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WO2016112849A1 - Nouvelle utilisation de l'interféron de type i et cellule souche mésenchymateuse dans la préparation d'un médicament antinéoplasique - Google Patents

Nouvelle utilisation de l'interféron de type i et cellule souche mésenchymateuse dans la préparation d'un médicament antinéoplasique Download PDF

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WO2016112849A1
WO2016112849A1 PCT/CN2016/070751 CN2016070751W WO2016112849A1 WO 2016112849 A1 WO2016112849 A1 WO 2016112849A1 CN 2016070751 W CN2016070751 W CN 2016070751W WO 2016112849 A1 WO2016112849 A1 WO 2016112849A1
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mscs
mesenchymal stem
type
interferon
ifnα
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时玉舫
陈箐
王莹
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Shanghai Institutes for Biological Sciences SIBS of CAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention belongs to the field of immunology, and more particularly, the present invention relates to a novel use of type I interferons and mesenchymal stem cells in the preparation of antitumor drugs.
  • MSCs Mesenchymal Stem Cells
  • the immunosuppressive effect of MSCs is the stimulation of inflammatory factors.
  • the core molecules that mediate the immunosuppressive effects of MSCs are species-differentiated, inducible nitric oxide synthase (iNOS) in mouse MSCs, and indoleamine-2,3 in human MSCs. - Dioxygenase.
  • iNOS inducible nitric oxide synthase
  • TNF ⁇ or IL-1 of which IFN ⁇ is essential.
  • interferon-type I interferon (IFN ⁇ and IFN ⁇ ) and type II interferon (IFN ⁇ ) and TNF ⁇ synergistically induce mouse macrophages to express iNOS.
  • type I interferon IFN ⁇ or IFN ⁇ is not a substitute for IFN ⁇ and TNF ⁇ to induce iNOS expression.
  • Interferon IFN
  • IFN Interferon
  • IFN can induce cell resistance to viral infection. It is the most important anti-viral infection and anti-tumor biological product by interfering with viral gene transcription or translation of viral protein components to prevent or limit viral infection. However, in the clinical application, type I interferon involves the problem of large side effects and short half-life, which limits its application value.
  • the object of the present invention is to provide a novel use of type I interferons and mesenchymal stem cells in the preparation of antitumor drugs.
  • a mesenchymal stem cell which is a type I interferon-pretreated mesenchymal stem cell or a mesenchymal stem cell overexpressing type I interferon.
  • the pre-processing comprises:
  • the type I interferon is mixed with the mesenchymal stem cells such that the final concentration of the type I interferon is 200-10000 U/ml; preferably 300-8000 U/ml; more preferably 500-5000 U/ml; for example 800U /ml, 1000U/ml, 15000U/ml, 2500U/ml.
  • the type I interferon-overexpressing mesenchymal stem cells are transformed with a polynucleotide encoding a type I interferon.
  • the type I interferon is selected from the group consisting of: IFN ⁇ or IFN ⁇ ; or the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells, or umbilical cord, fat, placenta, gums, etc. Mesenchymal stem cells.
  • the type I interferon is IFN ⁇ , and the amino acid sequence thereof is shown in GenBank Accession No. NP_034633.2.
  • the type I interferon is IFN ⁇ and the amino acid sequence thereof is shown in GenBank Accession No. AAA37891.1.
  • mesenchymal stem cell for the preparation of a medicament for inhibiting tumors.
  • the tumor is melanoma.
  • a pharmaceutical composition for inhibiting a tumor comprising any of the mesenchymal stem cells described above is provided.
  • a type I interferon for the preparation of a composition for increasing the expression of MHC I in a mesenchymal stem cell; or for the preparation of a reduced immunosuppressive ability of a mesenchymal stem cell (ie, a transfer chamber)
  • a mesenchymal stem cell ie, a transfer chamber
  • a type I interferon for preparing a composition for inhibiting DNA binding ability of STAT1 in mesenchymal stem cells; or for reducing inducible nitric oxide synthesis in mesenchymal stem cells
  • a composition for the expression of an enzyme for the expression of an enzyme.
