• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The present application provides a combination product for the treatment of cancer. The combination product comprises (i) a modified mesenchymal stem cell, and (ii) an antigenic substance. In addition, the present application also provides a method for the production of a modified mesenchymal stem cell comprising an intracellular parasite and a kit comprising (a) a mesenchymal stem cell, (b) an antigenic substance and (c) a receptor inhibitor. of type Toll 4 and/or receiver of type Toll 9. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2702618A1
    申请号:ES201731066
    申请日:2017-09-04
    公开日:2019-03-04
    发明作者:Castro Javier Garcia；Goyanes Teresa Cejalvo；Barrios Ana Judiht Perisé；Stefano Gambera；Molina Alvaro Morales
    申请人:Instituto de Salud Carlos III；
    IPC主号:
    专利说明:

    [0001]
    [0002] Combination product comprising a modified mesenchymal stem cell and an antigenic substance
    [0003]
    [0004] Technical field
    [0005]
    [0006] The present application relates to a combination product comprising a modified mesenchymal stem cell and an antigenic substance. The combination product can be used as a medicament, in particular, as a medicament for the treatment of cancer. The present application also relates to a method for producing a modified mesenchymal stem cell comprising an intracellular parasite and a kit comprising a mesenchymal stem cell, an antigenic substance and an inhibitor of the Toll-like receptor 4 (TLR4) and / or receiver type Toll 9 (TLR9).
    [0007]
    [0008] Previous technique
    [0009]
    [0010] Oncolytic virotherapy is an emerging treatment modality that uses replication-competent viruses to attack cancer cells. The main objective of this type of therapy is to use an oncolytic virus that is capable of infecting and eliminating cancer cells without causing damage to healthy tissue. Although the oncolytic activity of the virus is important, another important issue is the immune response that therapy can exert on a patient (Russell et al, 2012).
    [0011]
    [0012] Although interest in virotherapy has increased over the years (Litchy et al , 2014), the idea of using a virus to treat cancer is already 50 years old. The observation that the administration of certain viruses delayed the growth of tumors has already intrigued scientists 50 years ago (Kelly and Russell, 2007, Asada, 1974). However, in most cases the virotherapy remained ineffective because the virus was cleared by the immune system or the therapy produced unwanted side effects.
    [0013]
    [0014] These problems still limit the use of virotherapy today. The patient's own immune response to the virus can cause the virus to be eliminated and its therapeutic effects after the first dose. In addition, the inability of the virus to reach micrometastasis after systemic administration and clearance of the virus by the liver and spleen (Underhill and Ozinsky, 2002) also reduce the effectiveness of the virus in the treatment of metastases.
    [0015]
    [0016] The solution to the above limitations is based on the use of a carrier. The carrier must transport the virus to the tumor site while camouflaging the oncolytic virus. By means of this "Trojan Horse" strategy, it can be hidden from the oncolytic virus of the host immune system and the half-life of the oncolytic virus can be increased, and the use of cells that migrate to the tumor microenvironment increases the viral load in the tumor sites. relevant after the systemic administration of the carrier and the virus.
    [0017]
    [0018] It has been shown that mesenchymal stem cells (MSCs) migrate to tumors and sites of inflammation (Ling et al, 2010, Murphy et al, 2013, Ramírez et al, 2015). Therefore, MSCs have been implemented in cancer therapies. In one case, MSC has been used as a carrier for an oncolytic measles virus (ClinicalTrials.gov ID: NCT02068794) and in another case MSC has been used as a carrier for an oncolytic adenovirus (ClinicalTrials.gov ID: NCT01844661). "Celyvir" therapy comprises the use of mesenchymal stem cells as carriers for an engineered adenovirus and has been used to treat patients suffering from tumors that do not respond to treatment (Melen et al, 2016).
    [0019]
    [0020] Currently there is a need for a combination product that can be used to treat different types of cancer. An objective of the present application is to provide a combination product that can be used to treat different types of cancer.
    [0021]
    [0022] Bibliography
    [0023]
    [0024] Asada, T (1974). Treatment of human cancer with mumps virus. Cancer 34, 1907-1928.
    [0025]
    [0026] Kelly, EJ and Russell SJ (2007). History of oncolytic viruses: genesis to genetic engineering. Mol Ther 15 (4), 651-659.
    [0027]
    [0028] Ling, X, Marini F, Konopleva M, Schober W, Shi Y, Burks J, Clise-Dwyer K, Wang RY, Zhang W, Yuan X, Lu H, Caldwell L, and Andreeff M (2010). Mesenchymal stem cells overexpressing IFN-p inhibit breast cancer growth and metastases through Stat3 signaling in a syngeneic tumor model. Cancer Microenviron 3, 83-95.
    [0029] Litchy BD, Breitbach CJ, Stojdl DF and Bell JC (2014). Going viral with cancer immunotherapy. Nat Rev Cancer 14 (8), 559-567.
    [0030]
    [0031] Melen GJ, Franco-Luzon L, Ruano D, Gonzalez-Murillo A, Alfranca A, Casco F, Lassaletta A, Alonso M, Madero L, Alemany R, García-Castro J and Ramírez M (2016). Influence of carrier cells on the clinical outcome of children with neuroblastoma treated with high dose of oncolytic adenovirus delivered in mesenchymal stem cells. Cancer Lett 371, 161-170.
    [0032]
    [0033] Murphy MB, Moncivais K, and Caplan AI (2013). Mesenchymal stem cells: environmentally responsible therapeutics for regenerative medicine. Exp Mol Med 45: e54.
    [0034]
    [0035] Ramírez M, García-Castro J, Melen Gj, González-Murillo A and Franco-Luzón L (2015). Patient-derived mesenchymal stem cells as delivery vehicles for oncolytic virotherapy: novel state-of-the-art technology. Oncolytic Virotherapy 4, 149-155.
    [0036]
    [0037] Russell, SJ, Peng KW and Bell JC (2012). Oncolytic virotherapy. Nat Biotech 30 (7), 658 670.
    [0038]
    [0039] Underhill, DM and Ozinsky, A (2002). Phagocytosis of microbes: complexity in action. Ann Rev Immunology 20, 825-852.
    [0040]
    [0041] Figures
    [0042]
    [0043] Figure 1: Characterization of murine MSCs derived from adipose tissue (mMSC). Cultured mMSCs are characterized by a fibroblast-like morphology. The microscope image corresponds to mMSC BL6.
    [0044]
    [0045] Figure 2: Activation of the NF-kB pathway B. a) Murine MSCs (mMSC) were transduced with a lentivirus system to detect the activation of the NF-kB pathway by expression of luciferase after infection with ICOVIR -5. The darkest shades of gray correspond to Celyvir (infected cells). The expression of luciferase in TLR4 '/ _ cells was significantly higher after infection with ICOVIR-5. b) The ratio of luciferase expression in infected cells (Celyvir) to uninfected cells (mMSC) shows that the NF-kB pathway was activated in all cell types tested after infection with ICOVIR-5 . The expression ratio of the NF-kB pathway in TLR4 '/ _ cells after infection with ICOVIR-5 was significantly higher compared to BL6 and BL10 cells. It was considered that P <0.05 (*) and P <0.01 (**) were statistically significant.
    [0046]
    [0047] Figure 3: Detection of phosphorylated Akt and Jun. A western blot was performed using antibodies specific for pAkt and pJun in infected cells (Celyvir) 24 hours after infection and in uninfected cells (mMSC). Actin was used as charge control.
    [0048]
    [0049] Figure 4: Migration of infected and uninfected mMSCs to tumors derived from the CMT64 cell line. a) Microscope image of infected cells (Celyvir) and uninfected cells (mMSC) migrated, the field of view was 10X. b) Graph showing the number of Celyvir and mMSC that migrated to the CMT 64 tumor cells. The darker shades of gray correspond to Celyvir (infected cells). c) The proportion of cells migrated in Celyvir with respect to mMSC shows that migration was similar in all cell types tested after infection with ICOVIR-5. It was considered that P <0.05 (*) was statistically significant.
    [0050]
    [0051] Figure 5: Development of experiment to test the efficacy of treatments with Celyvir. a) Chronogram of the experiments performed in vivo. C57BL / 6J mice were inoculated with tumors derived from CMT64-Luc and treated every 5 days with Celyvir. After the fourth dose, treatment with Celyvir was stopped and the development of the tumor was monitored. b) In vivo visualization of CMT64-Luc tumors to establish homogeneous groups. The images are of 6 mice at random and serve as examples.
    [0052]
    [0053] Figure 6: Tumor development during treatment with low doses of Celyvir. a) Graph showing the increase in tumor volume over time. Celyvir using mMSC TLR4- / "and TLR9- /" as carriers slowed tumor growth compared to the other groups. b) Graph showing the increase in the volume of individual treated tumors over time in comparison with the mean tumor volume of the PBS group (dark blue line). c) Graph that shows the final volume of the tumors. The mMSC TLR4- / "and TLR9- /" reduced the final size of the tumor. d) Graph showing the final weight of the tumors. The weight of the tumors correlated with their volume.
