Pharmaceutical composition and application thereof

Abstract

The invention also discloses application of the pharmaceutical composition containing the oncolytic virus and the odontogenic stem cells in preparation of drugs for treating tumors, inhibiting tumor metastasis and / or preventing tumor recurrence. The medicine is an intravenous administration dosage form.

Description

Pharmaceutical composition and its application

[0001] This application claims priority to the Chinese patent application filed with the State Intellectual Property Office of China on December 30, 2022, with application number 202211725800.0 and invention name “Pharmaceutical Compositions and Their Applications”, the entire contents of which are incorporated by reference into this application.

[0002] Join by reference

[0003] All publications, patents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application were specifically set forth and individually incorporated herein by reference in its entirety. Technical Field

[0004] The present invention belongs to the field of medical technology and relates to a pharmaceutical composition comprising an oncolytic virus and dental stem cells, as well as the use of dental stem cells to carry the oncolytic virus. It also relates to a method for treating tumors, inhibiting tumor metastasis, and preventing tumor recurrence by intravenously administering dental stem cells carrying the oncolytic virus. Background Art

[0005] In my country, cancer is the second most common cause of death, and the number of cases continues to increase at an average annual rate of approximately 3%. Therefore, research on more effective cancer treatments is urgent. Currently, the most commonly used cancer treatments in clinical practice include traditional surgery, radiotherapy, chemotherapy, and new immunotherapy. Oncolytic virus therapy is one type of tumor immunotherapy. As early as the early 20th century, researchers discovered cases of remission or recovery in some patients with virally infected tumors, which aroused the curiosity of researchers and subsequently gave rise to the concept of oncolytic viruses and related research.

[0006] Oncolytic viruses are a class of natural or recombinant viruses that can selectively infect and kill tumor cells without damaging normal cells. Compared with traditional immunotherapy, oncolytic viruses have the advantages of good targeting, few adverse reactions, multiple ways to kill tumors, and are not prone to drug resistance. However, the administration of oncolytic viruses is limited to intratumoral administration, and systemic administration is temporarily not possible. The main reasons include the following: On the one hand, the neutralizing antibodies existing in the human body will recognize foreign viruses and play an immune clearance role. On the other hand, intravenous or intraperitoneal administration usually results in extremely low levels of virus reaching solid tumors, which cannot reach effective drug concentrations. In addition, adenoviruses tend to accumulate in the liver after systemic administration.

[0007] Mesenchymal stem cells are a type of multipotent stem cell that possess all the characteristics of stem cells, namely, self-renewal and multidirectional differentiation capabilities. In addition, their cell surface expresses a series of chemokine receptors, such as CXCR4 / 5 / 6, CCR1 / 4 / 7 / 9 / 10 and integrin-α4. Under the stimulation of chemokines, inflammatory mediators, specific cell secretions, etc., they can be induced to migrate to the site of injury and tumor (Ma, X. et al., J Cell Physiol 236, 3114-3128, 2021; Wang, H. et al., Stem cells 27, 1548-1558, 2009). Therefore, mesenchymal stem cells are often used as living cell drug delivery carriers, and are combined with drugs in various construction methods to achieve drug delivery to the target site (Chulpanova, D. Set al., Front Pharmacol 9, 259, 2018; Wang, X. et al., International journal of nanomedicine 13, 5231-5248, 2018); at the same time, mesenchymal stem cells do not express MHC Mesenchymal stem cells (MSCs) have low immunogenicity (Halm, D. et al., Biol Chem 402, 693-702, 2021), express MHC I, can inhibit T cell activity, and participate in immunosuppression and immunoregulation (Hu, CD, Kosaka, Y., Marcus, P., Rashedi, I. & Keating, A., Stem cells and development 28, 933-943, 2019). In recent years, the use of mesenchymal stem cells (MSCs) to carry various small molecules and bioactive drugs for anti-cancer research has become a research hotspot.

[0008] Currently, stem cell types used in anticancer efficacy studies combining stem cells with oncolytic viruses include neural stem cells, umbilical cord blood stem cells, and bone marrow and adipose-derived mesenchymal stem cells. Neural stem cells and umbilical cord blood stem cells are derived from patients undergoing cranial surgery and the umbilical cords of newborns. These sources are limited, raising ethical concerns, high cell culture costs, and low feasibility for mass production. Human bone marrow and adipose-derived mesenchymal stem cells require surgical extraction via bone puncture and liposuction, which are quite invasive.

[0009] In this case, it is still necessary to explore and develop different ways of administering oncolytic viruses in order to effectively exert the effects of oncolytic viruses.

[0010] Summary of the Invention

[0011] After a lot of research, the inventor unexpectedly discovered that the use of dental stem cells (DSCs) in combination with oncolytic viruses can effectively treat tumors. The inventor also found that the use of dental stem cells equipped with oncolytic viruses by intravenous administration has unexpected tumor treatment effects. The intravenous administration method does not require high technical skills of doctors, is easy to operate, and is easy to promote. Moreover, the oncolytic virus-dental stem cell composition administered intravenously can reach various parts of the body and can also effectively treat undiscovered tiny lesions. In addition, since the intravenously administered oncolytic virus-dental stem cell composition can reach other parts of the body, it also has the effect of inhibiting tumor metastasis. On this basis, the present invention is completed.

[0012] In a first aspect, the present invention provides a pharmaceutical composition comprising an oncolytic virus and dental stem cells. In one embodiment, the oncolytic virus and dental stem cells are contained in separate containers, and the oncolytic virus and dental stem cells are mixed before use. In another embodiment, at least a portion of the oncolytic virus is contained within the dental stem cells, that is, the dental stem cells serve as a carrier for the oncolytic virus.