  • the use of any of the foregoing, wherein the type I interferon is selected from the group consisting of: IFN ⁇ or IFN ⁇ .
  • IFN ⁇ alone or in combination with TNF ⁇ does not stimulate nitric oxide production in MSCs.
  • the content of nitric oxide in the Qing; Control is PBS.
  • the figure in the figure is means ⁇ SD.
  • IFN ⁇ can attenuate the immunosuppressive effects of MSCs.
  • (A) MSCs were co-cultured with CD3 antibody-activated splenocytes and treated with L-NMMA (1 mM) or IFN ⁇ (2500 U/ml) for 48 hours.
  • Nitric oxide in the culture supernatant was determined by the Greiss method, and the level of cell proliferation was measured by 3 H-TdR incorporation. Control is the PBS treatment group.
  • (B) MSCs of different densities were co-cultured with CD3 antibody-activated splenocytes (Spl) and treated with IFN ⁇ (2500 U/ml) or IFN ⁇ (2500 U/ml) for 48 hours.
  • the level of cell proliferation was measured by 3 H-TdR incorporation.
  • the figure in the figure is means ⁇ SD. Data are 1 representative result from 2 independent replicate experiments. Control is the PBS treatment group.
  • IFN ⁇ inhibits IFN ⁇ and TNF ⁇ -mediated iNOS production in MSCs.
  • AC and MSCs were stimulated with various cytokine combinations of IFN ⁇ (10 ng/ml), TNF ⁇ (10 ng/ml) or IFN ⁇ (2500 U/ml) for 24 hours, and the mRNA (A) and protein expression levels of iNOS were detected (B left). And the content of nitric oxide in the supernatant (B right); Control is the PBS treatment group.
  • the figure in the figure is means ⁇ SD. Data are 1 representative result from at least 3 independent replicate experiments.
  • IFN ⁇ does not affect the expression of most of the cytokines, chemokines, and the like detected by the present inventors.
  • MSCs were treated with IFN ⁇ (10 ng/ml) and TNF ⁇ (10 ng/ml) or IFN ⁇ (10 ng/ml), TNF ⁇ (10 ng/ml) and IFN ⁇ (2500 U/ml) for 24 hours.
  • the supernatant was detected by Bio-Plex; Control was PBS treatment group.
  • the figure in the figure is means ⁇ SD. Data are 1 representative result in at least 2 independent replicate experiments.
  • the abscissa below each column map applies to all graphs in the column as the abscissa.
  • (A) MSCs were treated with IFNy (10 ng/ml) and TNF ⁇ (10 ng/ml) or IFN ⁇ (10 ng/ml), TNF ⁇ (10 ng/ml) and IFN ⁇ (2500 U/ml) for 24 hours.
  • Total RNA collected was detected using Affymetrix mouse 4302.0 expression profile chip; Control was treated with PBS. The ordinate on the left is applied to all the maps in the row as the ordinate.
  • (B) MSCs were treated with IFN ⁇ (10 ng/ml) and TNF ⁇ (10 ng/ml) or IFN ⁇ (10 ng/ml), TNF ⁇ (10 ng/ml) and IFN ⁇ (2500 U/ml), and IFN ⁇ was detected by flow cytometry.
  • the level of expression of the body The values in the figure are means ⁇ SEM; Control is the PBS treatment group.
  • IFN ⁇ inhibits the binding activity of STAT1 in MSCs.
  • MSCs were harvested after treatment with various cytokines (TNF ⁇ (10 ng/ml), IFN ⁇ (10 ng/ml), IFN ⁇ (2500 U/ml) for 24 hours to collect total protein.
  • STAT1 and p65 were incubated and enriched with agarose microbeads ligated to their respective binding site oligonucleotide sequences, and then detected by western blotting.