    [0054]
    [0055] Figure 7: Development of the tumor during treatment with high doses of Celyvir. Graph showing the increase in tumor volume over time. The graph shows a 59.3% reduction in tumor volume in the group treated with Celyvir TLR4 - / - compared to the group administered with PBS. b) Graph showing the increase in the volume of individual treated tumors over time compared to the mean tumor volume of the PBS group (dark blue line).
    [0056]
    [0057] Figure 8: Analysis of the population of immune cells that infiltrated the tumor using flow cytometry. a) Percentage of population of CD45 + cells that infiltrated the tumor. b) Percentage of population of CD3 + cells that infiltrated the tumor. c) Percentage of population of CD4 + / CD8 + cells that infiltrated the tumor. The darker shades of gray correspond to CD4 + cells. d) Proportion of CD4 + / CD8 + cells that infiltrated the tumor. e) Percentage of population of NK cells that infiltrated the tumor. f) Percentage of population of dendritic cells that infiltrated the tumor. g) Percentage of population of macrophages (M1 is proinflammatory, M2 is anti-inflammatory) that infiltrated the tumor. The darker shades of gray correspond to M2 cells. h) Percentage of neutrophil population (N1 and N2) that infiltrated the tumor. The darker shades of gray correspond to N2 cells. It was considered that P <0.05 (*) was statistically significant.
    [0058]
    [0059] Figure 9: Tumor histology CMT64-Luc. a) Tumors treated with Celyvir TLR4 - / - had infiltrated lymphocytes in their periphery. b) Infiltrated eosinophils (arrows) were also observed in tumors treated with Celyvir TLR4 - / -. c) This image corresponds to a tumor that was not treated. No similar infiltration of immune cells was observed in this sample or in the rest of the samples studied. All samples were fixed and treated with hematoxylin / eosin stain.
    [0060]
    [0061] Figure 10: Quantification of secreted proinflammatory cytokines. a) Representative expression profile of proinflammatory cytokines in BL10 and TLR4 cells - / - in the basal state (mMSC) and after infection with ICOVIR-5 (Celyvir). In general, the secretion profile of BL10 cells is greater than that of TLR4 - / - cells. b) Semi-quantification of secreted proinflammatory cytokines in BL10 and TLR4 cells - / - in the basal state (mMSC) and after infection with ICOVIR-5 (Celyvir). c) Quantification of CXCL10 expression in BL10 and TLR4 - / - cells by ELISA in a basal state (mMSC) and after infection with ICOVIR-5 (Celyvir). The expression of CXCL10 in TLR4 - / - cells is significantly lower than in BL10 cells. Expression of CXCL10 by ELISA correlated with its semi-quantified expression in the cytokine matrix panel. He considered that P <0.05 (*) and P <0.01 (**) were statistically significant.
    [0062]
    [0063] Summary of the invention
    [0064]
    [0065] The present application provides a combination product, its use as a medicament and its use as a medicament for the treatment of cancer. The combination product comprises (i) a modified mesenchymal stem cell (MSC) in which TLR4 and / or TLR9 are inhibited, and (ii) an antigenic substance. The modified MSC and the antigenic substance can be marketed together or separately. In addition, the modified MSC can act as a carrier of the antigenic substance and the present application also comprises a method of producing modified MSCs comprising an intracellular parasite. The present application also provides a kit comprising an MSC, an antigenic substance and an inhibitor of TLR4 and / or TLR9.
    [0066]
    [0067] Detailed description of the invention
    [0068]
    [0069] Definitions
    [0070]
    [0071] The term "combination product" may refer to (i) a product composed of two or more regulated components that are combined or mixed in a physical, chemical or otherwise manner and produced as a single entity, (ii) two or more products separately packaged together in a single package or as a unit and composed of pharmacological and device products, device and biological products, or biological and pharmacological products; (iii) a pharmacological, device or biological product packaged separately according to your plan The proposed research or labeling is intended for use only with an individually specified and approved pharmacological, device or biological product in which both are required to achieve the intended use, indication or effect and in which after approval of the proposed product will need to change the labeling of the approved product, for example, to reflect a change in the intended use, dosage form, concentration, route of administration or significant dose change; or (iv) any separately packaged pharmacological, device or biological product that, according to its proposed labeling, is for use only with another individually specified pharmacological, device or biological research product in which both are required to achieve the intended use, indication or effect.
    [0072] The terms "treatment" and "therapy", as used in the present application, refer to a set of hygienic, pharmacological, surgical and / or physical means used with the intention of curing and / or alleviating a disease and / or symptoms with the objective of remedying the health problem The terms "treatment" and "therapy" include preventive and curative methods, since both are aimed at the maintenance and / or restoration of the health of an individual or animal. Symptoms, illness and disability, the administration of a suitable medication to alleviate and / or cure a health problem should be interpreted as a form of treatment or therapy within the context of this application.
    [0073]
    [0074] The term "therapeutically effective amount" refers to a quantity of matter of a combination product that has a therapeutic effect and that is capable of treating the cancer.
    [0075]
    [0076] The terms "individual", "patient" or "subject" are used interchangeably in the present application and are not intended to be limiting in any way. The "individual", "patient" or "subject" can be of any age, sex and physical condition. The term "animal", as used in the present application, refers to any multicellular eukaryotic heterotrophic being that is not a human.
    [0077]
    [0078] The term "modified" refers to any material that has been altered with respect to its original form. In the present application the term "modified" refers to any alteration that is based on human intervention.
    [0079]
    [0080] The term "mesenchymal stem cell" ("MSC") refers to multipotent progenitor cells derived from the mesoderm. They have the ability to differentiate into cells that make up adipose, bone, cartilage and muscle tissue. The minimum criteria established by the International Cell Therapy Society is that MSCs are positive for CD70, CD90 and CD105, and negative for CD34. They can be found in almost all tissues, preferably bone marrow, peripheral blood, menstrual blood, salivary gland, skin and foreskin, synovial fluid, endometrium, dental tissue, adipose tissue and tissues associated with newborns including placenta, umbilical cord, cord blood umbilical, amniotic fluid and amniotic membrane.
    [0081]
    [0082] The term "Toll-like receptor 4"("TLR4") refers to a transmembrane protein that can recognize lipopolysaccharides and initiate the intracellular route of NF-k B. find more information about this protein in humans in the UniProt database (UniProtKB - 000206, which was accessed on March 23, 2017).
    [0083]
    [0084] The term "Toll-like receptor 9" ("TLR9") refers to a transmembrane protein that can recognize viral and bacterial DNA and initiate the intracellular route of NF-k B. More information regarding this protein in humans can be found in the UniProt database (UniProtKB - Q9NR96, which was accessed on March 23, 2017).
    [0085]
    [0086] The term "Toll-like receptor 4 inhibitor" ("TLR4 inhibitor") refers to any material that can reduce the amount and / or activity of TLR4. This may include small-molecule bioactive inhibitors that inhibit receptor activation and / or their ability to initiate the NF-kB pathway, neutralizing antibodies that provoke the internalization of TLR4 and its degradation or that inhibit the ability of TLR4 to bind to an agonist blocking the extracellular binding site, or the use of siRNA or gene therapy to down regulate the amount of TLR4 present in the cell or to completely eliminate the presence of TLR4 in a cell, ie deactivation.
    [0087]
    [0088] The term "Toll-like receptor 9 inhibitor" ("TLR9 inhibitor") refers to any material that can reduce the amount and / or activity of TLR9. This may include small molecule bioactive inhibitors that inhibit receptor activation and / or their ability to initiate the NF-kB pathway, neutralizing antibodies that cause internalization of TLR9 and its degradation, or that inhibit the ability of TLR9 to bind to an agonist blocking the extracellular binding site, or the use of siRNA or gene therapy to down regulate the amount of TLR9 present in the cell or to completely eliminate the presence of TLR9 in a cell, ie deactivation.
    [0089]
    [0090] The term "antigenic substance" refers to any substance that can elicit an immune response, including immunogenic proteins, DNA, RNA, lipopolysaccharides, and any intracellular parasite that comprises antigenic substances, eg, modified MSC can be modified to express an antigenic protein. and can be modified with an emergency switch that is activated when the modified MSC is in a hypoxic environment such as the environment that is commonly found in solid tumors In an alternative example, the modified MSC can be infected with an intracellular parasite that is released in the tumor site to induce a targeted immune response.
    [0091] The term "intracellular parasite" refers to any microparasite that is capable of growing and replicating within a host cell Examples include: bacteria, viruses, protozoa and fungi.
    [0092]
    [0093] The term "oncolytic virus" refers to any virus that preferentially infects and destroys cancer cells Examples include: adenovirus, reovirus, measles virus, herpes simplex virus, Newcastle disease virus, vaccinia virus, Coxsackie virus and Seneca Valley virus.
