RU2846551C1 - Mesenchymal stem cells that provide improved tumour targeting and mass production of viruses - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention refers to biotechnology, particularly to using a pharmaceutical composition containing a mesenchymal stem cell and an oncolytic virus for treating cancer. Said mesenchymal stem cell contains the E1B55K gene, where the E1B55K gene is induced by an expression inducer, and said E1B55K gene is expressed after the introduction of the mesenchymal stem cell into the body. Method of treating cancer using said composition is also provided.

EFFECT: present invention provides an increase in the oncolytic virus production in the treatment of cancer.

8 cl, 41 dwg, 5 ex

Description

Field of technology to which the present invention relates

[1] The present invention relates to the production of mesenchymal stem cells that are designed to proliferate viral vectors and viruses and provide control over the timing of virus production and release so that tumor targeting can be improved and viruses can be mass-produced.

[2]

Prior art of the present invention

[3] In the case of vesicular stomatitis virus, the tumor reaching rate was reported to be 0.01% within 24 hours of the intravenously injected virus (input virus) (Non-patent Document 1), and the biggest limitation was the very low target delivery efficiency of the intravenous injection of the virus.

[4] Since most mortality in cancer patients is caused by metastatic tumors, it is essential that there is tumor tropism and that sufficient amounts of tumor-killing (oncolytic) virus relative to the injected virus are delivered to the tumors.

[5] However, when administered intravascularly, most viruses are taken up by Kupffer cells in the liver, are quickly removed by mechanical sedimentation, are easily destroyed by intravascular virus-neutralizing antibodies and complements, and there is also the problem that the proportion of intravascular viruses released into tumor foci is quite low. Because of these problems, in particular, it could not be expected that the administration of adenovirus alone by intravenous injection would be effective.

[6] Solutions to the above problems include genetic modification of viruses, the introduction of nanotechnology, the use of mesenchymal stem cells with tropism for tumor foci, and similar measures. In particular, around 2010, studies were conducted on tumor-tropic cells such as mesenchymal stem cells (Non-Patent Document 2). However, although mesenchymal stem cells (MSCs) have many advantages in terms of ethical and technical convenience, low immunogenicity, and genetic stability, MSCs have problems in reducing the infection rate and replication rate of adenoviruses.

[7] In order to become a carrier and amplifier of oncolytic adenovirus, MSCs need to maintain infection and replication at a certain level or higher, and despite these problems, the present inventors obtained genetically modified MSCs to register a patent in Korea (Patent Document 2). However, it was confirmed that the continuation of passages for these MSCs was not easy and the condition of the cell line deteriorated as the number of passages increased.

[8] In addition, Non-Patent Document 3 confirmed that intravascular delivery of MSCs to deliver oncolytic viruses is available for the treatment of multiple brain tumors, but there are problems such as the tumor growth ability of MSCs.

[9] Meanwhile, a method for creating a platform that temporarily releases a virus whose production increases at desired times after the virus migrates to tumors has been reported (Non-Patent Document 4). When MSCs are used as a viral vector, the host cells (MSCs) loaded with cell death-inducing genes are rapidly lysed before reaching the tumor site to release the virus, so this method leads to various problems that may be caused by the release of the virus, especially safety issues and the like.

[10] In fact, the majority of cancer deaths are caused by recurrence and metastasis, and therefore there is a need for a method that can overcome the major obstacles that have not yet been solved in the treatment of tumors with oncolytic viruses.

[11] Prior Art Documents

[12] Patent documents

[13] (Patent Document 1) Korean Patent No. 10-2169798 (October 26, 2020).

[14] Non-patent documents

[15] (Non-Patent Document 1) Silva, N et al., Double trouble for tumors: Exploiting the tumor microenvironment to enhance anticancer effect of oncolytic viruses, Cytokine & Growth Factor Reviews 21: 135, 2010.