  • C MSCs were treated with a combination of various cytokines (TNF ⁇ (10 ng/ml), IFN ⁇ (10 ng/ml), IFN ⁇ (2500 U/ml), and mRNA levels of IL-6 and iNOS were measured at different time points.
  • TNF ⁇ 10 ng/ml
  • IFN ⁇ 10 ng/ml
  • IFN ⁇ 2500 U/ml
  • mRNA levels of IL-6 and iNOS were measured at different time points.
  • the values in the figure are means ⁇ SEM. Data are 1 representative result from 3 independent replicates.
  • B16-F0 melanoma cells (1 ⁇ 10 6 /25 ⁇ l) were co-injected with IFN ⁇ (0.25 ⁇ g/g mouse body weight) treated MSCs, MSC-GFP or MSC-IFN ⁇ (1 ⁇ 10 6 /25 ⁇ l).
  • IFN ⁇ (0.25 ⁇ g/g mouse body weight) treated MSCs, MSC-GFP or MSC-IFN ⁇ (1 ⁇ 10 6 /25 ⁇ l).
  • MSCs were pretreated with IFN ⁇ (2500 U/ml) for 24 hours, and then co-injected into the leg muscles of C57BL/6 mice by the method of A with B16-F0 melanoma cells.
  • MSCs (2 ⁇ 10 6 ) pretreated with IFN ⁇ for 24 hours were injected into the tumor site every 3 days. After 14 days, the tumor was stripped from the legs of the sacrificed mice and weighed. The values in the figure are means ⁇ SEM. Data are 1 representative result from 2 independent replicate experiments.
  • type I interferon can inhibit the growth of tumor by inhibiting the expression of iNOS by mesenchymal stem cells (MSCs).
  • MSCs mesenchymal stem cells
  • type I interferons have signal transduction in MSCs. The results showed that type I interferon could up-regulate the expression of MHC I on MSCs, and the effect was not significantly different from that of type II interferon. However, type I interferons can eliminate the immunosuppressive effects of MSCs. In view of the same regulatory effects of IFN ⁇ and IFN ⁇ on MSCs, IFN ⁇ was used in subsequent experiments. Analysis of the signaling pathway revealed that IFN ⁇ did not affect IFN ⁇ receptor expression, phosphorylation of STAT1 and NF- ⁇ B, and their entry into the nucleus.
  • Luciferase assays and specific oligonucleotide binding experiments indicate that IFN ⁇ inhibits the binding capacity of STAT1, but not NF- ⁇ B. Therefore, IFN ⁇ reduces the expression of iNOS by inhibiting the binding ability of STAT1, thereby reducing the immunosuppressive ability of MSCs.
  • the exertion of immunosuppression of MSCs is also dependent on the high level of chemokines expressed by them. With chemokines, MSCs can recruit immune cells such as T cells to their surroundings, thereby exerting their powerful immunosuppressive effects.
  • microbead-based multi-channel cytokine results show that IFN ⁇ does not affect the expression of chemokines in MSCs.
  • IFNs on MSCs are mainly regulated by the regulation of iNOS expression, but there is no expression of chemokines. Significantly affected. More importantly, the inventors demonstrated in vivo tumor growth experiments that IFN ⁇ pretreated MSCs can inhibit the development of B16-F0 melanoma by down-regulating the expression of nitric oxide.
  • the present invention provides an MSCs which are pre-treated with type I interferon MSCs, or MSCs that overexpress type I interferons.
  • type I interferon-pretreated MSCs which are simple to prepare, do not require transgenic manipulation, do not involve insertion of foreign genes, and do not present a safety problem when administered.
  • the MSCs are MSCs pretreated with type I interferon; preferably, the pretreatment comprises: mixing type I interferons with mesenchymal stem cells to achieve a final concentration of type I interferon Can be 200-10000U/ml.