    [0094]
    [0095] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable diluent" means each and every solvent, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, compatible with the pharmaceutical administration. The use of such media and agents for pharmaceutically active principles is well known in the art. Acceptable carriers, excipients or stabilizers are not toxic to the recipient subjects at the dosages and concentrations employed and, without limiting the scope of the present invention, include: additional buffering agents; preservatives; cosolvents; antioxidants, including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers, such as polyesters; salt-forming counterions, such as sodium, polyhydric sugar alcohols; amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid and threonine; sugar alcohols or organic sugars, such as lactitol, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitol (eg, inositol), polyethylene glycol; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha] -monotioglycerol and sodium thiosulfate; low molecular weight proteins, such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; and hydrophilic polymers, such as polyvinylpyrrolidone.
    [0096]
    [0097] The term "pharmaceutically acceptable adjuvant" refers to each and every one of the substances that enhance the body's immune response to an antigen.Non-limiting examples of pharmaceutically acceptable adjuvants are: alum, incomplete Freund's adjuvant, MF59, synthetic analogs of DsRNA such as poly (I: C), bacterial lipopolysaccharides, bacterial flagellin, imidazolquinolines, oligodeoxynucleotides containing specific CpG motifs, fragments of bacterial cell walls such as muramyl dipeptide and Quil-A.
    [0098]
    [0099] The term "chemotherapeutic agent" refers to any drug that can be used to treat a disease In the present application, the term "chemotherapeutic agent" refers to any drug that can be used to treat cancer. Non-limiting examples include: actinomycin, all-trans-retinoic acid, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil , gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vemurafenib, vinblastine, vincristine, vindesine and vinorelbine.
    [0100]
    [0101] The term "cancer" refers to a group of diseases that involve abnormal cell growth with the potential to invade or spread to other parts of the body.Non-limiting examples include: acute granulocytic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, adenocarcinoma, adrenal cancer, anaplastic astrocytoma, angiosarcoma, cancer of the appendix, astrocytoma, basal cell carcinoma, B-cell lymphoma, bile duct cancer, bladder cancer, bone cancer, bone marrow cancer, bowel cancer, brain cancer, brain stem glioma, brain tumor, breast cancer, carcinoid tumors, cervical cancer, cholangiocarcinoma, chondrosarcoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial cancer, ependymoma, epithelioid sarcoma e, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid cancer, gastrointestinal stromal tumors, germ cell tumor, glioblastoma multiforme, glioma, tricholeukemia, head and neck cancer, hemangioendothelioma, Hodgkin's lymphoma, hypopharyngeal cancer, infiltrating ductal carcinoma, infiltrating lobular carcinoma, inflammatory breast cancer, intestinal cancer, intrahepatic bile duct cancer, cancer invasive / invasive breast cancer, islet cell cancer, jaw cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, leptomeningeal metastasis, leukemia, lip cancer, liposarcoma, liver cancer, lobular carcinoma in situ, low grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, mesenchyme, mesothelioma, metastatic breast cancer, metastatic melanoma, squamous neck cancer metastatic, mixed gliomas, mouth cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, fungal mycosis, myelodysplastic syndrome, nasal cavity cancer, nasopharyngeal cancer, neck cancer, neuroblastoma, neuroendocrine tumors, non-Hodgkin's lymphoma, cancer non-small cell lung, oat grain cell cancer, ocular cancer, ocular melanoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteogenic sarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian cancer germ cells of the ovaries, primary peritoneal carcinoma of the ovaries, tumor of the stroma of sexual cords of the ovary, Paget's disease, pancreatic cancer, papillary carcinoma, paranasal sinus cancer, parathyroid gland cancer, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma , tumor of the pineal region, pituitary cancer, primary lymphoma of the central nervous system, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis cancer, rhabdomyosarcoma, cancer of the salivary gland, sarcoma, bone sarcoma, sarcoma soft tissue, uterine sarcoma, breast cancer, skin cancer, small cell lung cancer, small bowel cancer, spinal cancer, cancer of the spine, spinal cord cancer, spinal tumor, squamous cell carcinoma, cancer of stomach, synovial sarcoma, T-cell lymphoma, testicular cancer, throat cancer, thymoma / carcinoma of the thymus, thyroid cancer, tongue cancer, tonsil cancer, transitional cell cancer, triple negative breast cancer, tubal cancer, tubular carcinoma, urethral cancer, uterine adenocarcinoma, uterine cancer, vaginal cancer and vulvar cancer.
    [0102]
    [0103] The term "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or liquid zones.Non-limiting examples include adenocarcinoma, adrenal cancer, anaplastic astrocytoma, angiosarcoma, appendix cancer, astrocytoma, basal cell carcinoma, lymphoma. of B cells, bile duct cancer, bladder cancer, bone cancer, bone marrow cancer, bowel cancer, brain cancer, brainstem glioma, brain tumor, breast cancer, carcinoid tumors, cervical cancer, cholangiocarcinoma, chondrosarcoma, colon cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial cancer, ependymoma, epithelioid sarcoma, esophageal cancer, Ewing sarcoma, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid cancer, stromal tumors gastrointestinal, germ cell tumor, glioblastoma multiforme, glioma, head and neck cancer, hemangioendothelioma, Hodgkin's lymphoma, hypopharyngeal cancer, infiltrating ductal carcinoma, infiltrating lobular carcinoma, inflammatory breast cancer, intestinal cancer, intrahepatic bile duct cancer, cancer invasive / invasive breast cancer, islet cell cancer, jaw cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, leptomeningeal metastasis, lip cancer, liposarcoma, liver cancer, lobular carcinoma in situ, astrocytoma low grade, lung cancer, lymph node cancer, lymphoma, c male breast cancer, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, mesenchyme, mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous neck cancer, mixed gliomas, mouth cancer, mucinous carcinoma , mucosal melanoma, multiple myeloma, fungal mycosis, myelodysplastic syndrome, cancer of the nasal cavity, nasopharyngeal cancer, neck cancer, neuroblastoma, neuroendocrine tumors, non-Hodgkin's lymphoma, non-small cell lung cancer, cell cancer in grain of oats, ocular cancer, ocular melanoma, oligodendroglioma, oral cancer, cancer of the oral cavity, oropharyngeal cancer, osteogenic sarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, germ cell tumor of the ovaries, primary peritoneal carcinoma of the ovaries, tumor of the stroma of the sexual cords of the ovary, Paget's disease, cancer p ancreatic, papillary carcinoma, paranasal sinus cancer, parathyroid gland cancer, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal region tumor, pituitary cancer, Primary lymphoma of the central nervous system, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis cancer, rhabdomyosarcoma, cancer of the salivary gland, sarcoma, bone sarcoma, soft tissue sarcoma, uterine sarcoma, breast cancer, cancer skin cancer, small cell lung cancer, small bowel cancer, spinal cancer, spinal cancer, spinal cord cancer, spinal tumor, squamous cell carcinoma, stomach cancer, synovial sarcoma, T-cell lymphoma, testicular cancer, throat cancer, thymoma / thymic carcinoma, thyroid cancer, tongue cancer, tonsil cancer, cancer of transitional cells, triple negative breast cancer, tubal cancer, tubular carcinoma, urethral cancer, uterine adenocarcinoma, uterine cancer, vaginal cancer and vulvar cancer.
    [0104]
    [0105] The term "chemotherapy" refers to the use of a chemotherapeutic agent to treat a disease. In the present application, the term "chemotherapy" refers to the use of a chemotherapeutic agent to treat cancer.
    [0106]
    [0107] The term "radiotherapy" refers to a therapy that uses ionizing radiation to control or destroy malignant cells and is generally used as part of a cancer treatment.
    [0108]
    [0109] Combination product
    [0110]
    [0111] In a first aspect, the present application provides a combination product comprising: (i) a modified MSC wherein TLR4 and / or TLR9 are inhibited, and (ii) an antigenic substance.
    [0112]
    [0113] In a preferred embodiment, the combination product is a composition. In the composition, the combination product composed of (i) the modified MSC in which TLR4 and / or TLR9 are inhibited and (ii) the antigenic substance are mixed and produced as a single entity. Preferably, the modified MSC in which TLR4 and / or TLR9 are inhibited is a carrier of the antigenic substance.
    [0114]
    [0115] The term "carrier" in the present application can refer to an organism carrying an antigenic substance. In a preferred embodiment, the modified MSC in which TLR4 and / or TLR9 are inhibited is infected, transduced and / or transformed with the antigenic substance.
    [0116]
    [0117] In a preferred embodiment a modified MSC in which TLR4 and / or TLR9 are inhibited and an antigenic substance can be marketed separately or together. If the modified MSC and the antigenic substance are marketed together, they may be marketed in a single vial or in two separate vials packaged in a container and may include instructions on how to combine the modified MSC with the antigenic substance. If the modified MSC and the antigenic substance are sold separately, they are marketed with a label that explains briefly that the two products should be used together and / or with instructions that summarize the use of the two products together.
