的方法或本领域已知的其他将间充质干细胞扩展至治疗性水平的方法活化和扩展的细胞，与任何数量的有关治疗形式结合(例如，之前、同时或之后)施用给患者，所述治疗形式包括但不限于用以下试剂进行治疗：所述试剂诸如抗病毒疗法、西多福韦和白细胞介素-2、阿糖胞苷(也已知为ARA-C)或对MS患者的那他珠单抗治疗或对牛皮癣患者的厄法珠单抗治疗或对PML患者的其他治疗。在进一步的实施方式中，本发明的间充质干细胞可与以下结合使用：化疗、辐射、免疫抑制剂，诸如，环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯和FK506，抗体或其他免疫治疗剂。在进一步的实施方式中，本发明的细胞 组合物与骨髓移植、利用化疗剂诸如氟达拉滨、外部光束放射疗法(XRT)、环磷酰胺结合(例如，之前、同时或之后)而施用给患者。例如，在一个实施方式中，对象可经历高剂量化疗的标准治疗，之后进行外周血干细胞移植。在一些实施方式中，在移植后，对象接受本发明的扩展的间充质干细胞的注入。在一个额外的实施方式中，扩展的细胞在外科手术前或外科手术后施用。
施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施用的剂量比例可根据本领域接受的实践实施。通常，每次治疗或每个疗程，可将1×10 3个至1×10 10个本发明间充质干细胞，通过例如静脉回输的方式，施用于患者。
本发明的技术方案，具有如下有益效果：
1.本发明间充质干细胞在体内可从多种组织中获取，在体外易于培养扩增和利用基因工程的方法改造，免疫原性较低。
2.与大多数免疫疗法的相比，本发明方法不依赖于肿瘤浸润淋巴细胞的存在，在临床中也适用于极低或抵制淋巴细胞浸润的肿瘤的治疗。
3.本发明间充质干细胞过表达CCL3、CCL19、CCL21、XCL1、CXCL9、OX40L、4-1BBL、GITRL、CD40L中的一种或多种时，在肿瘤部位能够特异地吸引并激活杀伤肿瘤组织的免疫细胞，具有更高效且副作用低的免疫疗效。
4.本发明间充质干细胞过表达CXCL9和OX40L时，具有相互协同作用，对肿瘤组织杀伤能力显著增强，尤其是结直肠癌细胞。此方法对MHC-I阴性的具有传统免疫疗法(如CAR-T或PD-1/PD-L1抗体)抗性的肿瘤细胞也具有杀伤作用。
5.本发明与其他临床中使用的免疫疗法如CAR-T或PD-1/PD-L1抗体联用时，也可增强这些免疫疗法的功效。本发明的间充质干细胞和PD-1抗体的联合治疗比两者的单独治疗有更显著的肿瘤抑制作用，二者表现出下协同作用。
下面结合具体实施，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
材料和方法
细胞系
CT26细胞是源自BALB/c小鼠的结肠腺癌细胞，MC38是源自C57BL/6小鼠的结肠腺癌细胞系。CT26和MC38细胞分别培养在含有10％胎牛血清和1％青霉素/链霉素的RPMI 1640和DMEM中。
抗体
流式细胞分析使用的抗体来着BD Biosciences,BioLegend或eBioscience公司。用于蛋白免疫印迹分析的抗体包括anti-CXCL9(Abcam)，anti-Myc-tag(Cell Signaling Technology)，anti-OX40L(Abcam)和anti-GAPDH(Abcam)。免疫荧光染色的抗体包括anti-GFP(Abcam)，anti-CD8a(BioLegend)和anti-NKp46(CD335)(BioLegend)。
免疫检查点阻断抗体anti-PD-1(clone RMP1-4)和anti-CTLA-4(clone 9D9)购自Bio X Cell公司。这两种抗体(anti-PD-1:200μg/小鼠；anti-CTLA-4:100μg/小鼠)通过腹腔注射给小鼠。
从小鼠脂肪组织中分离、培养和鉴定间充质干细胞