  • the type I interferon is a known protein (polypeptide), which may be a recombinant polypeptide or a synthetic polypeptide.
  • the type I interferon is IFN ⁇ , which may have the amino acid sequence shown by GenBank Accession No. NP_996753.1.
  • the nucleotide sequence of the polynucleotide encoding IFN ⁇ may be GenBank Accession No. NC_000070.6.
  • the type I interferon is IFN ⁇ , which may have the amino acid sequence set forth in GenBank Accession No. AAA37891.1.
  • Type I interferon variants or biologically active fragments formed by substitution, deletion or addition of one or more amino acid residues but retaining the same function as the type I interferon used in the examples of the invention are also included in the present invention.
  • Type I interferon variants or biologically active fragments include a replacement sequence for a portion of a conserved amino acid that does not affect its activity or retains its partial activity. Proper replacement of amino acids is a technique well known in the art that can be readily implemented and ensures that the biological activity of the resulting molecule is not altered. These techniques have taught one in the art that, in general, altering a single amino acid in a non-essential region of a polypeptide does not substantially alter biological activity. See Watson et al, Molecular Biology of The Gene, Fourth Edition, 1987, The Benjamin/Cummings Pub. Co. P224.
  • any biologically active fragment of type I interferon can be used in the present invention.
  • the meaning of the biologically active fragment of type I interferon refers to a polypeptide which still retains all or part of the function of the full-length type I interferon.
  • the biologically active fragment retains at least 50% of the activity of the full length type I interferon.
  • the active fragment is capable of maintaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% activity of the full length type I interferon.
  • Type I interferons modified or modified may also be employed in the present invention, for example, Type I interferons modified or modified to promote their half-life, effectiveness, metabolism, and/or potency of the protein. That is, any variation that does not affect the biological activity of the type I interferon can be used in the present invention.
  • a polynucleotide sequence encoding an overexpressed type I interferon can be inserted into a recombinant expression vector to transform MSCs.
  • Any plasmid and vector can be used in the present invention as long as it can be replicated and stabilized within the MSCs.
  • An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing a type I interferon polynucleotide sequence and a suitable transcriptional/translational control signal. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • the transformation vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the tumor may be various benign or malignant tumors, such as melanoma, liver cancer, breast cancer, gastric cancer, prostate cancer, lung cancer, brain tumor, ovarian cancer, bone tumor, colon, thyroid tumor, mediastinal tumor, small intestine tumor, kidney Tumor, adrenal tumor, bladder tumor, testicular tumor, malignant lymphoma, multiple myeloma, nervous system tumor, etc.
  • benign or malignant tumors such as melanoma, liver cancer, breast cancer, gastric cancer, prostate cancer, lung cancer, brain tumor, ovarian cancer, bone tumor, colon, thyroid tumor, mediastinal tumor, small intestine tumor, kidney Tumor, adrenal tumor, bladder tumor, testicular tumor, malignant lymphoma, multiple myeloma, nervous system tumor, etc.
  • MSCs have chemotaxis, which is capable of accumulating in tumor regions. Therefore, the interferon-pretreated MSCs or MSCs overexpressing type I interferons can achieve anti-tumor function at the tumor site.
  • type I interferons is also provided for preparing a composition for improving the expression of MHC I in mesenchymal stem cells; or for preparing an immunosuppressive ability for reducing mesenchymal stem cells (ie, mobilization room) a composition in which a mesenchymal stem cell exerts an immune action to enhance an anti-tumor immune response; or a composition for inhibiting DNA binding ability of STAT1 in mesenchymal stem cells; or a preparation for reducing inducible type in mesenchymal stem cells Composition for expression of nitric oxide synthase
  • the present invention also provides a composition
  • a composition comprising an effective amount (e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably 0.0001-10% by weight) of the interferon-pretreated MSCs or Type I interferon-expressing MSCs, and a pharmaceutically acceptable carrier.