[0013] The dental stem cells are mesenchymal stem cells with self-renewal ability and multi-differentiation potential derived from the tooth or periodontal tissue, such as dental pulp stem cells (DPSCs), stem cells from exfoliated deciduous teeth (SHED), stem cells from apical papilla (SCAP), periodontal ligament stem cells (PDLSCs), alveolar bone-derived mesenchymal stem cells (ABMSCs), tooth germ progenitor cells (TGPCs), gingival mesenchymal stem cells (GMSCs), dental follicle precursor cells (DFPCs), etc.

[0014] In one embodiment, the dental stem cells are dental pulp stem cells. Preferably, the dental stem cells are of human origin.

[0015] In another embodiment, the oncolytic virus is a weakened wild-type virus strain, such as reovirus and Newcastle disease virus; or, the oncolytic virus is a genetically engineered virus, such as an engineered adenovirus, herpes simplex virus, vaccinia virus, and measles virus. Preferably, the oncolytic virus is an oncolytic adenovirus.

[0016] In another embodiment, the oncolytic virus is an oncolytic virus that expresses interferon, and preferably, the nucleotide sequence encoding the interferon is as shown in SEQ ID NO: 2-4. For example, an oncolytic virus prepared according to patent application publication CN111363726A.

[0017] In another embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The pharmaceutical composition can be formulated into a dosage form suitable for intravenous administration, such as an injection.

[0018] In another embodiment, the content of oncolytic virus carried by dental stem cells in the pharmaceutical composition is 0.1-100 vp / cell (virus particle per cell), for example, 0.2-50 vp / cell, 0.3-20 vp / cell, 0.4-10 vp / cell, 0.5-5 vp / cell or 0.6-2 vp / cell. The oncolytic virus can be inside the dental stem cells or adsorbed / attached to the surface of the dental stem cells. The specific content can be prepared and used according to actual clinical needs.

[0019] In a second aspect, the present invention provides use of the pharmaceutical composition of the first aspect in the preparation of a medicament for treating tumors. In one embodiment, the pharmaceutical composition can be formulated into a dosage form suitable for intravenous administration, such as an injection. The tumors include, but are not limited to, osteosarcoma, pancreatic cancer, bile duct cancer, vulvar cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, melanoma, lymphoma, gastric cancer, esophageal cancer, ovarian cancer, head and neck squamous cell carcinoma, squamous cell carcinoma, bladder cancer, glioma, cervical cancer, and renal cancer.

[0020] The present invention also provides a composition for treating tumors, comprising the pharmaceutical composition of the first aspect. In one embodiment, the composition can be formulated into a dosage form suitable for intravenous administration, such as an injection. The tumors include, but are not limited to, osteosarcoma, pancreatic cancer, bile duct cancer, vulvar cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, melanoma, lymphoma, gastric cancer, esophageal cancer, ovarian cancer, head and neck squamous cell carcinoma, squamous cell carcinoma, bladder cancer, glioma, cervical cancer, and renal cancer.

[0021] In a third aspect, the present invention provides a method for treating a tumor, comprising administering the pharmaceutical composition of the first aspect to a subject in need thereof. In one embodiment, the pharmaceutical composition is administered by intravenous administration. In another embodiment, the pharmaceutical composition is administered both by intravenous administration and by intratumoral administration, i.e., a combination of the two. In another embodiment, the pharmaceutical composition is administered both by intravenous administration and by intraperitoneal administration, i.e., a combination of the two. In yet another embodiment, the pharmaceutical composition is administered in combination by intravenous administration, intraperitoneal administration, and intratumoral administration. The tumors include, but are not limited to, osteosarcoma, pancreatic cancer, bile duct cancer, vulvar cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, melanoma, lymphoma, gastric cancer, esophageal cancer, ovarian cancer, head and neck squamous cell carcinoma, squamous cell carcinoma, bladder cancer, glioma, cervical cancer, and renal cancer.

[0022] The inventors also unexpectedly discovered that using the pharmaceutical composition described in the first aspect of the present invention to treat tumors significantly reduces the amount of virus required to achieve therapeutic effects, reducing or avoiding potential side effects of oncolytic viruses. In certain embodiments, the oncolytic virus content of dental stem cells is 0.1-100 vp / cell, for example, 0.2-50 vp / cell, 0.3-20 vp / cell, 0.4-10 vp / cell, 0.5-5 vp / cell, or 0.6-2 vp / cell.

[0023] In a fourth aspect, the present invention provides a method for preventing tumor recurrence, comprising administering the pharmaceutical composition of the first aspect to a subject in need thereof. In one embodiment, the present invention also provides use of the pharmaceutical composition of the first aspect in the preparation of a medicament for preventing tumor recurrence. In another embodiment, the present invention also provides a composition for preventing tumor recurrence, comprising the pharmaceutical composition of the first aspect.

[0024] In a fifth aspect, the present invention provides a method for inhibiting tumor metastasis, comprising administering the pharmaceutical composition of the first aspect to a subject in need thereof. In one embodiment, the present invention also provides use of the pharmaceutical composition of the first aspect in the preparation of a medicament for inhibiting tumor metastasis. In another embodiment, the present invention also provides a composition for inhibiting tumor metastasis, comprising the pharmaceutical composition of the first aspect.

[0025] The technical solution provided by the present invention has at least one of the following beneficial effects: improving the limitations of intratumoral administration, realizing systemic administration, broadening the indications and improving the convenience of operation; reducing the dosage of virus administration and increasing safety; having a good systemic tumor inhibition effect; and providing an adequate source of dental stem cells, which can greatly reduce the cost of cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 shows the morphology of human dental pulp stem cells under a microscope (100×) (A) and the growth curve (B).