    [0118] TLR4 can be inhibited by using a TLR4 inhibitor. In a preferred embodiment, TLR4 is inhibited using: (i) siRNA, (ii) gene therapy vectors, preferably gene therapy vectors comprising the CRISPR / Cas9 system, (iii) a bioactive small molecule inhibitor of the Toll-like receptor 4 selected from the group consisting of 3,4,6-triacetate isopropyl-2- (acetylamino) -2-deoxy-aD-glucopyranoside, resatorvid, eritorán and a peptide with the sequence KYSFKLILAEYRRRRRRRRR (SEQ ID NO: 1), and / or (iv) a neutralizing antibody that binds to the extracellular region of TLR4. Preferably, TLR4 is inhibited using: (i) small interfering RNA and / or (ii) vectors for gene therapy, preferably gene therapy vectors comprising the CRISPR / Cas9 system. Most preferably, TLR4 is inhibited using vectors for gene therapy, preferably gene therapy vectors comprising the CRISPR / Cas9 system.
    [0119]
    [0120] TLR9 can be inhibited by using a TLR9 inhibitor. In a preferred embodiment, TLR9 is inhibited using: (i) small interfering RNA, (ii) gene therapy vectors, preferably gene therapy vectors comprising the CRISPR / Cas9 system, (iii) a bioactive small molecule inhibitor of the receptor of Toll type 9 selected from the group consisting of a nucleotide comprising the sequence TTAGGG, ODN 2088, ODN 4084-F, ODN INH-1, ODN-INH-18, ODN A151, G-ODN, 3- [4- ( 6- (3- (dimethylamino) propoxy) benzo [d] oxazol-2-yl) phenoxy] -N, N-dimethylpropan-1-amine, 6- [3- (pyrrolidin-1-yl) propoxy) -2- (4- (3- (pyrrolidin-1-yl) propoxy) phenyl] benzo [d] oxazole, and / or (iv) a neutralizing antibody that binds to the extracellular region of TLR9 Preferably, TLR9 is inhibited using: ( i) small interfering RNA and / or (ii) vectors for gene therapy, preferably gene therapy vectors comprising the CRISPR / Cas9 system.Most preferably, TLR9 is inhibited using vectors for gene therapy, preferably p-vectors. for gene therapy comprising the CRISPR / Cas9 system.
    [0121]
    [0122] The term "small interfering RNA"("siRNA") refers to a class of RNA molecules, usually 20-25 base pairs in length, that can down-regulate the translation of a protein. Methods for designing suitable siRNAs that can downregulate TLR4 and / or TLR9 are known in the art (Birmingham et al, 2007. Nat. Protocol 2 (9): 2068-78).
    [0123] The term "vectors for gene therapy" refers to any DNA molecule used as a vehicle to artificially transport foreign genetic material into another cell, in which it can replicate and / or express itself, in which the genetic material foreign is able to modify the genome of the host cell. Examples of foreign genetic material capable of modifying the host cell genome include genetic material comprising zinc finger nucleases (Porteus and Carroll, 2005. Nat. Biotech, 23 (8): 967-73) and / or the system CRISPR / Cas9 (Ran et al, 2013. Nat. Protocol 8 (11): 2281-308). In a preferred embodiment, the vector for gene therapy comprises the CRISPR / Cas9 system. Methods for designing a suitable CRISPR / Cas9 system that can downregulate TLR4 and / or TLR9 are known in the art (Ran et al, 2013. Nat. Protocol 8 (11): 2281-308; Chari et al, 2015. Nat. Met. 12 (9): 823-6; Aach et al, 2014. CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomes, BioRxiv).
    [0124]
    [0125] The antigenic substance can be any substance that can be produced by the modified MSC, produces an immunogenic response and can be released at the tumor site to produce a localized immunogenic response. In a preferred embodiment, the antigenic substance is an intracellular parasite. Preferably, the intracellular parasite is selected from the group consisting of bacteria, viruses, protozoa and fungi. More preferably, the antigenic substance is an oncolytic virus.
    [0126]
    [0127] The oncolytic virus can be any virus that preferentially infects and destroys cancer cells. In a preferred embodiment, the oncolytic virus is selected from the group consisting of adenovirus, reovirus, measles virus, herpes simplex virus, Newcastle disease virus, vaccinia virus, Coxsackie virus and Seneca valley virus. Preferably, the oncolytic virus is an adenovirus. More preferably, the oncolytic virus is ICOVIR-5.
    [0128]
    [0129] The term "ICOVIR-5" refers to a modified oncolytic adenovirus having an activity that depends on the amount of free E2F in the cell (Cascallo et al, 2007. Mol.Ther.15 (9): 1607-15). A method for producing ICOVIR-5 is known in the art (Cascallo et al, 2007. Mol.Ther.15 (9): 1607-15).
    [0130]
    [0131] In a preferred embodiment, the MSCs are derived from bone marrow, placenta, umbilical cord, amniotic membrane, menstrual blood, peripheral blood, salivary gland, skin and foreskin, synovial fluid, amniotic fluid, endometrium, adipose tissue, umbilical cord blood and / or dental tissue. Preferably, the MSCs derive from adipose tissue and / or bone marrow. More preferably, MSCs are derived from bone marrow. In the state of the art methods for obtaining MSC from the previous tissues are known (Ullah et al. al, 2015. Biosci. Rep. 35 (2): e00191).
    [0132]
    [0133] In a preferred embodiment, the MSC is allogeneic or autologous with respect to the individual suffering from or at risk of suffering from cancer. Preferably, the MSC is autologous.
    [0134]
    [0135] In a second aspect, the present application provides a pharmaceutical composition comprising a composition and a pharmaceutically acceptable carrier or diluent and / or a pharmaceutically acceptable adjuvant, wherein the composition comprises a modified MSC wherein TLR4 and / or TLR9 are inhibited , and (ii) an antigenic substance.
    [0136]
    [0137] TLR4 can be inhibited by using a TLR4 inhibitor. In a preferred embodiment, TLR4 is inhibited using: (i) siRNA, (ii) gene therapy vectors, preferably gene therapy vectors comprising the CRISPR / Cas9 system, (iii) a bioactive small molecule inhibitor of the Toll-like receptor 4 selected from the group consisting of 3,4,6-triacetate isopropyl-2- (acetylamino) -2-deoxy-aD-glucopyranoside, resatorvid, eritorán and a peptide with the sequence KYSFKLILAEYRRRRRRRRR (SEQ ID NO: 1), and / or (iv) a neutralizing antibody that binds to the extracellular region of TLR4. Preferably, TLR4 is inhibited using: (i) small interfering RNA and / or (ii) vectors for gene therapy, preferably gene therapy vectors comprising the CRISPR / Cas9 system. Most preferably, TLR4 is inhibited using vectors for gene therapy, preferably gene therapy vectors comprising the CRISPR / Cas9 system.
    [0138]
    [0139] TLR9 can be inhibited by using a TLR9 inhibitor. In a preferred embodiment, TLR9 is inhibited using: (i) small interfering RNA, (ii) gene therapy vectors, preferably gene therapy vectors comprising the CRISPR / Cas9 system, (iii) a bioactive small molecule inhibitor of the receptor of Toll type 9 selected from the group consisting of a nucleotide comprising the sequence TTAGGG, ODN 2088, ODN 4084-F, ODN INH-1, ODN-INH-18, ODN A151, G-ODN, 3- [4- ( 6- (3- (dimethylamino) propoxy) benzo [d] oxazol-2-yl) phenoxy] -N, N-dimethylpropan-1-amine, 6- [3- (pyrrolidin-1-yl) propoxy) -2- (4- (3- (pyrrolidin-1-yl) propoxy) phenyl] benzo [d] oxazole, and / or (iv) a neutralizing antibody that binds to the extracellular region of TLR9 Preferably, TLR9 is inhibited using: ( i) small interfering RNA and / or (ii) vectors for gene therapy, preferably gene therapy vectors comprising the CRISPR / Cas9 system.Most preferably, TLR9 is inhibited using vectors for gene therapy, preferably p-vectors. for gene therapy that comprise the system CRISPR / Cas9.
    [0140]
    [0141] The antigenic substance can be any substance that can be produced by the modified MSC, produces an immunogenic response and can be released at the tumor site to produce a localized immunogenic response. In a preferred embodiment, the antigenic substance is an intracellular parasite. Preferably, the intracellular parasite is selected from the group consisting of bacteria, viruses, protozoa and fungi. More preferably, the antigenic substance is an oncolytic virus.
    [0142]
    [0143] The oncolytic virus can be any virus that preferentially infects and destroys cancer cells. In a preferred embodiment, the oncolytic virus is selected from the group consisting of adenovirus, reovirus, measles virus, herpes simplex virus, Newcastle disease virus, vaccinia virus, Coxsackie virus and Seneca valley virus. Preferably, the oncolytic virus is an adenovirus. More preferably, the oncolytic virus is ICOVIR-5.