  • an effective amount e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably 0.0001-10% by weight
  • a pharmaceutically acceptable carrier e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably 0.0001-10% by weight
  • the cells can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium.
  • the pH is usually from about 5 to about 8, preferably, the pH is from about 6 to about 8.
  • the term “contains” means that the various ingredients can be used together in the mixture or composition of the invention. Therefore, the terms “consisting essentially of” and “consisting of” are encompassed by the term “contains.”
  • the term “effective amount” or “effective amount” refers to an amount that can produce a function or activity on a human and/or animal and that can be accepted by a human and/or animal.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, having a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • Fluorescently labeled flow antibody and isotype control antibody used in flow cytometry were purchased from eBioscience; antibodies for immunoblotting: antibodies against iNOS, STAT1, p-STAT1, p65, p-p65, GAPDH, respectively, were purchased from Cell Signaling Technology the company.
  • RNA extraction kits and reverse transcription kits were purchased from Tiangen biotech; RIPA lysates were purchased from Millipore; Nucleofector kit V was purchased from Amaxa; and the culture broth components used to culture MSCs were all purchased from Gibco.
  • MSCs isolated and purified from mouse bone marrow.
  • the isolated MSCs were cultured in low glucose DMEM medium.
  • the IFN ⁇ protein obtained from PBL assay science for cell processing is used, and the amino acid sequence thereof is shown as the first position in GenBank Accession No. NP_996753.1.
  • the overexpressed IFN ⁇ in MSCs has a polynucleotide sequence as shown in GenBank Accession No. NC_000070.6 or its degenerate sequence.
  • the IFN ⁇ has an amino acid sequence as shown in GenBank Accession No. AAA37891.1.
  • mice were purchased from the Slack Shanghai Laboratory Animal Center of the Chinese Academy of Sciences and were raised in the SPF barrier environment of the Laboratory Animal Science Department of Shanghai Jiao Tong University School of Medicine. In all experiments, experiments were performed using mice of gender and age, and all animal procedures were reviewed by animal ethics.
  • Protein sample treatment Take the sample from -80 ° C, melt on ice, determine the total protein concentration by BCA method, and dilute the total protein of each sample to the same concentration for use; add 3X SDS loading buffer, incubate at 100 ° C After 10 min, cool on ice for 2 min, centrifuge, and mix.
  • 1X10 6 cells were resuspended in 100 ⁇ l of FACS buffer, mixed with fluorescently labeled antibody, and incubated at 4 ° C for 30 min in the dark. After centrifugation, the supernatant was discarded and washed three times with FACS buffer. Finally, the cells were resuspended in 300 ⁇ l of FACS buffer and assayed using a FACS Calibur flow cytometer. Data analysis with FCS Express software.
  • a 2X SIE plasmid containing a specific binding site for STAT1 (see Wu, TR et al. (2002).
  • SHP-2 is a dual-specificity phosphatase involved in Stat1 dephosphorylation at both tyrosine and serine residues in nuclei.
  • J Biol Chem 277, 47572-47580 Transfected into MSCs by Amaxa Nuleofector device, inoculated into 24-well plates and cultured overnight. After treatment with different cytokines, cells were harvested at different time points (6 hr, 12 hr and 24 hr), respectively, and luciferase activity was measured using a luciferase assay kit (Promega).
  • 3 H-methyl-thymidine method First, a certain number of MSCs were inoculated in a 96-well plate overnight. The supernatant was aspirated and 2 ⁇ 10 6 mouse spleen cells were inoculated per well (activated with 1 ⁇ g/ml anti-CD3 and 1 ⁇ g/ml anti-CD28, respectively). After mixing with MSCs for 2-3 days, 1 ⁇ Ci of 3 H-methyl-thymidine was added per 100 ⁇ l of cell culture solution for the last 6 hours. After 6 hours, the plate was placed in a -80 ° C refrigerator, and the cells were repeatedly frozen and thawed twice before the measurement. The cells were transferred to a special filter by vacuum adsorption, scintillation fluid was added, and T cell proliferation was measured using a Wallac Microbeta scintillation counter.