[0028] Figure 2 shows the multidirectional differentiation potential of human dental pulp stem cells. Dental pulp stem cells were induced to differentiate into adipocytes, as shown by Oil Red O staining (A); and dental pulp stem cells were induced to differentiate into osteoblasts, as shown by Alizarin Red staining (B).

[0029] FIG3 shows fluorescence expression images of human dental pulp stem cells after being infected with different concentrations of oncolytic virus (expressing EGFP) for 72 hours.

[0030] FIG4 shows the crystal violet staining results of human dental pulp stem cells infected with oncolytic virus YSCH01 at different concentrations for 72 hours.

[0031] Figure 5 shows the cell growth activity curves of human dental pulp stem cells (A) and tumor cells SCC152 (B) after oncolytic virus infection.

[0032] FIG6 shows the results of the Transwell assay, wherein (A) shows glossopharyngeal carcinoma squamous epithelial cells SCC152, and (B) shows bladder cancer epithelial cells SW780.

[0033] Figure 7 shows the anti-cancer effect of intravenous injection of YSCH01-hDPSC in a humanized SCC152 tumor-bearing mouse model. (A) Mouse body weight curve; (B) Tumor growth curve.

[0034] Figure 8 shows the anti-cancer effect of intravenous YSCH01 / hDPSC in a nude mouse SW780 tumor-bearing model. (A) Schematic diagram of injection frequency; (B) Mouse body weight curve; (C) Tumor weight isolated from the administration side of each group on day 23 after administration; (D) Tumor growth curve of the administration side.

[0035] Figure 9 shows the anti-cancer effect of intravenous injection of YSCH01 / hDPSC in a nude mouse SCC152 tumor-bearing model. (A) Schematic diagram of injection frequency; (B) Tumor growth curve. DETAILED DESCRIPTION

[0036] Other aspects and advantages of the present invention will be readily apparent to those skilled in the art based on the detailed description below. It should be noted that the following detailed description (including the accompanying drawings) is intended to be illustrative only and not limiting of the present invention. It will be readily understood that the present invention may be implemented in various other embodiments by adjusting and varying certain specific details, without departing from the present invention.

[0037] Unless otherwise specified, terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which they belong.

[0038] The term "oncolytic virus" refers to a virus that can selectively replicate in cancerous or hyperproliferative cells, thereby slowing their growth or causing their death, while having little or no effect on normal cells. Exemplary oncolytic viruses include vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), herpes simplex virus (HSV), reovirus, measles virus, retrovirus, influenza virus, Sindbis virus, vaccinia virus and adenovirus, etc.

[0039] In the present invention, the unit of viral dose is vp (viral particle), which represents the number of viral particles contained in 1 ml of viral solution, and is the viral particle titer, usually expressed as vp / ml. In some specific embodiments, it can also be expressed as vp / cell (viral particles per cell) or other suitable expressions.

[0040] The term "dental stem cells" (DSCs) refers to a type of stem cell isolated from teeth or periodontal tissues. They are a mesenchymal stem cell (MSC)-like cell population with self-renewal and multipotential differentiation capabilities. The main DSCs reported so far include dental pulp stem cells (DPSCs), stem cells from exfoliated deciduous teeth (SHED), stem cells from apical papilla (SCAP), periodontal ligament stem cells (PDLSCs), alveolar bone-derived mesenchymal stem cells (ABMSCs), tooth germ progenitor cells (TGPCs), gingival mesenchymal stem cells (GMSCs), and dental follicle precursor cells (DFPCs).

[0041] Dental stem cells, a type of mesenchymal stem cell, offer advantages such as rapid and easy collection, complete autologous retrieval, shortened treatment cycles, and minimal host response, making them promising clinical anti-tumor agents. Previous studies have shown that genetically engineered gingival mesenchymal stem cells expressing the interferon-β gene can effectively inhibit the growth of glossopharyngeal carcinoma (Du, L., Liang, Q., Ge, S., Yang, C. & Yang, P. Stem Cell Res Ther 10, 224, 2019).

[0042] Human dental pulp stem cells can be obtained from a wide range of sources, including healthy adult third molars, orthodontic teeth, and children's deciduous teeth. 50% of the population has third molars, and most wisdom teeth require extraction, providing an ample tissue source for in vitro mass production of these stem cells. Furthermore, dental pulp stem cells can be isolated from ex vivo teeth obtained after tooth extraction. This procedure is minimally invasive, time-consuming, convenient, and relatively low-cost, making it a highly humane sampling method for both healthy organ donors and patients requiring autologous cell transplantation. Furthermore, compared to bone marrow mesenchymal stem cells, dental pulp stem cells possess enhanced proliferation, clonogenicity, and multidirectional differentiation, significantly reducing the cost and time of stem cell culture.

[0043] The term "pharmaceutical composition" refers to a combination of at least one drug and, optionally, a pharmaceutically acceptable carrier or excipient, that is combined to achieve a specific purpose. In certain embodiments, the pharmaceutical composition includes a combination that is separated in time and / or space, as long as they can work together to achieve the purposes of the present invention. For example, the ingredients contained in the pharmaceutical composition can be administered to the subject as a whole or separately. When the ingredients contained in the pharmaceutical composition are administered to the subject separately, the ingredients can be administered to the subject simultaneously or sequentially. Preferably, the pharmaceutically acceptable carrier is water, a buffered aqueous solution, an isotonic saline solution such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, or a polyalkylene glycol such as polypropylene glycol, triglycerides, etc. The type of pharmaceutically acceptable carrier used depends, among other things, on whether the composition according to the present invention is formulated for oral, nasal, intratumoral, perfusion, intradermal, subcutaneous, intramuscular, or intravenous administration. The composition according to the invention may contain lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring substances, flavoring substances and / or aromatic substances as additives.