    [0144]
    [0145] In a preferred embodiment, the MSCs are derived from bone marrow, placenta, umbilical cord, amniotic membrane, menstrual blood, peripheral blood, salivary gland, skin and foreskin, synovial fluid, amniotic fluid, endometrium, adipose tissue, umbilical cord blood and / or dental tissue. Preferably, the MSCs derive from adipose tissue and / or bone marrow. More preferably, MSCs are derived from bone marrow. In the state of the art, methods for obtaining MSCs are known from the previous tissues (Ullah et al, 2015. Biosci, Rep. 35 (2): e00191).
    [0146]
    [0147] In a preferred embodiment, the MSC is allogeneic or autologous with respect to the individual suffering from or at risk of suffering from cancer. Preferably, the MSC is autologous.
    [0148]
    [0149] In a preferred embodiment, the pharmaceutical composition may further comprise a chemotherapeutic agent.
    [0150]
    [0151] For use as a medicine or for use in the treatment of cancer
    [0152]
    [0153] In a third aspect, the combination product and / or the pharmaceutical composition of the present application can be used as a medicament. In a fourth aspect, the combination product and / or the pharmaceutical composition of the present application can be used in the treatment of a solid tumor.
    [0154] In a preferred embodiment, the solid tumor is selected from the group consisting of adenocarcinoma, adrenal cancer, anaplastic astrocytoma, angiosarcoma, appendix cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, bile duct cancer, bladder cancer, bone cancer, bone marrow cancer, bowel cancer, brain cancer, brainstem glioma, brain tumor, breast cancer, carcinoid tumors, cervical cancer, cholangiocarcinoma, chondrosarcoma, colon cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial cancer, ependymoma, epithelioid sarcoma, esophageal cancer, Ewing sarcoma, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, fibrosarcoma, cancer of the gallbladder, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid cancer, tumore Gastrointestinal stroma, germ cell tumor, glioblastoma multiforme, glioma, head and neck cancer, hemangioendothelioma, Hodgkin's lymphoma, hypopharyngeal cancer, infiltrating ductal carcinoma, infiltrating lobular carcinoma, inflammatory breast cancer, intestinal cancer, bile duct cancer intrahepatic, invasive / invasive breast cancer, islet cell cancer, jaw cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, leptomeningeal metastasis, lip cancer, liposarcoma, liver cancer, lobular carcinoma in situ , low grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, mesenchyme, mesothelioma, metastatic breast cancer, melanoma metastatic, metastatic squamous neck cancer, mixed gliomas, cancer of the oca, mucinous carcinoma, mucosal melanoma, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, nasal cavity cancer, nasopharyngeal cancer, neck cancer, neuroblastoma, neuroendocrine tumors, non-Hodgkin's lymphoma, non-small cell lung cancer, cancer of oat grain cells, ocular cancer, ocular melanoma, oligodendroglioma, oral cancer, cancer of the oral cavity, oropharyngeal cancer, osteogenic sarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, germ cell tumor of the ovaries, carcinoma primary ovarian peritoneum, ovarian sex cord stromal tumor, Paget's disease, pancreatic cancer, papillary carcinoma, paranasal sinus cancer, parathyroid gland cancer, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal region tumor, cancer of the hip diaphysis, primary lymphoma of the central nervous system, prostate cancer, rectal cancer, renal cell carcinoma, cancer of renal pelvis, rhabdomyosarcoma, cancer of the salivary gland, sarcoma, bone sarcoma, soft tissue sarcoma, uterine sarcoma, breast cancer, skin cancer, small cell lung cancer, small bowel cancer, spinal cancer, cancer the spine, spinal cord cancer, spinal tumor, squamous cell carcinoma, stomach cancer, synovial sarcoma, T-cell lymphoma, testicular cancer, throat cancer, thymoma / thymic carcinoma, thyroid cancer, tongue cancer Tonsil cancer, transitional cell cancer, triple negative breast cancer, tubal cancer, tubular carcinoma, urethral cancer, uterine adenocarcinoma, uterine cancer, vaginal cancer and vulvar cancer. Preferably, the solid tumor is a brain cancer, lung cancer, kidney cancer, ovarian cancer and / or liver cancer.
    [0155]
    [0156] In a preferred embodiment, the combination product and / or the pharmaceutical composition of the present application can be used in the treatment of brain cancer and / or lung cancer. Preferably, brain cancer is a neuroblastoma and / or lung cancer is a lung carcinoma.
    [0157]
    [0158] In a preferred embodiment, the combination product and / or the pharmaceutical composition of the present application can be used in the treatment of brain cancer. Preferably, brain cancer is a neuroblastoma.
    [0159]
    [0160] In a preferred embodiment, the combination product and / or the pharmaceutical composition of the present application can be used in the treatment of lung cancer. Preferably, lung cancer is a lung carcinoma.
    [0161]
    [0162] In a preferred embodiment, the combination product and / or the pharmaceutical composition of the present application can be used in combination with chemotherapy and / or radiotherapy.
    [0163]
    [0164] Administration
    [0165]
    [0166] The combination product and / or the pharmaceutical composition can be administered to animals and / or humans. Preferably, the combination product and / or the pharmaceutical composition can be administered to a human individual. More preferably, the combination product and / or the pharmaceutical composition can be administered to an individual who is a child. In a preferred embodiment, the combination product is administer to an individual who suffers or is at risk of developing brain cancer, lung cancer, kidney cancer, ovarian cancer and / or liver cancer.
    [0167]
    [0168] In a preferred embodiment, the combination product is administered to prevent the onset of cancer. For example, the combination product can be administered to an animal or individual that is at risk of developing cancer. In a preferred embodiment, the combination product is administered to treat cancer. For example, the combination product can be administered to an animal or individual suffering from cancer.
    [0169]
    [0170] In a preferred embodiment, the combination product is prepared for intravenous, intramuscular, subcutaneous and / or transdermal routes of administration, preferably intravenous routes.
    [0171]
    [0172] In a preferred embodiment, the combination product is administered once a week, month or year. Preferably, the combination product is administered at least once a week.
    [0173]
    [0174] Method for the production of modified MSCs comprising an intracellular parasite
    [0175]
    [0176] In a fifth aspect, the present application provides a method for the production of modified mesenchymal stem cells comprising an intracellular parasite, comprising the steps of:
    [0177]
    [0178] (1) isolate mesenchymal stem cells from an isolated sample;
    [0179]
    [0180] (2) cultivate mesenchymal stem cells;
    [0181]
    [0182] (3) inhibiting the Toll-like receptor 4 and / or the Toll-like receptor 9 of the isolated mesenchymal stem cells; Y
    [0183]
    [0184] (4) infect mesenchymal stem cells with an intracellular parasite.
    [0185]
    [0186] In a preferred embodiment, the method further comprises a step of X-ray irradiation between steps (3) and (4).
    [0187]
    [0188] In a preferred embodiment, MSCs are isolated from bone marrow, placenta, cord umbilical, amniotic membrane, menstrual blood, peripheral blood, salivary gland, skin and foreskin, synovial fluid, amniotic fluid, endometrium, adipose tissue, umbilical cord blood and / or isolated dental tissue. Preferably, the MSCs are isolated from isolated adipose tissue and / or bone marrow. More preferably, the MSCs are isolated from isolated bone marrow. In the state of the art, methods for obtaining MSCs are known from the previous tissues (Ullah et al, 2015. Biosci, Rep. 35 (2): e00191).
    [0189]
    [0190] In a preferred embodiment, the isolated MSCs are autologous or allogeneic. Preferably, the isolated MSCs are autologous. In a preferred embodiment, the MSCs are isolated from an isolated autologous bone marrow tissue, wherein the bone marrow tissue was obtained from the patient suffering from or at risk of suffering from cancer.
    [0191]
    [0192] In a preferred embodiment, the MSCs are cultured in a cell culture plate. Preferably, the medium is Eagle's medium modified by Dulbecco. More preferably, the medium is also supplemented with fetal bovine serum, preferably 10% (w / v). Most preferably, the cells are cultured at 37 ° C in a humidified atmosphere with 5% CO2.
    [0193]
    [0194] TLR4 can be inhibited by using a TLR4 inhibitor. In a preferred embodiment, TLR4 is inhibited using: (i) siRNA, (ii) gene therapy vectors, preferably gene therapy vectors comprising the CRISPR / Cas9 system, (iii) a bioactive small molecule inhibitor of the Toll-like receptor 4 selected from the group consisting of 3,4,6-triacetate isopropyl-2- (acetylamino) -2-deoxy-aD-glucopyranoside, resatorvid, eritorán and a peptide with the sequence KYSFKLILAEYRRRRRRRRR (SEQ ID NO: 1), and / or (iv) a neutralizing antibody that binds to the extracellular region of TLR4. Preferably, TLR4 is inhibited using: (i) small interfering RNA and / or (ii) vectors for gene therapy, preferably gene therapy vectors comprising the CRISPR / Cas9 system. Most preferably, TLR4 is inhibited using vectors for gene therapy, preferably gene therapy vectors comprising the CRISPR / Cas9 system.