  • RNA extraction Total RNA in cells was extracted with RNAprep pure Cell/Bacteria Kit (TianGen).
  • RNA extracted in the previous step was reverse transcribed into cDNA using TaqMan Reverse Transcription Reagents.
  • c) The cDNA obtained above was stored at -20 ° C or diluted to 100 ⁇ l with sterile ddH 2 O for use.
  • the mouse type I interferon IFN ⁇ coding sequence was amplified from the bone marrow-derived DC cell cDNA by PCR, and the primer sequences were: 5'ACGCTCGAGGCCACCATGGCTAG ACTCTGTGC 3' (SEQ ID NO: 1) and 5'ACGTCTAGATCACTCCTTCTCTTCACT CAG 3 '(SEQ ID NO: 2).
  • the resulting fragment was digested with XhoI and XbaI, and the digested product was inserted into the lentiviral shuttle plasmid pLVX-IRES-zsGreen1 (with GFP, purchased from Clontech Laboratories) to obtain plasmid pLVX-IRES-zsGreen1-mIFN ⁇ .
  • Viral packaging was performed using a three-plasmid system, including pMD2G, pSPAX2, and a shuttle plasmid (pLVX-IRES-zsGreen1 or pLVX-IRES-zsGreen1-mIFN ⁇ ).
  • Three plasmids were transfected into 293FT cells together using Lipofectamine 2000. After 48 hours, the cell culture supernatant was collected and centrifuged at 800 g for 10 min to remove cell debris. The virus was concentrated using an Ultra-15 ultrafiltration tube equipped with an Ultracel-100 membrane and the virus titer was determined.
  • GFP and GFP together with IFN ⁇ -infected MSCs were named MSC-GFP and MSC-IFN ⁇ , respectively.
  • Example 1 IFN ⁇ can not induce MSCs to produce iNOS but can upregulate MHC class I molecules
  • IFN ⁇ nitrogen oxide
  • TNF ⁇ nitrogen oxide
  • IFN ⁇ and IFN ⁇ can synergize with TNF ⁇ to induce mass production of bone marrow-derived macrophages.
  • Raw NO the inventors have found that unlike the effect of IFNy in MSCs, the combination of IFN ⁇ and TNF ⁇ does not induce the production of NO by MSCs (Fig. 1A). Almost all types of cells express type I interferon receptors, and the inventors' gene chip data also indicate that MSCs express IFN ⁇ receptors: IFNAR1 and IFNAR2.
  • IFN ⁇ immunomodulatory effects of MSCs.
  • the present inventors added IFN ⁇ to an immunosuppressive system in which MSCs were co-cultured with activated mouse spleen cells (splenocytes) to study their effects.
  • the present inventors have found that, similar to the iNOS inhibitor L-NMMA, the addition of IFN ⁇ not only abolished the inhibitory effect of MSCs on the activation of spleen cells, but increased the proliferation of spleen cells compared with the control group (Fig. 2A left panel).
  • the inventors examined the amount of NO in the co-culture system and found that IFN ⁇ is similar to L-NMMA, and both inhibited NO production by MSCs (Fig. 2A right panel).
  • IFN ⁇ Another molecule of the type I interferon family, IFN ⁇ , also abolished the immunosuppressive effects of MSCs (Fig. 2B), which also means that the body's anti-tumor immune response can be improved. IFN ⁇ was used in the subsequent examples.
  • Example 3 IFN ⁇ inhibits IFN ⁇ and TNF ⁇ -stimulated MSCs to produce iNOS
  • IFN ⁇ can attenuate the immunosuppressive effects of MSCs and reduce the amount of NO in the co-culture system. Therefore, the inventors hypothesized that IFN ⁇ may inhibit the expression of iNOS or block the function of iNOS, thereby reducing the production of NO.