使用Ⅰ型胶原酶消化小鼠皮下脂肪组织分离出AT-MSC。将细胞培养在含有10％胎牛血清和1％青霉素/链霉素的α-MEM培养基中。细胞贴壁培养三代之后利用流式细胞法鉴定细胞表面标记蛋白的表达。
慢病毒的产生和转导
将cDNA克隆到慢病毒载体中。慢病毒的包装和滴度测定由和元生物技术(上海)股份有限公司完成。在6μg/ml聚凝胺(Sigma-Aldrich)存在下，用感染复数(MOI)为60的慢病毒感染间充质干细胞。
肿瘤细胞增殖实验
根据说明书的步骤，利用CCK-8试剂盒(Dojindo Molecular Technologies)测定肿瘤细胞的增殖。使用酶标仪(Tecan)测量吸光度。
蛋白免疫印迹法(Western Blotting)
收获细胞并用细胞裂解缓冲液(RIPA缓冲液+1％蛋白酶抑制剂)(ThermoFisher Scientific)处理以制备细胞裂解物。利用BCA试剂盒(ThermoFisher Scientific)测定细胞裂解物中的蛋白质浓度。将15至30微克蛋白质上样到5％至15％SDS-PAGE蛋白胶(ThermoFisher Scientific)上，然后转移到PVDF膜(Millipore)上。用TBST缓冲液中配置的5％脱脂牛奶封闭膜，并在4℃下与Myc标签，OX40L，CXCL9和GAPDH的抗体一起孵育过夜。用TBS-T缓冲液洗涤膜，并在室温下与辣根过氧化物酶偶联的二抗一起孵育1小时。用增强的化学发光 试剂盒(Millipore)显影膜并暴露于膜。
酶联免疫吸附实验(ELISA)
收集慢病毒转导的AT-MSC的上清液并保存在-80℃的冰箱中直至测量。使用来自Abcam的ELISA试剂盒根据说明书测试CXCL9的分泌。
小鼠皮下移植瘤模型
分别在8至10周龄BALB/c或C57BL/6小鼠的右下背皮下注射CT26或MC38(0.5或1×10 6/小鼠)细胞。当肿瘤最大直径达到0.5至0.7cm时，将小鼠随机分配至实验组。每只动物尾静脉注射250μl PBS或含有5×10 5AT-MSC的250μl PBS悬液来进行全身给药。每三天用游标卡尺测量肿瘤大小一次，并使用下式计算肿瘤体积：V＝L×W 2/2，其中L和W分别是肿瘤的长径和短径。监测小鼠的肿瘤大小和存活率。当肿瘤体积达到2cm 3或肿瘤变成溃疡或小鼠濒临死亡时，处死小鼠。
流式分析
为了鉴定AT-MSC，使用20μM EDTA消化分离第3至第5代的贴壁细胞，然后用PBS洗涤两次并用抗体染色。
为了分析肿瘤浸润性免疫细胞，将皮下植入的肿瘤解剖并转移到RPMI培养基中，用剪刀剪碎，置于含有0.25mg/ml Liberase TL(Roche)和50μg/ml的DNase I(Sigma-Aldrich)无血清RPMI培养基中，使用gentleMAC Octo Dissociator(Milteniy Biotec)在37℃下消化，并通过40μm细胞过滤器(BD Biosciences)分散。进一步洗涤单细胞，并用抗体染色。通过Zombie Fixable Viability Kit(BioLegend)染色排除死细胞。对于细胞因子的胞内染色，在收获样品之前4-6小时给每只小鼠腹膜内注射0.25mg布雷菲德菌素A(BFA)(Selleck)。在5μg/ml BFA存在下进行表面染色后，用胞内固定和透化缓冲液组(eBioscience)进行胞内染色。进行表面染色后用Foxp3转录因子染色缓冲液(eBioscience)进行核染色。
在BD LSRFortessa细胞分析仪(BD Biosciences)上获得流式数据，并使用FlowJo软件分析。用于流式细胞术的所有抗体均购自BD Biosciences，BioLegend或eBioscience。
AOM/DSS诱导小鼠结直肠癌模型