    [0195]
    [0196] TLR9 can be inhibited by using a TLR9 inhibitor. In a preferred embodiment, TLR9 is inhibited using: (i) small interfering RNA, (ii) gene therapy vectors, preferably gene therapy vectors comprising the CRISPR / Cas9 system, (iii) a bioactive small molecule inhibitor of the receptor of Toll 9 type selected from the group consisting of a nucleotide comprising the sequence TTAGGG, ODN 2088, ODN 4084-F, ODN INH-1, ODN-INH-18, ODN A151, G-ODN, 3- [4- (6- (3- (dimethylamino) propoxy) benzo [d] oxazol-2-yl ) phenoxy] -N, N-dimethylpropan-1-amine, 6- [3- (pyrrolidin-1-yl) propoxy) -2- (4- (3- (pyrrolidin-1-yl) propoxy) phenyl] benzo [ d] oxazole, and / or (iv) a neutralizing antibody that binds to the extracellular region of TLR9 Preferably, TLR9 is inhibited using: (i) small interfering RNA and / or (ii) vectors for gene therapy, preferably vectors for gene therapy comprising the CRISPR / Cas9 system.Most preferably, TLR9 is inhibited using vectors for gene therapy, preferably gene therapy vectors comprising the CRISPR / Cas9 system.
    [0197]
    [0198] In a preferred embodiment, the isolated MSC is infected using a multiplicity of infection (MOI) of between 1 and 1000. Preferably, the MOI is between 10 and 500, 50 and 300, 150 and 250 or the MOI is 200. Preferably , the MOI is at least 1, 10, 50, 150 or 200 and the MOI is at most 1000, 500, 300 or 250. More preferably, the MOI is 200.
    [0199]
    [0200] In a preferred embodiment, the intracellular parasite is selected from the group consisting of bacteria, viruses, protozoa and fungi. More preferably, the antigenic substance is an oncolytic virus.
    [0201]
    [0202] The oncolytic virus can be any virus that preferentially infects and destroys cancer cells. In a preferred embodiment, the oncolytic virus is selected from the group consisting of adenovirus, reovirus, measles virus, herpes simplex virus, Newcastle disease virus, vaccinia virus, Coxsackie virus and Seneca valley virus. Preferably, the oncolytic virus is an adenovirus. More preferably, the oncolytic virus is ICOVIR-5.
    [0203]
    [0204] Kit
    [0205]
    [0206] In a sixth aspect, the present application provides a kit comprising (a) a mesenchymal stem cell, (b) an antigenic substance and (c) an inhibitor of TLR4 and / or TLR9 selected from the group consisting of: (i) siRNA , (ii) vectors for gene therapy, preferably gene therapy vectors comprising the CRISPR / Cas9 system, (iii) a bioactive small molecule inhibitor of the Toll-like receptor 4 selected from the group consisting of 3,4,6-triacetate of isopropyl-2- (acetylamino) -2-deoxy-aD-glucopyranoside, resatorvid, eritorán and a peptide with the sequence KYSFKLILAEYRRRRRRRRR (SEQ ID NO: 1), and / or a bioactive small molecule inhibitor of the Toll-like receptor 9 selected from the group consisting of a nucleotide comprising the sequence TTAGGG, ODN 2088, ODN 4084-F, ODN INH-1 , ODN-INH-18, ODN A151, G-ODN, 3- [4- (6- (3- (dimethylamino) propoxy) benzo [d] oxazol-2-yl) phenoxy] -N, N-dimethylpropan-1 -amine, 6- [3- (pyrrolidin-1-yl) propoxy) -2- (4- (3- (pyrrolidin-1-yl) propoxy) phenyl] benzo [d] oxazole, and (iv) a neutralizing antibody which binds to the extracellular region of the Toll-like receptor 4 and / or the Toll-like receptor 9.
    [0207]
    [0208] 3,4,6-isopropyl-2- (acetylamino) -2-deoxy-aD-glucopyranoside triacetate is available from Sigma (product no .: SML0832, CAS number: 40592-88-9). Resatorvid is marketed by Cayman Chemical (product no .: SML0832, CAS number: 243984-11-4). Eritorán is marketed by Eisai Co. (product no .: E5564, CAS number: 185955-34-4). ODN 2088 is sold by InvivoGen (product no .: tlrl-2088-1). ODN 4084-F is sold by InvivoGen (product no .: tlrl-4084). ODN INH-1 is marketed by InvivoGen (product no .: tlrl-inh1). ODN-INH-18 is sold by InvivoGen (product no .: tlrl-inh18). ODN A151 is sold by InvivoGen (product no .: tlrl-ttag151). G-ODN is marketed by InvivoGen (product no .: tlrl-godn). 3- [4- (6- (3- (Dimethylamino) propoxy) benzo [d] oxazol-2-yl) phenoxy] -N, N-dimethylpropan-1-amine can be synthesized by Eisai Inc. (AT791; Lamphier et al. , 2014. Mol.Pharmacol. 85 (3): 429-40). 6- [3- (Pyrrolidin-1-yl) propoxy) -2- (4- (3- (pyrrolidin-1-yl) propoxy) phenyl] benzo [d] oxazole can be synthesized by Eisai Inc. (E6446; Lamphier et al. al, 2014. Mol.Pharmacol. 85 (3): 429-40).
    [0209]
    [0210] In a preferred embodiment, the inhibitor of TLR4 and / or TLR9 is selected from the group consisting of: (i) siRNA and (ii) gene therapy vectors, preferably gene therapy vectors comprising the CRISPR / Cas9 system. Preferably, the inhibitor of TLR4 and / or TLR9 are vectors for gene therapy, preferably gene therapy vectors comprising the CRISPR / Cas9 system.
    [0211]
    [0212] The antigenic substance can be any substance that can be produced by the modified MSC, produces an immunogenic response and can be released at the tumor site to produce a localized immunogenic response. In a preferred embodiment, the antigenic substance is an intracellular parasite. Preferably, the intracellular parasite is selected from the group consisting of bacteria, viruses, protozoa and fungi. More preferably, the antigenic substance is an oncolytic virus.
    [0213] The oncolytic virus can be any virus that preferentially infects and destroys cancer cells. In a preferred embodiment, the oncolytic virus is selected from the group consisting of adenovirus, reovirus, measles virus, herpes simplex virus, Newcastle disease virus, vaccinia virus, Coxsackie virus and Seneca valley virus. Preferably, the oncolytic virus is an adenovirus. More preferably, the oncolytic virus is ICOVIR-5.
    [0214]
    [0215] Examples
    [0216]
    [0217] Example 1: Isolation and characterization of mMSC
    [0218]
    [0219] MMSC was obtained from abdominal and inguinal adipose tissue of mice of different breeds: C57BL / 6J mice (mMSC BL6), C57BL / 10J (mMSC BL10), C57BL / 10TLR4 '/' (mMSC TLR4 - / -) and C57BL / 10TLR9 - / - (mMSC TLR9 - / -). Adipose tissue was digested with collagenase B (2 mg / ml; Roche) for 45 min at 37 ° C and filtered through a sterile 70 μM nylon mesh cell sieve (Fisher Scientific). Cells were seeded in a cell culture dish and cultured in complete Dulbecco's modified Eagle's medium (DMEM) (Lonza): DMEM supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), streptomycin (100 mg / ml; Lonza), penicillin (100 U / ml; Lonza) and glutamine (2 mM; Lonza) at 37 ° C in a humidified atmosphere with 5% CO2. It was characterized that the mMSC had a fibroblast type morphology (see Figure 1).
    [0220]
    [0221] Example 2: Viral infection with ICOVIR-5
    [0222]
    [0223] The mMSC were infected with ICOVIR-5 (an oncolytic adenovirus) at a multiplicity of infection (MOI) of 200 in 1 ml per 1x106 cells for 2 h at 37 ° C. The infection was performed in suspension in DMEM without FBS. The cells were washed 3 times with phosphate buffered saline (PBS) and centrifuged at 1200 rpm for 5 min to remove the virus from the cell culture supernatant. Infected mMSCs are called Celyvir.
    [0224]
    [0225] Example 3: Activation of the NF-k B pathway, luciferase assay
    [0226]
    [0227] The activation of NF-k P was evaluated using a luciferase indicator system. MMSC 1x105 were seeded in a 6 - well plate 700. ^ L per well of DMEM supplemented with 10% FBS and transduced with a non-replicative lentiviral vector overnight. The lentiviral vector contains the plasmid pHAGE NF-kB-TA-LUC-UBC-GFP-W (Addgene plasmid No. 49343), whose structure codes for the consensus binding sequence to NF-kP in the sense of 'of the minimal herpes simplex virus TA promoter followed by the firefly luciferase gene as well as the eGFP gene upstream of the ubiquitin-C promoter. The efficiency of transduction was evaluated by quantification of GFP expression by flow cytometry. The transduced cells were infected with ICOVIR-5 as described above and 1x104 cells were seeded per well in 96-well plates with DMEM supplemented with 10% FBS. After 24 h, cell lysis was performed for total protein extraction and the luciferase activity was assayed with the luciferase assay system following the manufacturer's instructions (Promega Corporation, Madison, WI, USA) .