[0044] The pharmaceutical composition comprising oncolytic virus and dental stem cells referred to herein may comprise the oncolytic virus and dental stem cells contained in separate containers and mixed before use; or the oncolytic virus may be contained within the dental stem cells or attached / adsorbed onto the surface of the dental stem cells, i.e., the dental stem cells serve as carriers for the oncolytic virus.

[0045] The term "oncolytic virus-dental stem cell composition" (sometimes referred to herein as "oncolytic virus-dental stem cell complex") refers to dental stem cells equipped with an oncolytic virus. In some embodiments, the oncolytic virus enters the dental stem cells through infection, causing the dental stem cells to carry the oncolytic virus. The oncolytic virus can be replicative or non-replicative. In some embodiments, the oncolytic virus-dental stem cell composition is an oncolytic virus-human dental pulp stem cell composition.

[0046] The terms "treat" and "treatment" include both therapeutic and prophylactic treatments. Those in need of treatment include those already suffering from a particular medical condition as well as those at risk of eventually developing that condition. Treating tumors includes eliminating or inhibiting tumor growth, inhibiting tumor metastasis, and removing residual tumors after or concurrently with surgery or other treatments.

[0047] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably.

[0048] In certain embodiments, disorders and diseases include tumors and cancers, for example, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma and other hematological malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T cell / histiocyte-rich B cell lymphoma, EBV-positive and EBV-negative PTLD, and EBV-associated diffuse large B-cell lymphoma. The main targets of the present invention are: primary lymphoma, DLBCL, plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma, HHV8-related primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal cord tumors, and brainstem glioma.

[0049] The term "administering" or "administering" means providing a substance, such as a pharmaceutical composition, to a subject in a pharmacologically usable manner.

[0050] The term "intravenous administration" refers to the administration of medications into a vein via injection or infusion. The intravenous route is the fastest way to deliver medications and fluids to the body because they are introduced directly into the circulatory system and are therefore rapidly distributed throughout the body. Common methods of intravenous administration include intravenous bolus (a one-time dose) and intravenous infusion (a drip), which are determined by the physician based on the medication and the patient's treatment needs.

[0051] The dosage of a pharmaceutical composition provided to a subject is that dosage sufficient to demonstrate a benefit to the subject to which it is administered, and may also be referred to herein as a "pharmaceutically effective amount" or "effective amount." The actual amount administered, as well as the rate and time course of administration, will depend on the individual condition and severity of the condition being treated. The prescription of treatment (e.g., determination of dosage, etc.) is ultimately the responsibility of and is relied upon by physicians and other medical practitioners, who typically consider the disease being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to physicians.

[0052] As used herein, unless otherwise expressly stated, a noun is not preceded by an article or is modified by "the" to indicate that the term can be one or more. It should also be noted that as used in this specification, a singular form includes a plural form of its referent unless it is clearly and unequivocally limited to one referent.

[0053] The term "about" or "approximately" generally means within a range of error for a particular value as determined by one of ordinary skill, which depends in part on the manner in which the measurements are made, i.e., on the limitations of the measurement system. For example, "about" can refer to one or more standard deviations, depending on the practice in a particular field. In particular, "about" can refer to values ​​that are within 20%, 10%, 5%, or 1% of a given value.

[0054] Terms such as "comprises," "comprising," "including," and "include" as used herein are not intended to be limiting. Furthermore, "or," "alternatively," means "and / or" unless otherwise stated.

[0055] The following is a clear and complete description of the technical solution in conjunction with the embodiments of the present invention. Obviously, the described embodiments are merely illustrative and not an exhaustive list of all possible implementations of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0056] Example

[0057] Example 1: Extraction and identification of human dental pulp stem cells

[0058] 1. Primary Isolation of Dental Pulp Stem Cells

[0059] Healthy third molars and orthodontic teeth aged 18-30 years were extracted and placed in a culture medium containing 1% double antibiotics (penicillin and streptomycin) and rinsed. A turbine was used to grind a small groove along the neck of the tooth to separate the crown and root, exposing the pulp cavity. The pulp was removed with a pulp needle, and the apical 1 / 3 was discarded. The pulp tissue was rinsed three times with PBS containing 1% double antibiotics and then minced into a minced meat. A mixed enzyme solution (3 mg / ml type I collagenase and 4 mg / ml type II protease) was added and cultured at 37°C and 5% CO2.

[0060] 2. Screening and Culture of Dental Pulp Stem Cells

[0061] Fibronectin is used to screen out dental pulp stem cells with good growth conditions. The screening method is as follows:

[0062] a. One day before screening, prepare fibronectin solution in PBS to a 10 mg / ml working solution. Add 1 ml of the working solution to each well of a six-well plate and incubate at 4°C overnight.

[0063] b. On the day of screening, P1 dental pulp stem cells were obtained and prepared into a cell suspension using serum-free a-MEM.

[0064] c. Take out the pre-laid six-well plate, carefully aspirate and discard the supernatant, and plate the P1 cells at 4000 cells / cm 2 The density is spread in the perforated plate.