BALB/c小鼠腹膜内注射AOM(12.5mg/kg体重；Sigma-Aldrich)(14)。1周后，给小鼠喝含有3％DSS(MP Biomedicals)的饮用水7天，然后是引用2周的正常水。DSS继续诱导两个周期，并且从DSS诱导周期的最后一周开始，通过尾静脉注射五 次MSC后处死小鼠。在DSS治疗期间记录体重。从小鼠中取出结肠，用冰PBS冲洗，纵向打开，在室温下在4％多聚甲醛溶液(Sigma-Aldrich)中固定过夜，并石蜡包埋。在固定之前，使用数字卡尺进行尺寸测量。
免疫荧光
用10％正常羊血清封闭组织切片，然后在4℃下与一抗一起孵育过夜，在室温下与二抗孵育1小时。将载玻片安装在具有DAPI(ThermoFisher Scientifitic)的抗褪色封固剂中，并在Nikon Eclipse Ti荧光显微镜下观察。用于免疫荧光的抗体是GFP抗体(Abcam)，CD8a抗体(BioLegend)和NKp46抗体(CD335)(BioLegend)。
统计
所有结果表示为平均值±SEM。通过Student t检验评估差异，或者当比较两组以上的平均值时，通过双向ANOVA，然后进行Bonferroni多重比较检验。用Prism软件(GraphPad)进行数据分析。统计学显着性设定在p<0.05的水平。
研究批准
所有动物程序均经上海交通大学动物护理和使用委员会批准。
实施例1 脂肪间充质干细胞向肿瘤迁移的特性
从小鼠皮下脂肪提取了间充质干细胞，经过流式细胞法检测，这些细胞表达特异的间充质干细胞标志分子，不表达其他细胞类型的标志分子(图1A)，证实了用于实验的脂肪间充质干细胞的纯度。利用慢病毒转染使脂肪间充质干细胞表达GFP，再将5×10 5个细胞通过尾静脉注射到荷瘤小鼠(CT26肠癌皮下瘤)，7天之后对肿瘤及其他脏器进行组织切片的免疫荧光染色，结果表明GFP阳性的间充质干细胞只在肿瘤中驻留，在其他脏器如肝、脾、肾中均未发现(图1B)。利用流式细胞法对肿瘤中GFP阳性的间充质干细胞进行检测，在细胞注入后14天依然能检测到一定数量的细胞(图1C)。这些结果都证明了脂肪间充质干细胞能够特异地向肿瘤部位迁移并驻留较长时间的特性，支持了其作为治疗分子的载体的潜能。
实施例2 CXCL9和OX40L的抗肿瘤特性
为了探寻具有较高功效的免疫激活型治疗分子，选取了有潜在抗肿瘤功能的趋化因子以及细胞因子，将基因克隆到慢病毒载体中包装出携带这些基因的慢病毒。将转导了这些载有基因或者空白对照的慢病毒的CT26肠癌细胞系，进行皮下移植瘤实验检测这些分子的抗肿瘤功效。将具有潜在抗肿瘤功效的趋化因子CCL3 和CXCL9在CT26中过表达(图2A)之后，细胞的体外增殖均不受影响(图2B)，而体内皮下移植瘤的生长受到了显著性的抑制(图2C)，说明这些趋化因子可能是通过体内免疫系统的作用实现了对肿瘤生长的抑制。其中CXCL9的抗肿瘤效果最为显著。通过流式细胞法检测肿瘤中的免疫细胞的构成揭示了CXCL9的确能提高CD8、CD4和NK等抗肿瘤的免疫细胞的浸润(图2D)。
在过表达两种免疫激活型细胞因子IL36β和OX40L的CT26细胞中(图3A&B)，同样发现过表达这两种细胞因子对肿瘤细胞的体外增殖没有影响(图3C)，而且皮下移植瘤的生长有着明显的抑制作用(图3D)，也说明可能是通过体内免疫系统起到了抗肿瘤的功效。其中OX40L表现出了较强的抗肿瘤功效。
实施例3 过表达趋化因子CXCL9与细胞因子OX40L的间充质干细胞的抗肿瘤功效
利用慢病毒感染建立了过表达CXCL9和OX40L的脂肪间充质干细胞体系。通过蛋白免疫印迹与ELISA技术鉴定了CXCL9的成功表达与分泌(图4A&B)，蛋白免疫印迹与流式细胞法证实了OX40L在细胞膜上的成功表达(图4C&D)。之后建立了同时过表达CXCL9和OX40L的MSC(图5A&B)在CT26皮下移植瘤模型中，通过尾静脉每次给小鼠注射5×10 5个间充质干细胞或者PBS，经过每次间隔四天共三次的治疗，同时携带有CXCL9和OX40L的间充质干细胞表现出了最强的抗肿瘤功效(图5C)。流式细胞法分析发现肿瘤中淋巴细胞特别是抗肿瘤的CD8T细胞和NK细胞的比例显著上升(图5D)，说明该疗法有效地激活了抗肿瘤免疫应答。