  • the present inventors stimulated MSCs together with IFN ⁇ or IFN ⁇ and TNF ⁇ , and collected RNA and protein 24 hours later, and detected the expression of iNOS at mRNA and protein levels by real-time quantitative PCR and Western blotting, respectively. The results showed that IFN ⁇ partially inhibited the expression of iNOS at the mRNA level (Fig. 3A), while the inhibition of its protein level was more pronounced (Fig. 3B).
  • MSCs also need to produce a large amount of chemotaxis when exerting immunosuppressive effects.
  • Factor recruiting immune cells to migrate to the vicinity of MSCs, so that NO can play a role in inhibiting cell proliferation in a short distance.
  • IFN ⁇ also have a role in producing chemokines in MSCs?
  • the present inventors co-stimulated MSCs with IFN ⁇ and TNF ⁇ or IFN ⁇ , IFN ⁇ and TNF ⁇ for 24 hr, and collected supernatants, and detected the expression of chemokines using the Bio-Plex suspension chip system. It was found that IFN ⁇ not only significantly inhibited the expression of NO by MSCs but also promoted the expression of IL-6, which had no significant effect on the expression of other chemokines (Fig. 4).
  • IFN ⁇ does not affect the expression of IFN ⁇ receptor on MSCs and the expression, phosphorylation level and nuclear importing ability of STAT1 and p65
  • IFN ⁇ inhibits iNOS expression of MSCs It has been reported in the literature that IFN ⁇ / ⁇ inhibits macrophage activation by down-regulating the expression of IFN ⁇ receptor IFNGRs on the surface of macrophages, making the host susceptible to Listeria monocytogenes.
  • the inventors' chip data analysis showed that the expression of IFN ⁇ receptor IFNGR1/2 was not affected by IFN ⁇ , and as a positive control, interferon could up-regulate the expression of H2-D1 and H2-K1 (Fig. 5A).
  • Example 5 IFN ⁇ reduces iNOS expression in MSCs by inhibiting the binding ability of STAT1
  • IFN ⁇ does not affect the expression of IFN ⁇ receptor and the expression, phosphorylation and nuclear importing ability of STAT1 and p65 on MSCs.
  • activated STAT1 can enter the nucleus and bind to specific recognition sites in the promoter of a specific gene (such as iNOS) to exercise its ability to regulate the transcriptional activity of its gene.
  • the inventors used an oligonucleotide sequence capable of specifically binding to a transcription factor (STAT1: 5'GATCCTTCTGGGAATTCCTAGATC 3' (SEQ ID NO: 3); p65: 5' AGTTG AGGGGACTTTCCCAGG 3' (SEQ ID NO: 4)
  • STAT1 5'GATCCTTCTGGGAATTCCTAGATC 3'
  • p65 5' AGTTG AGGGGACTTTCCCAGG 3'
  • the linked agarose beads precipitate STAT1 or NF- ⁇ B to detect their DNA binding ability, respectively. It was found that the STAT1 binding ability of IFN ⁇ -treated MSCs was significantly decreased, while the binding ability of NF- ⁇ B was not significantly affected (Fig. 6A).
  • MSCs stimulated by IFN ⁇ /TNF ⁇ and IFN ⁇ /TNF ⁇ /IFN ⁇ were collected at different time points, total RNA was extracted, and the expression pattern of iNOS and IL-6 in 24 hr was detected by real-time PCR. Results Consistent with previous bio-plex results, IFN ⁇ inhibited iNOS expression and promoted IL-6 expression (Fig. 6C).
  • Stromal cells in the tumor microenvironment play an important role in the development of tumors. Previous reports have shown that MSCs can be isolated from tumors, and the regulation mechanism of nitric oxide production is very similar to that of bone marrow-derived MSCs. In vitro experiments have shown that tumor stromal cells can suppress immune responses by producing nitric oxide. In vivo experiments have also demonstrated that inhibition of nitric oxide production by tumor stromal cells can significantly alleviate tumor development.