[0065] d. Incubate at 37°C for 20 min, wash twice with PBS, discard unattached cells, add α-MEM (Hyclone, SH30265.01) medium containing 10% FBS and continue culturing. Change the medium every 2-3 days and subculture when the cell density reaches 80-90%.

[0066] e. Subculture at a density of 1:3 to reserve sufficient dental pulp stem cells as subsequent cell carriers. P3-P6 cells were used for in vitro experiments, and P6-P10 cells were used for in vivo experiments.

[0067] Dental pulp stem cell growth culture medium: α-MEM (Hyclone, catalog number: SH30256.01) containing 10% FBS (Sciencell, catalog number: 0500) and 1% double antibody (Sigma, catalog number: V900929).

[0068] The morphology of the cultured human dental pulp stem cells is shown in Figure 1A , where the cells are spindle-shaped and fibrous.

[0069] The growth curve is shown in Figure 1B. It can be seen that the hDPSC cell growth curve is basically "S"-shaped. Within 1 day after passaging, the cells grow slowly; from days 2 to 4, they enter the logarithmic growth phase and grow rapidly; from days 5 to 7, the cell growth slows down and tends to be gentle.

[0070] 3. Identification of Dental Pulp Stem Cells

[0071] Flow cytometry was used to identify the cell surface markers of stem cells (CD146, CD90, CD44, STRO-1), and it was found that more than 99% of the cells positively expressed Stro-1, CD90, and CD44, which are the dental pulp mesenchymal stem cells we need.

[0072] 4. Identification of Multidirectional Differentiation of Dental Pulp Stem Cells

[0073] Human dental pulp stem cells were induced into adipogenic and osteogenic forms using adipogenic induction medium (α-MEM (Hyclone, SH30265.01) containing 10% FBS, 1 μM dexamethasone, 10 μg / ml insulin, 200 μM indomethacin, and 0.5 mM IBMX) and osteogenic induction medium (α-MEM (Hyclone, SH30265.01) containing 10% FBS, 10 mmol / L sodium β-glycerophosphate, 0.05 mmol / L vitamin C, and 100 mmol / L dexamethasone), respectively. The medium was changed every 2-3 days. Osteogenic induction lasted 14-21 days, and adipogenic induction lasted 21-28 days. Mineralized nodules and lipid granules were then identified using Alizarin Red staining and Oil Red staining, respectively. Alizarin Red stains mineralized nodules, while Oil Red stains lipid granules. The results are shown in Figure 2.

[0074] Figure 2A shows an Oil Red O staining of hDPSCs after 28 days of culture in adipogenic induction medium. Red, plump fat granules are visible, indicating that these stem cells can differentiate into adipocytes. Furthermore, after 18 days of culture in mineralization induction medium, microscopic observation reveals the formation of mineralized nodules, which appear as heterogeneous red masses after Alizarin Red staining (Figure 2B), demonstrating that hDPSCs can differentiate into osteoblasts and odontocytes. These results demonstrate that human dental pulp stem cells can be induced to differentiate into both adipocytes and osteoblasts, demonstrating their multipotential differentiation potential.

[0075] Example 2 Preparation of Oncolytic Virus-Human Dental Pulp Stem Cell Composition

[0076] 1. Oncolytic virus infection of human dental pulp stem cells (hDPSCs)

[0077] 1) Plating: hDPSCs at passage P5 were plated in 24-well plates at 1E+5 cells per well in 1 ml of culture medium.

[0078] 2) Infection: 18 hours after plating, dilute the OncoMul-V2-EGFP virus stock solution (the control empty virus of YSCH01, carrying the EGFP gene, for preparation methods, see patent application publication CN111363726A) in serum-free α-MEM medium to a concentration of 6.9E+9 vp / ml. Add the virus dilution solution to the 24-well plate according to the preset dose;

[0079] 3) Photography: 72 hours after infection, the expression of green fluorescence in hDPSCs was observed under an inverted fluorescence microscope and images were recorded (Figure 3). The results showed that as the amount of virus added increased, the number of human dental pulp stem cells infected by the virus also increased.

[0080] 2. Tolerance of human dental pulp stem cells to oncolytic viruses

[0081] a. Crystal violet staining

[0082] 1) Pre-wash a 24-well plate with α-MEM serum-free medium;

[0083] 2) Digesting P4 hDPSCs to prepare a cell suspension containing P5 hDPSCs;

[0084] 3) Plate the cells at a density of 1E+5 cells per well, add 1 ml of culture medium to each well, let the cells rest for 30 minutes, and then place them in an incubator overnight.

[0085] 4) Add OncoMul-V2-EGFP and YSCH01 virus solutions (preparation methods, see patent application publication CN111363726A) at different concentrations and volumes to a 24-well plate, mix thoroughly, and continue culturing.

[0086] 5) Crystal violet staining after 72 hours: After discarding the cell supernatant, the cells were fixed with 4% paraformaldehyde (PFA) for 20 minutes, washed three times with PBS, and stained with 2% crystal violet solution (0.5g crystal violet powder dissolved in 25ml of 20% methanol) for 15 minutes. The dye was rinsed with running water, allowed to dry, and photographed, as shown in Figure 4. The results showed that hDPSCs had good tolerance to the oncolytic adenovirus YSCH01. Within the infection dose range of 2000 vp / cell, there was no significant difference in the number of viable hDPSC cells, indicating that the infection dose of 2000 vp / cell did not affect the normal proliferation of hDPSCs.

[0087] b. CCK 8 assay to detect cell viability

[0088] 1) Digest well-grown P4 hDPSCs with trypsin (Sigma-Adrich T4049) at 37°C. After 3 minutes, add growth medium (α-MEM supplemented with 10% FBS and 1% double-antibody) to terminate the digestion. Prepare a cell suspension at 5E+4 cells / ml and plate in a 96-well plate, 100 μl per well.