为了进一步探究间充质干细胞免疫治疗体系的功效，利用AOM/DSS诱导出炎症导致的原位肠癌模型，在第三个DSS处理循环的最后一周开始，对小鼠进行为期4周共5次治疗(图6A)，每次通过尾静脉注入PBS或5×10 5个间充质干细胞，载有CXCL9和OX40L的间充质干细胞使小鼠的肠肿瘤有了极为显著的减少(图6B&C)。免疫荧光染色证明抗肿瘤的CD8T细胞和NK细胞的浸润显著上升(图6D)，同移植瘤模型中观测到的结果一致。
相比较，在AOM/DSS诱导的原位肠癌模型中联合使用PD-1抗体(200μg/小鼠)和CTLA-4(100μg/小鼠)抗体对小鼠进行治疗(图7A)，肿瘤生长并没有显著变化(图7B)。
上述结果均可表明，过表达CXCL9和OX40L的脂肪间充质干细胞在小鼠皮下移植瘤与炎症诱导的原位发生的肠癌模型中均展现出了极为显著的治疗效果。同时携带有CXCL9和OX40L的间充质干细胞，表达CXCL9和OX40L，两者具有协同作用， 抗肿瘤效果明显好于单独表达CXCL9或OX40L的间充质干细胞的抗肿瘤效果。而且在炎症诱导的原位发生的肠癌模型中间充质干细胞治疗的功效明显优于免疫检查点阻断疗法。
实施例4 过表达趋化因子CXCL9与细胞因子OX40L的间充质干细胞提高PD-1阻断疗法的治疗功效
在CT26皮下移植瘤小鼠模型中，给小鼠注射共计四次的过表达CXCL9和OX40L的间充质干细胞(MSC)、PD-1阻断抗体(αPD-1)或者两者(图8A)。
结果如图8B所示，MSC和αPD-1各自单独均能显著抑制肿瘤生长，而MSC和αPD-1联合治疗比两种单独治疗更有效地抑制肿瘤生长，说明MSC疗法对于PD-1阻断疗法有明显的增强作用。
实施例5 过表达趋化因子CXCL9与细胞因子OX40L的间充质干细胞对于MHC-I缺失的肿瘤的抑制作用
利用CRISPR技术敲除MC38中的B2m基因(sgB2m)成功建立了MHC-I缺失的肿瘤模型(图9A)。在此基础上过表达OVA基因使肿瘤细胞表达特异的OVA抗原。相对于对照sgLacZ，MHC-I缺失的MC38-sgBm生长更快并且对OVA多肽疫苗治疗不敏感(图9B)。而过表达CXCL9和OX40L的间充质干细胞依然可以显著地抑制MHC-I缺失的MC38-sgBm肿瘤的生长(图9C)。说明在肿瘤MHC-I缺失的情况下，间充质干细胞治疗比肿瘤疫苗有更大优越性。
实施例6 过表达其他趋化因子或细胞因子的间充质干细胞的抗肿瘤功效
在CT26移植瘤模型中，给小鼠通过尾静脉注射共计六次的表达不同细胞因子或趋化因子的间充质干细胞(5×10 5个/小鼠/次)或PBS，过表达IL15的间充质干细胞没有表现出明显的抗肿瘤功效，而过表达GITRL、4-1BBL、CD40L的间充质干细胞均展现出了高效的抗肿瘤作用(图10A)。同样的，过表达其他几种趋化因子CCL3、CCL19、CCL21、XCL1均表现出了显著的抗肿瘤功效(图10B)。
讨论
免疫疗法使得癌症的治疗产生了革新。尽管一些细胞因子以及免疫检查点阻断剂等在肿瘤的临床治疗中表现出显著的疗效，全身性地系统使用这些药物会非特异地激活免疫系统而影响大多数的器官。为了解决全身性给药带来的副作用，选取了间充质干细胞作为药物载体。研究结果表明了脂肪间充质干细胞可主动向肿瘤部位迁移，并且不在肝、脾、肾等脏器中富集，充分支持了间充质干细胞作为肿瘤治疗药物载体的特异性和安全性，为在肿瘤中局部激活免疫反应避免全身性副作用提 供了有效的手段。