[16] (Non-Patent Document 2) Xia, X et al, Mesenchymal stem cells as carriers and amplifiers in CRAd delivery to tumors, Molecular Cancer 10: 134, 2011.

[17] (Non-patent document 3) Kerrigan BCP, Shimizu Y, Andreeff M, Lang FF. Mesenchymal stromal cells for the delivery of oncolytic viruses in gliomas. Cytotherapy 2017; 19: 445-457.

[18] (Non-patent document 4) Nakashima H, Kaur B, Chiocca EA. Directing systemic oncolytic viral delivery to tumors via carrier cells. Cytokine Growth Factor Rev 2010; 21: 119–126.

[19]

Brief summary of the present invention

Technical task

[20] Accordingly, the present inventors conducted research and made efforts to solve the above-described problems, and as a result developed mesenchymal stem cells capable of controlling the timing of virus production and release to improve the targeting of mesenchymal stem cells to a tumor for the proliferation of viral vectors and viruses and to ensure mass production of viruses, thereby completing the present invention.

[21] Accordingly, the aim of the present invention is to create mesenchymal stem cells for delivering an oncolytic virus, characterized by improved tumor targeting ability, into which the glucose-regulated protein 78 (GRP78) gene is introduced.

[22] Furthermore, another object of the present invention is to create mesenchymal stem cells for delivery of an oncolytic virus, into which the E1B55K gene is introduced in such a way that its expression is induced by an expression inducer.

[23] Furthermore, another object of the present invention is to create mesenchymal stem cells for delivering an oncolytic virus, comprising the glucose-regulated protein 78 (GRP78) gene and the E1B55K gene, in which the E1B55K gene is expressed by an expression inducer.

[24] Furthermore, another object of the present invention is to provide a composition for delivering an anti-cancer gene comprising mesenchymal stem cells and an oncolytic virus.

[25] Furthermore, another object of the present invention is to provide an anti-cancer pharmaceutical composition comprising mesenchymal stem cells and an oncolytic virus.

[26] Furthermore, another object of the present invention is to provide a composition for diagnosing cancer, including mesenchymal stem cells and an oncolytic virus.

[27] Furthermore, another object of the present invention is to provide a method for treating cancer, wherein the method comprises administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising mesenchymal stem cells and an oncolytic virus.

[28]

Technical solution

[29] Three major difficulties associated with methods of treating tumors with a virus alone are as follows.

[30] First, destruction by circulating antiviral antibodies against oncolytic adenovirus, second, non-specific adsorption by other non-tumor tissues such as the liver and spleen, and third, virus outflow from blood vessels into tumor sites rarely occurs.

[31] To overcome these fundamental problems of virotherapy, mesenchymal stem cells were used as a cellular carrier to facilitate systemic transport of oncolytic viruses in the blood, and in addition to virus delivery, a genetically modified mesenchymal stem cell (MSC) line was developed for effective antitumor activity caused by the large amount of virus produced during migration to the target tumor site.

[32] The present invention relates to the production of mesenchymal stem cells that are designed to proliferate viral vectors and viruses and provide control over the timing of virus production and release so that tumor targeting can be improved and viruses can be mass-produced.

[33]

Beneficial effects

[34] Because most of the mortality in cancer patients is related to metastatic tumors, the migration into tumors compared with the administered virus and the existing reduction in the replication level of adenoviruses in MSCs were significantly improved, so that sufficient amount of oncolytic virus could be delivered into tumors, and, at the same time, MSCs with improved tumor targeting were developed.

[35] The MSCs of the present invention can provide innovative improvements in stability and anti-cancer efficacy while minimizing side effects due to the significant improvement in tumor targeting and simultaneously a significant increase in the amount of infectious viral particles/viral particles (IVP/VP) by using MSCs as virus factories with an efficiency comparable to existing virus-producing cell lines. That is, it is possible to simultaneously achieve maximization of efficacy and minimization of toxicity of oncolytic virus-loaded MSCs by solving the most difficult problems of virus-based anti-cancer drugs, such as tumorigenesis, tumor tropism, tumor migration ability, and control of virus production/release at the appropriate time.