[0089] 2) 6 hours after plating, remove a 96-well plate and add 10 μl of CCK8 reagent (Company: Yisheng; Catalog No.: 40203ES80) to each well. Incubate at 37°C for 3.5 hours. Read the OD value at 450 nm using a spectrophotometer. Verify that there is no significant difference in cell viability between wells before proceeding to subsequent experiments. Otherwise, replate the plate.

[0090] 3) 24 hours after plating, discard the original culture medium and add 100 μl of freshly prepared culture medium containing different concentrations of virus particles (YSCH01) and continue culturing;

[0091] 4) 24 hours, 48 ​​hours, 72 hours, 96 hours, and 120 hours after virus addition, remove a 96-well plate and add 10 μl of CCK8 reagent to each well. The CCK8 incubation time is 3.5 hours and the OD value is read at 450 nm using a spectrophotometer.

[0092] Similar experiments were performed using tumor cells SCC152.

[0093] Based on the data obtained, a cell growth activity curve was drawn, see Figure 5, where Figure 5A is human dental pulp stem cells and Figure 5B is tumor cells SCC152. It can be seen from the curve in Figure 5A that within 24 hours of infection, the activity of hDPSC dental pulp stem cells is not affected when the dose is less than 1000vp / cell; it will be affected only after 48 hours of infection. This shows that dental pulp stem cells infected with the virus can still maintain normal cell activity for a sufficient period of time and can be used as a carrier to carry oncolytic viruses. Subsequent experiments mostly use an infection time of 4 hours to prepare oncolytic virus-human dental pulp stem cell compositions. For tumor cells, the results in Figure 5B show that a YSCH01 concentration of 50vp / cell can exert a significant tumor cell killing effect within 24 hours, and tumor cells are significantly more sensitive to oncolytic viruses than human dental pulp stem cells.

[0094] 3. Construction of Oncolytic Virus-Human Dental Pulp Stem Cell (OV-hDPSC) Composition

[0095] a. Take dental pulp stem cells from the logarithmic growth phase of P6-P10 and calculate the density of the stem cells according to the formula of 4.5E+5 cells / cm 2 18 hours after plating, dental pulp stem cells were infected with oncolytic virus YSCH01 at a dose of 1000 vp / cell and mixed;

[0096] b. Infect at 37°C, 5% CO2 for 4 hours to obtain an oncolytic virus-dental pulp stem cell composition.

[0097] The construction and production of the oncolytic virus YSCH01 can be found in patent application publication CN111363726A, which is an oncolytic adenovirus that expresses interferon.

[0098] The amino acid sequence of interferon carried by the oncolytic virus YSCH01 is:

[0099] Preferably, the nucleotide sequence encoding interferon carried by the oncolytic virus YSCH01 is selected from SEQ ID NO: 2, 3 and 4. In this embodiment, the oncolytic virus carries SEQ ID NO: 2.

[0100] Example 3: Tumor tropism of oncolytic virus-human dental pulp stem cell composition

[0101] 1. Transwell assay

[0102] a. P5 dental pulp stem cells were obtained and plated at a density of 4E+5 hDPSCs per 6 cm culture dish;

[0103] b. Tumor cells were seeded in the lower chamber of a Transwell plate at a density of 1.5E+6 cells per well and resuspended in 650 μl of culture medium. The tumor cells used in this experiment were: SCC152 cells and SW780 cells.

[0104] c. Digest hDPSCs and count them. Seed 2E+4 hDPSCs per well in the upper chamber and resuspend in 200 μl of serum-free / serum-containing medium.

[0105] d. After 16 hours of seeding the upper chamber of the Transwell plate, discard the supernatant and fix the cells in the upper chamber with 4% paraformaldehyde for 20 minutes. Wash three times with PBS and stain with 2% crystal violet for 15 minutes. Then, wash away the excess dye with running water.

[0106] e. Allow to dry for 24 hours and photograph under a microscope (objective lens 10x). Take 5 fields of view for each well (top, bottom, left, right, and center), count, and perform statistical analysis.

[0107] The results are shown in Figure 6. Group A had no serum concentration gradient between the upper and lower chambers and served as a negative control for the experiment. Group B had a serum concentration gradient between the upper and lower chambers. Compared with Group A, a large number of hDPSC dental pulp stem cells migrated to the lower chamber, validating the establishment of the transwell experimental system. Tumor cells were cultured in the lower chamber of Group C. The results of the Transwell in vitro experiment showed that hDPSC dental pulp stem cells could migrate toward tumor cells, whether they were glossopharyngeal carcinoma squamous epithelial cells SCC152 (Figure 6A) or bladder cancer epithelial cells SW780 (Figure 6B).

[0108] Example 4: Determination of viral load

[0109] Determination of actual viral load

[0110] a. Extraction of intracellular virus from YSCH01-hDPSCs

[0111] 1) Plate cells at a density of 4E+5 cells / well in a six-well plate. 18 hours after plating, add YSCH01 virus to infect hDPSCs.

[0112] 2) 4 hours after infection, discard the supernatant, wash twice with PBS, add 2 ml of serum-free medium to each well, freeze-thaw repeatedly three times to lyse the cells and release the virus. After centrifugation, remove the supernatant, dilute to 2 ml, and store at -80°C as the sample solution.

[0113] b. Immunochemical cell staining

[0114] Hexon is the largest and most abundant structural protein in the adenovirus capsid. Hexon staining can be used to detect the presence and titer of adenovirus. TM Virus titer was detected using the Adenovirus Titer Immunoassay Kit (CELL BIOLABS, catalog number VPK-109).