T细胞和NK细胞在肿瘤中的浸润是实体瘤的免疫治疗功效的一个关键决定因素。肿瘤可以通过不同机制限制淋巴细胞的浸入。T细胞浸润较多的肿瘤通常也高表达能够吸引T细胞的趋化因子，包括CCL3、CCL4和CXCL10等。本发明意外地发现，利用间充质干细胞为载体表达CXCL9并运输到肿瘤部位继而吸引抗肿瘤的淋巴细胞，表现出更强的T细胞和NK细胞吸引能力，解决了淋巴细胞难以进入实体瘤的难题。此外，间充质干细胞体系中搭载OX40L也能够更高效地激活T细胞和NK细胞。OX40受体的激活型抗体已经进入临床阶段(ClinicalTrials.gov)，但潜在的问题也是系统用药带来的副作用。OX40的配体OX40L是一种膜蛋白，在间充质干细胞中表达既可以定向运输到肿瘤部位也不会像分泌性细胞因子一样分泌出来而转移到其他部位，减少了分泌带来的扩散，可在肿瘤部位激活已有的或者新迁移来的淋巴细胞。本发明还意外地发现，过表达CXCL9和OX40L的脂肪间充质干细胞在小鼠皮下移植瘤与炎症诱导的原位发生的肠癌模型中均展现出了极为显著的治疗效果。
总地来看，本发明建立的过表达趋化因子CXCL9和细胞因子OX40L的脂肪间充质干细胞治疗体系具有特异靶向肿瘤部位的特性，能够吸引并激活T细胞和NK细胞从而达到理想的抗肿瘤功效。与大多数免疫疗法的相比，该疗法不依赖于肿瘤浸润淋巴细胞的存在，在临床中也适用于极低或抵制淋巴细胞浸润的肿瘤的治疗。脂肪或者脐带间充质干细胞易于提取和培养，易应用于个体化治疗。其低免疫源性也使异体间使用具有可行性。因此，建立的基于间充质干细胞的免疫疗法具有极高的临床转化价值。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种间充质干细胞，其特征在于，所述间充质干细胞表达免疫刺激因子，所述免疫刺激因子选自下组：CCL3、CCL19、CCL21、XCL1、CXCL9、OX40L、4-1BBL、GITRL、CD40L、或其组合。
  2. 如权利要求1所述的间充质干细胞，其特征在于，所述免疫刺激因子包括至少一种趋化因子，所述趋化因子包括：CCL3、CCL19、CCL21、XCL1、CXCL9、或其组合；和/或
    所述免疫刺激因子包括至少一种细胞因子，所述细胞因子包括：OX40L、4-1BBL、GITRL、CD40L、或其组合。
  3. 如权利要求1所述的间充质干细胞，其特征在于，所述免疫刺激因子为CCL3、CCL19、CCL21、XCL1之中的一个或两个和CD40L组合；或者
    所述免疫刺激因子为OX40L、4-1BBL、GITRL之中的一个或两个和CXCL9组合。
  4. 如权利要求1所述的间充质干细胞，其特征在于，所述间充质干细胞包括：脂肪间充质干细胞、脐带间充质干细胞、或其组合。
  5. 一种制备权利要求1所述的间充质干细胞的方法，其特征在于，包括以下步骤：
    (1)提供一待改造的间充质干细胞；和
    (2)将含有编码免疫刺激因子的核酸序列的外源核酸导入到所述待改造的间充质干细胞中，从而获得权利要求1所述的间充质干细胞；
    其中，所述免疫刺激因子选自下组：CCL3、CCL19、CCL21、XCL1、CXCL9、OX40L、4-1BBL、GITRL、CD40L、或其组合。
  6. 一种制剂，其特征在于，所述制剂含有权利要求1所述的间充质干细胞，以及药学上可接受的载体、稀释剂或赋形剂。
  7. 如权利要求1所述的间充质干细胞的用途，其特征在于，用于制备预防和/或治疗癌症或肿瘤的药物或制剂。
  8. 如权利要求7所述的用途，其特征在于，在另一优选例中，所述肿瘤为实体瘤，较佳地为结直肠癌。
  9. 一种药盒，其特征在于，所述药盒含有
    (1)第一容器，以及装于该第一容器中的权利要求1所述的间充质干细胞；和
    (2)第二容器，以及装于该第二容器中的抗肿瘤免疫治疗剂。
  10. 如权利要求9所述的药盒，其特征在于，所述抗肿瘤免疫治疗剂为免疫检查点抗体。
PCT/CN2020/102473 2020-07-16 2020-07-16 间充质干细胞靶向运输趋化因子和细胞因子的免疫疗法 WO2022011651A1 (zh)

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