    [0228]
    [0229] Activation of the NF-k P pathway is the main initial immunological event that occurs in response to a pathogen. As can be seen in Figure 2, the mMSC TLR4- / "cells showed an increased amount of luciferase activity after infection with ICOVIR-5, therefore infection of the mMSC TLR4- /" cells with ICOVIR-5 activated the route of NF-kB.
    [0230]
    [0231] Example 4: Western blotting
    [0232]
    [0233] 5x105 mMSC or Celyvir were seeded per well in 6-well plates with DMEM supplemented with 10% FBS. After 3 and 24 h of treatment, the total proteins were extracted with SDS sample buffer (62.5 mM Tris, pH 6.8, 2% sodium dodecylsulfate (SDS), 10% glycerol, phenylmethylsulfonyl fluoride 1 mM (PMSF), 5 mM sodium fluoride (NaF), 20 mM p-glycerophosphate, Na3VO40.1 mM and protease inhibitor cocktail 1: 100 (Sigma-Aldrich)) and the total lysates were frozen. The samples were boiled at 95 ° C for 5 min and sonicated three times for 30 s each time on ice. The protein concentration was determined using the DC protein assay (Bio-Rad). 100 mM dithiothreitol (DTT) and 0.1% bromophenol blue (BPB) were added to the samples and boiled at 95 ° C for 5 min.
    [0234] Proteins were separated by electrophoresis in polyacrylamide gel electrophoresis with 10% sodium dodecyl sulfate (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA) and blocked with 2% milk in tris-buffered saline (TBS). Antibodies Primary antibodies that were used were anti-c-Jun mouse monoclonal antibody at a dilution of 1: 1000, anti-p-actin antibody (clone AC-15, Sigma-Aldrich) at 1: 100000 and anti-phospho rabbit monoclonal antibody -Akt (Ser473, clone 2118; Epitomics) at a dilution of 1: 1000. The membranes were washed three times with TBS with 0.1% Tween 20 (TBS-T) for 10 min. Secondary antibodies were polyclonal goat anti-rabbit and anti-mouse / HRP (Dako) immunoglobulins at a dilution of 1: 3000, and incubated for 1 h at RT. The HRP signal was detected with Western Immobilon type chemiluminescent HRP substrate (Millipore, Darmstadt, Germany) and was revealed using an X-ray Hyperoprocessor (Amersham Pharmacia Biotech). The membranes were digitalized and protein expression was quantified by measuring the mean gray value of the bands in the Image J software. The bottom of each membrane was subtracted from the values obtained.
    [0235]
    [0236] Akt and Jun are phosphorylated as a result of the activation of TLR4 and TLR9. Akt and Jun were phosphorylated in all cells 24 hours after infection with ICOVIR-5 (see Figure 3). The absence of TLRs in mMSCs does not seem to modify the response of the cells to an infection with ICOVIR-5.
    [0237]
    [0238] Example 5: In vitro migration assay
    [0239]
    [0240] The migration capacity of mMSC and Celyvir was studied using an in vitro assay . Transwell 24-well plates (filters with 8 .mu.m pores, BD Biosciences) were coated with 0.1% gelatin (Sigma-Aldrich) and seeded 5x104 cells per well with DMEM without FBS in the upper chamber. 1x105 CMT 64 cells were seeded per well with DMEM without FBS in the lower chamber as stimuli. Inserts were incubated for 24 h at 37 ° C in a humidified atmosphere with 5% CO2. A negative control assay was performed incubating mMSC in the upper chamber in the presence of DMEM without cells in the lower chamber. After 24 h, the Transwell plates were washed and the non-migrating cells were removed with a swab humidified with PBS. The migrated cells were fixed with 10% formaldehyde for 10 min and stained with crystal violet. Cells from 13 high-power fields (HPF) were counted for each condition.
    [0241]
    [0242] BL6 and BL10 cells infected with ICOVIR-5 had a greater tendency to migrate to CMT 64 cells. This effect could not be measured in TL4- / "or TLR9- /" cells that migrated to CMT64 cells regardless of whether they were infected or no (see figure 4).
    [0243] Example 6: Tumor induction and antitumor treatment
    [0244]
    [0245] The CMT 64 cell line, a murine non-small cell lung carcinoma, was obtained from the general ECACC collection (ECACC 10032301) and cultured in complete DMEM at 37 ° C in a humidified atmosphere with 5% CO2. For in vivo monitoring , CMT 64 cells were transduced with a non-replicative lentiviral vector containing a firefly luciferase cassette (CMT 64-Luc). For in vivo experiments , subcutaneous tumors of 64-Luc CMT cells were established by injection of 1 × 10 6 cells diluted in 100 μl of PBS in 7-week-old female C57BL / 6J mice. After 6 days from tumor inoculation, the luminescence of the tumors was measured and five homogeneous groups were established (see Figure 5b) as follows: PBS (n = 4), Celyvir BL6 (n = 6), Celyvir BL10 (n = 5), Celyvir TLR4 - / - (n = 4) and Celyvir TLR9 - / - (n = 6).
    [0246]
    [0247] A total of two in vivo experiments were performed, each with different doses: low-dose Celyvir (3x104 cells / mouse) and high-dose Celyvir (5x105 cells / mouse) resuspended in 200 μl of PBS and injected by intraperitoneally (ip). A total of 4 doses were administered, 7 days apart from each other. Tumor growth was monitored by measuring the length (L), width (W) and height (H) with a caliber every 3-5 days. The tumor volume was calculated as (LxWxH) rc / 6, assuming that the tumors were approximately semi-ellipsoid. After 30 days from the tumor inoculation, the mice were sacrificed and the tumors were excised and processed for flow cytometry and histology analysis.
    [0248]
    [0249] The study received the approval of the ethics committee of animal experimentation in compliance with the directive of the European Union 86/609. The care and the experiments with animals were carried out according to the guidelines detailed in Royal Decree 1201/2005 of Spain.
    [0250]
    [0251] The groups treated with Celyvir were compared with the control groups treated with PBS. Celyvir BL6 was used as a positive control since its efficacy had been demonstrated previously. In the experiments performed with low doses of Celyvir (see Figure 6), Celyvir TLR4- / "and Celyvir TLR9- /" presented the best yields. Both of these infected cell lines decreased both the volume and the weight of the tumors. Similar results were obtained with Celyvir TLR4- / "when high doses were used (see Figure 7).
    [0252] Example 7: Flow cytometry
    [0253]
    [0254] Tumors extracted with collagenase IV (1 mg / ml) were digested under agitation for 1 h at 37 ° C and mechanically homogenized using a PTFE pestle from Potter-Elvehjem when necessary. The spleens were crushed mechanically and used as a positive control for the different populations of immune cells. Cell suspensions were filtered through a sterile 70 [mu] M nylon mesh cell sieve using the rubber end of a 3 ml syringe in ice cold PBS and the red blood cells were lysed by incubation with Quicklysis buffer (Cytognos) . Cell suspensions were blocked with mouse FcR blocking reagent (Miltenyi) for 20 min and incubated with the following mouse monoclonal antibodies diluted in PBS for 20 min at 4 ° C: anti-CD45 antibody (clone 30-F11) , anti-CD3 antibody (clone 145-2C11), anti-CD4 antibody (clone GK1.5), CD8 (clone 53-6.7), CD11b (clone M1 / 70), CD11c (clone N418), CD206 (clone C068C2) , MHCII (clone M5 / 114.15.2), Ly6C (clone AL-21), Ly6G (clone 1A8-Ly6g) and NK1.1 (clone PK136), all of them from eBioScience. After incubation, the cells were washed and labeled with the viability marker 7-aminoactinomycin (7AAD) (Fisher Scientific, Waltham, MA) for 10 min at RT. Samples were acquired with the MACSQuant® analyzer cytometer and analyzed using MACSQuantify analysis software (Miltenyi Biotec). Immune populations were defined as follows: leukocytes (CD45 +); total T lymphocytes (CD45 + CD3 +), subclassified as helper T lymphocytes (CD4 +) and cytotoxic T lymphocytes (CD8 +); NK cells (NK1.1 +); dendritic cells (NK1.1- CD11b + CD11c +); macrophages (CD45 + CD11b + MHCII + Ly6G-), subclassified as M1 proinflammatory (CD206-) and M2 anti-inflammatory (CD206 +); and neutrophils (CD45 + CD11b + MHCII- Ly6G +), subclassified as N1 proinflammatory (CD206-) and N2 antiinflammatory (CD206 +).