[0115] 1) 293A cell plating: 24-well plate, 2.5E+5 cells per well;

[0116] 2) 100 μl of the test sample solution was added 24 hours after plating, and the cells were infected for 48 hours;

[0117] 3) Fixation: Fix in -20°C pre-cooled methanol for 20 min, then wash three times with PBS;

[0118] 4) Blocking: Block with 1% BSA for 1 hour;

[0119] 5) Primary antibody: dilute hexon primary antibody (Santa Cruz, sc-58651) at a concentration of 1:500, add 200 μl to each well, dilute with 1% BSA, and incubate at 37°C for 1 hour;

[0120] 6) Wash 3 times with PBS;

[0121] 7) Secondary Antibody: Dilute HRP-labeled secondary antibody at a concentration of 1:1000, add 200 μl to each well in 1% BSA diluent, and incubate at 37°C for 1 hour.

[0122] 8) Wash 3 times with PBS;

[0123] 9) Staining: Prepare fresh DAB staining solution (Solution A and Solution B at a ratio of 1:1) and add 250 μl to each well. Observe the color development under a microscope. After 10-15 minutes, discard the staining solution, rinse twice with water, and add PBS.

[0124] 10) Photography: Images were taken using a 10x objective lens, with nine fields of view each for each well, including the upper left, upper right, left, center, right, lower left, lower right, and lower left fields. The number of positive viral particles was counted and the titer was calculated using the following formula: (ifu / m) = 135.2 × average number of fields × dilution factor. The titer was calculated according to the kit manual. The results are shown in Table 1.

[0125] Table 1

[0126] As can be seen from Table 1, the starting dose of virus-infected stem cells is 1000 viruses / cell. After 4 hours of infection, the average number of active viruses carried by the cells was detected to be 0.77 viruses / cell, which is only about 0.7% of the initial infection dose. This result shows that the use of human dental pulp stem cells as a carrier greatly reduces the number of oncolytic viruses that directly enter the body. The following examples show that the obtained oncolytic virus-human dental pulp stem cell composition has a significant anti-tumor effect.

[0127] Example 5: Anti-tumor effect of YSCH01-hDPSC in humanized SCC152 tumor-bearing mice

[0128] 1. Construction of SCC152 humanized tumor-bearing mice: 8-14 week old female fully humanized Hu-HSC-NPG mice (purchased from Beijing Weitongda Biotechnology Co., Ltd.) were implanted with 5E+6 human glossopharyngeal carcinoma SCC152 tumor cells subcutaneously on both sides, and the tumor volume reached 80-100mm 3 Follow-up experiments were conducted.

[0129] 2. Intravenous injection of oncolytic virus-dental pulp stem cell composition

[0130] a. Dental pulp stem cells infected with oncolytic virus for 4 hours were prepared into a cell suspension of 2E+6 cells / 100 μl;

[0131] b. Disinfect the injection site of tumor-bearing mice with alcohol and inject the cell suspension through the tail vein of the mice. Each injection should be 100 μl. The administration should be repeated 4 times with an interval of 4 days between each injection. The tumor size should be measured regularly.

[0132] 3. Control Group

[0133] The intratumoral injection group (IT) served as the positive control group: the injection site of tumor-bearing mice was disinfected with alcohol, and 100 μl of YSCH01 virus dilution (1.5E+10 vp / injection) was injected directly into the center of the tumor. The administration was repeated four times, with an interval of 3 days between each administration, and the tumor was measured regularly.

[0134] The results showed (see Figure 7) that intravenous injection of YSCH01-hDPSCs had a significant bilateral tumor-suppressing effect on glossopharyngeal tumors SCC152 in a humanized mouse model, comparable to the effect of intratumoral YSCH01 injection. It is noteworthy that, based on the data measured in Example 4 and calculated based on the actual stem cell intracellular viral load of 0.77 vp / cell, the actual intracellular viral load of the YSCH01 / hDPSC-IV group was far lower than the initial viral infection dose used when infecting dental pulp stem cells. The actual viral load per administration was: 0.77 vp / cell × 2E+6 cells = 1.54 x 10 6 The amount of virus injected into the tumor is 1.5E+10vp / injection×4=6E+10VP. Therefore, it is calculated that the virus dose injected intravenously into YSCH01-hDPSC is less than one ten-thousandth of that injected into the tumor.

[0135] Example 6: Anti-tumor effect of YSCH01-hDPSC in nude mice bearing SW780 tumors

[0136] 1. Construction of nude mice bearing SW780 tumors: 6-8 week old Balb / c mice were implanted with human bladder cancer cells SW780 in the axilla. Other procedures were the same as in Example 5. The mice were loaded with tumors with a volume of 80-100 mm. 3 There were 5 mice in each group, and the tumor formation period was about 7-14 days.

[0137] 2. Preparation of Cells for Therapy

[0138] a. One day before administration: Replace the P6-P9 generation hDPSCs with those for plating at a density of 6E+6 cells / T175 cell flask;

[0139] b. On the day of administration: 18 hours after plating, remove three T175 cell culture flasks and add the oncolytic adenovirus YSCH01 at an infection dose of 1000 vp / cell. Mix the supernatant and culture medium. Leave the remaining T175 cell culture flasks untreated as the stem cell-only experimental group.