[36]

Brief description of the figures

[37] Fig. 1 shows confirmation of TERT expression of human adipose tissue MSC-TERT by Western blot.

[38] Figure 2 shows confirmation of the efficiency of continued passage of human adipose tissue MSC-TERT even at passage 30 using Western blot.

[39] Figure 3 shows confirmation of the infectivity of MSC-TERT adenovirus in human adipose tissue using fluorescence microscopy.

[40] Figure 4 shows that there is a decrease in p53 accumulation and survival-related signals upon induction of E1B55K expression in human bone marrow and adipose tissue-derived MSCs, as confirmed by Western blot.

[41] Figure 5 shows confirmation of the increase in p53 promoter activity due to E1B55K expression in the presence of doxycycline in the Tet-One inducible expression system by luminescence intensity.

[42] Figure 6 shows that the increase in p53 promoter activity by E1B55K is reduced by E4orf6, as evidenced by the luminescence intensity.

[43] Figure 7 shows that the E4orf6 gene in the dl324-BstBI adenoviral backbone was amplified by PCR, transferred into the pFlag-CMV2 vector, and sequenced to confirm 100% fidelity.

[44] Fig. 8 shows the insertion of the E1B55K gene into pCA14, which is an adenoviral shuttle vector, as confirmed by DNA electrophoresis.

[45] Fig. 9 shows the sequence confirmation of the E1B55K gene in pCA14-E1B55K.

[46] Fig. 10A shows confirmation that detection of E1B55K by anti-E1B55K polyclonal antibody is normal.

[47] Fig. 10B shows that the detection result of replication-incompetent virus expressing purified E1B55K at MOI confirms that normal expression has been achieved.

[48] Fig. 11 shows confirmation of transfection of MSC-TERT with E1B55K-expressing plasmid and improved virus production upon infection with oncolytic adenovirus by virus titration.

[49] Figure 12 shows confirmation of improved virus production according to increasing MOI with additional infection with oncolytic adenovirus following infection of MSC-TERT with replication-incompetent virus expressing purified E1B55K for each MOI of virus titration.

[50] Figure 13 illustrates the protocol for the Tet-One inducible expression system.

[51] Figure 14 illustrates the PCR conditions during construction of pRetro-x-tetone-puro-E1B55K.

[52] Figure 15 shows the confirmation of the plasmid construction in which E1B55K was properly inserted into pRetro-x-Tetone using the In-Fusion exogenous gene transfer method.

[53] Figure 16 shows the cleavage map of the pRetro-x-Tetone-puro-E1B55K vector used for construction.

[54] Figure 17 shows confirmation of final insertion by sequence analysis of the E1B55K plasmid inserted into pRetro-x-Tetone.

[55] Figure 18 shows confirmation of induction of E1B55K, which is a gene inserted into pRetro-x-Tetone, by doxycycline for expression as a protein.

[56] Figure 19 shows the expression of E1B55K protein in pRetro-x-tetone-puro-E1B55K as a function of doxycycline amount during doxycycline treatment.

[57] Figure 20 shows a comparison of virus production with and without doxycycline treatment following oncolytic adenovirus infection following transfection of human adipose tissue MSC-TERT with pRetro-Tetone-E1B55K.

[58] Fig. 21 shows the results of Western blot, where the MSC-TERT-tetoneE1B55K cell line is derived from MSC-TERT.

[59] Fig. 22 shows the results of confirmation of p53 accumulation by Western blotting in the presence of doxycycline (2.5 μg/ml) for the MSC-TERT-tetoneE1B55K cell line.

[60] Fig. 23 shows the confirmation of the possibility of eliminating tumorigenesis of the MSC-TERT-tetoneE1B55K cell line.