    [0255]
    [0256] In general, mice treated with Celyvir TLR4- / "showed greater leukocyte infiltration at the tumor site (Figure 8a). Similarly, the percentage of CD4 + cells within the population of CD3 + cells appears to be increased compared to the other groups (Figure 8c) and the proportion of CD4 + / CD8 + cells increases markedly (Figure 8d) As shown in Figure 8e, the groups treated with Celyvir have a greater tendency to contain a higher percentage of NK cells in The sample, especially the groups treated with Celyvir TLR9 '/ _ The percentage of dendritic cells also increases in the groups treated with Celyvir, especially the group treated with Celyvir TLR4- / "(see Figure 8f).
    [0257] Macrophages and monocytes are heterogeneous populations comprising a proinflammatory M1 subtype and an anti-inflammatory M2 subtype. Although the levels of M1 and M2 increased in the group treated with Celyvir BL10, the proportion between the two subtypes appears to be similar to that found in the group treated with PBS (figure 8g). On the other hand, in the group treated with Celyvir TLR4 - / - the proportion of M1 cells increases, while in the group treated with TLR9 - / - the opposite seems to be the case. In the groups treated with Celyvir that are deficient in TLR, there is a tendency to observe an increase in the percentage of neutrophils (figure 8h), in which the proportion of N1 / N2 is maintained in the group treated with Celyvir TLR4 - / - and the proportion decreased in the group treated with Celyvir TLR9 - / - (figure 8h).
    [0258]
    [0259] Therefore, it seems that therapy with Celyvir is based on the induction of an immune response in the tumor site. Therefore, it is plausible that mMSCs comprising other intracellular parasites or antigens to treat tumors can also be used.
    [0260]
    [0261] Example 8: Histology of excised tumors
    [0262]
    [0263] Samples of excised tumors were fixed in 10% buffered formaldehyde solution overnight, washed with PBS and immersed in 30% sucrose solution, embedded in medium of optimal cut-off temperature (OCT) (Tissue-Tek). and they froze. Sections of 8. ^ M thick were obtained in a cryostat and then stained with hematoxylin and eosin staining following conventional staining protocols.
    [0264]
    [0265] It was validated that a greater degree of leukocyte infiltration could be observed at the tumor site in the group treated with Celyvir TLR4 - / - (see Figure 9). In the samples, the infiltration of what appeared to be eosinophils could even be observed, which was not observed in the untreated tumor samples.
    [0266]
    [0267] Example 9: Cytokine microarray
    [0268]
    [0269] The pattern of proinflammatory cytokines in Celyvir and mMSC BL10 and TLR4 - / - was analyzed by cytokine matrix assay. 4 × 10 4 cells were seeded per well in 24-well plates with 1 ml of DMEM supplemented with 10% FBS. After 24 h, the supernatants were collected and the pattern of proinflammatory cytokines was analyzed using a mouse matrix panel kit A Proteome Profiler according to the indications of the manufacturer (R & D Systems, Minneapolis, MN). Matrix images were digitalized and cytokine profile quantitation was measured by pixel density of duplicate spots using a protein matrix analyzer for the ImageJ software.
    [0270]
    [0271] The quantification of cytokine CXCL10 secreted by mMSC and Celyvir was measured by ELISA. 4 × 10 4 cells were seeded per well in 24-well plates in 1 ml of DMEM supplemented with 20% FBS. After 24 h, the supernatants were collected and the CXCL10 protein levels were measured using the Mouse ELISA CRG-2 / IP-10 Ray Bio kit (RayBiotech, Norcross, GA) and following the manufacturer's instructions. Quantitation was measured by reading the absorbance at 450 nm using the Infinite® 200 reader (Tecan, Zurich, Switzerland).
    [0272]
    [0273] As can be seen in Figure 10, mMSC TLR4- / "and Celyvir TLR4- /" secreted generally fewer proinflammatory cytokines. Of the cytokines studied, there was a change in the expression levels of IL-6, CXCL10, CXCL1, CCL2, CXCL2, CCL5, TIMP-1 and CXCL12. Only CXCL12 was further secreted in mMSC TLR4- / "and Celyvir TLR4 - /
    [0274]
    [0275] Therefore, it seems that the inhibition / elimination of TLR4- / "makes the infected mMSC less likely to be detected by the immune system before reaching the tumor site." In the tumor site, the mMSC releases the virus containing the antigen, what induces a directed immune response.
    权利要求:
    Claims (18)
    [1]
    1. Combination product comprising:
    (i) a modified mesenchymal stem cell in which the Toll-like receptor 4 and / or the Toll-like receptor 9 are inhibited; Y
    (ii) an antigenic substance.
    [2]
    2. The combination product according to claim 1, wherein the Toll-like receptor 4 is inhibited.
    [3]
    3. The combination product according to claim 1, wherein the Toll-like receptor 9 is inhibited.
    [4]
    4. The combination product according to any one of claims 1-3, wherein the antigenic substance is an intracellular parasite.
    [5]
    The combination product according to claim 4, wherein the intracellular parasite is an oncolytic virus, preferably the oncolytic virus is selected from the group consisting of adenovirus, reovirus, measles virus, herpes simplex virus, Newcastle, vaccinia virus, Coxsackie virus and Seneca valley virus.
    [6]
    6. The combination product according to claim 5, wherein the oncolytic virus is ICOVIR-5.
    [7]
    Combination product according to any one of the preceding claims, wherein the mesenchymal stem cell is derived from bone marrow, placenta, umbilical cord, amniotic membrane, menstrual blood, peripheral blood, salivary gland, skin and foreskin, synovial fluid, amniotic fluid , endometrium, adipose tissue, umbilical cord blood and / or dental tissue.
    [8]
    8. Combination product according to any one of the preceding claims, wherein the combination product is a composition.
    [9]
    9. Pharmaceutical composition comprising the composition according to claim 8, and a pharmaceutically acceptable carrier or diluent, and / or a pharmaceutically acceptable adjuvant.
    [10]
    10. The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition further comprises a chemotherapeutic agent.
    [11]
    11. The combination product according to claims 1-8 or pharmaceutical composition according to claims 9 or 10 for use as a medicament.
    [12]
    The combination product according to claims 1-8 or pharmaceutical composition according to claims 9 or 10 for use in the treatment of a solid tumor.
    [13]
    13. A combination product or pharmaceutical composition for use according to claim 12, wherein the solid tumor is a brain cancer, lung cancer, kidney cancer, ovarian cancer and / or liver cancer.
    [14]
    14. A combination product or pharmaceutical composition for use according to any one of claims 12-13, wherein the treatment is used in combination with chemotherapy and / or radiotherapy.
    [15]
    15. Method for the production of modified mesenchymal stem cells comprising an intracellular parasite, comprising the steps of:
    (1) isolate mesenchymal stem cells from an isolated sample;
    (2) cultivate mesenchymal stem cells;
    (3) inhibiting the Toll-like receptor 4 and / or the Toll-like receptor 9 of the isolated mesenchymal stem cells; Y
    (4) infect mesenchymal stem cells with an intracellular parasite.
    [16]
    The method of claim 15, further comprising a step of irradiating with X-rays between steps (3) and (4).
    [17]
    17. Kit comprising:
    (a) a mesenchymal stem cell;
    (b) an antigenic substance; Y
    (c) an inhibitor of the Toll-like receptor 4 and / or the Toll-like receptor 9 selected from the group consisting of:
    (i) small interference RNA;
    (ii) vectors for gene therapy, preferably gene therapy vectors comprising the CRISPR / Cas9 system;
    (iii) a bioactive small molecule inhibitor of the Toll-like receptor 4 selected from the group consisting of isopropyl-2- (acetylamino) -2-deoxy-aD-glucopyranoside 3,4,6-triacetate, resatorvid, eritorán and a peptide with the sequence KYSFKLILAEYRRRRRRRRR (SEQ ID NO: 1), and / or a bioactive small molecule inhibitor of the Toll-like receptor 9 selected from the group consisting of a nucleotide comprising the sequence TTAGGG, ODN 2088, ODN 4084-F, ODN INH-1, ODN-INH-18, ODN A151, G-ODN, 3- [4- (6- (3- (dimethylamino) propoxy) benzo [d] oxazol-2-yl) phenoxy] -N, N -dimethylpropan-1-amine, 6- [3- (pyrrolidin-1-yl) propoxy) -2- (4- (3- (pyrrolidin-1-yl) propoxy) phenyl] benzo [d] oxazole;
    (iv) a neutralizing antibody that binds to the extracellular region of the Toll-like receptor 4 and / or the Toll-like receptor 9.
    [18]
    18. Kit according to claim 17, wherein the antigenic substance is an intracellular parasite.
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    同族专利:
    公开号 | 公开日
    ES2702618B2|2019-10-31|
    WO2019043282A1|2019-03-07|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    CA3098145A1|2018-05-02|2019-11-07|Tonix Pharma Holdings Limited|Stem cells comprising synthetic chimeric vaccinia virus and methods of using them|
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