[0140] c. In vivo administration: Four hours after infection, cells were trypsinized and resuspended in PBS phosphate buffer to prepare a cell suspension of 2E+7 cells / mL. To avoid animal death caused by injection of aggregated cell clumps, the cell suspension was filtered through a 70 μm cell strainer before injection. According to the experimental design, hDPSCs and YSCH01 / hDPSCs were injected intravenously, with 100 μl injected per animal, resulting in a cell count of 2E+6 cells. Dosing was performed once every four days, labeled Q4D, for a total of four times (Figure 8A).

[0141] During the above experiment, the mice were weighed every 3 days to monitor their growth and the tumor volume was also monitored. The tumor volume was calculated as follows: volume (mm 3 )=Length (mm)×Width 2 (mm 2 ) / 2, body weight was in g, and the tumor growth curve and body weight curve were drawn using Graphpad Prism software, and statistical analysis was performed. *, p < 0.05 indicates a significant difference, **, p < 0.01 indicates an extremely significant difference.

[0142] Mouse weights are shown in Figure 8B, demonstrating that intravenous administration is safe in the SW780 model. Tumor weight (Figure 8C) and volume (Figure 8D) measurements showed that, compared with intravenous injection of dental pulp stem cells alone, intravenous injection of dental pulp stem cells (YSCH01-hDPSCs) loaded with oncolytic virus significantly inhibited the growth of bladder cancer SW780 in a nude mouse model. Combined with the data measured in Example 4, it can be calculated that the viral dose required for intravenous injection of YSCH01-hDPSCs is less than one ten-thousandth of that required for intratumoral injection (see Example 5).

[0143] Example 7: Antitumor Effect of YSCH01-hDPSC in Human Glossopharyngeal Squamous Cell Carcinoma SCC152-Bearing Mice

[0144] Establishment of an SCC152 bilateral tumor-bearing mouse model: The experimental procedures were the same as in Example 6, with the subcutaneous implantation of human glossopharyngeal carcinoma cell line SCC152. According to the experimental design, hDPSCs and YSCH01 / hDPSCs were intravenously injected, with 100 μl of solution per mouse, containing 2E+6 cells. Dosing was performed every four days, designated Q4D, for a total of five doses (see Figure 9A).

[0145] The animal dosing experimental procedures were essentially the same as those in Example 6. In addition, a positive control group was established, in which YSCH01 oncolytic adenovirus was injected intratumorally at 2E+9 vp / injection, also five times in total. The left tumor was the injection site, and the right tumor was not injected with oncolytic virus.

[0146] From the tumor volume measurement results (Figure 9B), it can be seen that in the nude mouse SCC152 tumor-bearing mouse model, intravenous injection of dental pulp stem cells YSCH01-hDPSC carrying oncolytic virus can significantly inhibit tumor growth.

[0147] Based on the results of Examples 6 and 7, it can be seen that YSCH01 / hDPSC has significant anticancer effects on both human glossopharyngeal squamous cell carcinoma and bladder cancer when administered intravenously, indicating that its anticancer effect is not dependent on the tissue type of the cancer and may have a good broad-spectrum anticancer effect.

[0148] The above description is only a partial embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included in the scope of protection of the present invention.

Claims

1. Use of a pharmaceutical composition in the preparation of a drug for treating tumors, inhibiting tumor metastasis and / or preventing tumor recurrence, wherein the pharmaceutical composition comprises an oncolytic virus and dental stem cells.

2. The use according to claim 1, wherein the oncolytic virus and dental stem cells are contained in separate containers.

3. The use according to claim 1, wherein at least a portion of the oncolytic virus is contained in the dental stem cells.

4. The use according to any one of claims 1 to 3, wherein the dental stem cells are mesenchymal stem cells derived from tooth or periodontal tissue with self-renewal ability and multi-differentiation potential, such as dental pulp stem cells, exfoliated deciduous tooth stem cells, apical papilla stem cells, periodontal ligament stem cells, alveolar bone mesenchymal stem cells, tooth germ progenitor cells, gingival mesenchymal stem cells and dental follicle precursor cells. The use according to claim 4 , wherein the dental stem cells are dental pulp stem cells.

6. The use according to any one of claims 1 to 5, wherein the dental stem cells are of human origin.

7. The use according to any one of claims 1-6, wherein the oncolytic virus is a attenuated wild-type virus strain, such as reovirus and Newcastle disease virus; or, the oncolytic virus is a genetically engineered virus, such as an engineered adenovirus, herpes simplex virus, vaccinia virus and measles virus.

8. The use according to any one of claims 1-7, wherein the oncolytic virus is an oncolytic adenovirus.

9. The use according to any one of claims 1-8, wherein the oncolytic virus is an oncolytic virus expressing interferon, preferably, the nucleotide sequence encoding the interferon is as shown in SEQ ID NO: 2-4.

10. The use according to any one of claims 1 to 9, further comprising a pharmaceutically acceptable carrier. The composition according to claim 10 , which is formulated for intravenous administration.

12. The use according to any one of claims 1-11, wherein the content of the oncolytic virus carried by the dental stem cells is 0.1-100 vp / cell, such as 0.2-50 vp / cell, 0.3-20 vp / cell, 0.4-10 vp / cell, 0.5-5 vp / cell or 0.6-2 vp / cell.

13. The use according to any one of claims 1 to 12, wherein the pharmaceutical composition is formulated as a dosage form for intravenous administration.

14. The use according to any one of claims 1 to 13, wherein the tumor is selected from osteosarcoma, pancreatic cancer, bile duct cancer, vulvar cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, melanoma, lymphoma, gastric cancer, esophageal cancer, ovarian cancer, head and neck squamous cell carcinoma, squamous cell carcinoma, bladder cancer, glioma, cervical cancer and renal cancer.

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