  • this study first used MSCs (MSC-IFN ⁇ ) overexpressing IFN ⁇ , and co-injected it with B16-F0 melanoma cells in mouse leg muscles; injection of PBS+B16-F0, injection of IFN ⁇ +B16-F0, Mice injected with MSC-GFP + B16-F0 served as controls.
  • MSC-IFN ⁇ significantly inhibited the growth of melanoma (Fig. 7A).
  • the inventors In order to rule out the direct inhibition of tumor growth by IFN ⁇ , the inventors also adopted a method of co-injection of MSCs pretreated with IFN ⁇ for 24 hours and B16-F0. The results showed that IFN ⁇ pretreated MSCs still inhibited tumor growth (Fig. 7B). Therefore, the present inventors have shown that type I interferon can inhibit tumor growth by inhibiting the production of nitric oxide in MSCs and affecting its immunosuppressive effect in the tumor microenvironment.

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  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des cellules souches mésenchymateuses (CSM) prétraitées par l'interféron (IFN) de type I ou surexprimant l'IFN de type I ainsi que l'utilisation de CSM dans la préparation d'un médicament antinéoplasique. L'invention concerne également une utilisation de l'IFN de type I dans la préparation d'une composition pour améliorer l'expression du CMH I dans les CSM, diminuer la capacité immunosuppressive des CSM, améliorer la réponse immune antitumorale de l'organisme, inhiber l'aptitude de combinaison à l'ADN de STAT1 dans les CSM et diminuer l'expression d'une oxyde nitrique synthase inductible (iNOS) dans les CSM.
PCT/CN2016/070751 2015-01-16 2016-01-13 Nouvelle utilisation de l'interféron de type i et cellule souche mésenchymateuse dans la préparation d'un médicament antinéoplasique Ceased WO2016112849A1 (fr)

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CN201510023359.5A CN105838671A (zh) 2015-01-16 2015-01-16 I型干扰素及间充质干细胞在制备抗肿瘤药物中的新用途
CN201510023359.5 2015-01-16

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
EP4269567A4 (fr) * 2020-12-23 2025-01-08 SCM Lifescience Co., Ltd. Composition d'amorçage de cellule souche et cellule souche amorcée

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109868259A (zh) * 2019-02-27 2019-06-11 广东美赛尔细胞生物科技有限公司 一种重组间充质干细胞及其用途
CN110819594A (zh) * 2019-11-04 2020-02-21 四川大学 持续过表达IFN-γ的间充质干细胞及其用途

Citations (1)

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WO2014093948A1 (fr) * 2012-12-14 2014-06-19 Rutgers, The State University Of New Jersey Procédés permettant la modulation de l'effet immunorégulateur des cellules souches

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WO2014093948A1 (fr) * 2012-12-14 2014-06-19 Rutgers, The State University Of New Jersey Procédés permettant la modulation de l'effet immunorégulateur des cellules souches

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DJOUAD, F. ET AL.: "Transcriptional Profiles Discriminate Bone Marrow-derived and Synovium-derived Mesenchymal Stem Cells", ARTHRITIS RESEARCH & THERAPT, vol. 7, no. 6, 20 September 2005 (2005-09-20), pages R1304 - R1315, XP021011672, ISSN: 1478-6354 *
STUDENY, M. ET AL.: "Bone Marrow-derived Mesenchymal Stem Cells as Vehicles for Interferon-beta Delivery into Tumors", CANCER RESEARCH, vol. 62, no. 13, 1 July 2002 (2002-07-01), pages 3603 - 3608, ISSN: 0008-5472 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4269567A4 (fr) * 2020-12-23 2025-01-08 SCM Lifescience Co., Ltd. Composition d'amorçage de cellule souche et cellule souche amorcée

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