[61] Figure 24A shows evidence that the expression of all factors acting at each stage of MSC homing is increased by increasing GRP78 levels.

[62] Fig. 24B shows the confirmation of the increase in GRP78 mRNA expression by introducing pcDNA3.1-GRP78 into human MSC-TERT to confirm the robust expression of GRP78.

[63] Fig. 25 shows the result of confirmation of the GRP78 gene insertion into the pcDNA3.1-hygro vector (C: pcDNA3.1-hygro).

[64] Fig. 26 shows that the liver cancer cell line was transfected with the plasmid derived from the pcDNA3.1-GRP78 clone, and then the increase in GRP78 protein expression was confirmed by Western blot to confirm the gene introduction.

[65] Fig. 27 shows the result of base sequence analysis of the introduced GRP78 gene from the plasmid obtained from the pcDNA3.1-GRP78 clone.

[66] Figure 28 illustrates the cleavage map of the LNCXneo vector with an increased number of restriction enzyme recognition sites in the LNCX vector.

[67] Figure 29 shows the results of screening plasmid clones in which the GRP78 gene was inserted into LNCXneo by restriction enzyme digestion.

[68] Fig. 30 shows the results of screening GRP78-expressing clones by introducing a retrovirus expressing the GRP78 gene into the MSC-TERT cell line.

[69] Fig. 31 shows the results clearly confirming that the MSC-TERT-GRP78 entry site is located in tumor tissue sites without adsorption to other organs within a very short period of time.

[70] Fig. 32 shows the results of tumor targeting verification confirmation (rapid and precise tumor targeting) of the final type MSC-TERT-tetoneE1B55K-GRP78, which completed gene transfer, in lung cancer (a), liver cancer (b), and pancreatic cancer (c) cell lines.

[71] Fig. 33 shows the results of confirming the enhancement of intratumoral infiltration of final-type MSC-TERT-tetoneE1B55K-GRP78 at 24 hours (a), 48 hours (b), and 72 hours (c) after injection.

[72] Fig. 34 shows the expression of adenovirus-associated proteins in tumor tissue in the presence of doxycycline during infection of final-type MSC-TERT-tetoneE1B55K-GRP78 with oncolytic viruses.

[73] Fig. 35 shows the results of obtaining cell lines of clones 21, 24, 25 and 26 of the final type MSC-TERT-tetone E1B55K-GRP78 cell line.

[74] Fig. 36 shows the result of confirming the efficient and sufficient induction of E1B55K induction by doxycycline in the final clone 21 without leakage even at a very low concentration.

[75] Figure 37 shows the amount of virus produced when MSC-TERT-tetoneE1B55K-GRP78 final-type cells were infected with oncolytic virus in the presence of doxycycline or when E4orf6 was expressed.

[76] Figure 38 shows confirmation of tumor homing marker expression as a function of increasing MSC-TERT passage.

[77] Fig. 39 shows a graph confirming the antitumor effect of MSC-TERT-tetoneE1B55K-GRP78 infected with oncolytic adenovirus on the change in tumor size in mice.

[78] Fig. 40A shows the confirmation of the antitumor effect of MSC-TERT-tetoneE1B55K-GRP78 infected with oncolytic adenovirus by the change in tumor size after tumor removal in mice.

[79] Fig. 40B shows the confirmation of the antitumor activity of MSC-TERT-tetoneE1B55K-GRP78 infected with oncolytic adenovirus by the changes in tumor size in mice in photographs of mice.

[80] Fig. 41 illustrates the relationship between virus adsorption on tumor (A) and on other organs [liver (B), lung (C), spleen (D), kidney (E), and heart (F)].

[81]

Detailed disclosure of the present invention

[82] The present invention will now be described in more detail.

[83] The present invention relates to mesenchymal stem cells for delivery of an oncolytic virus into which the human glucose-regulated protein 78 (GRP78) gene has been introduced (GenBank NM 005347).

[84] Furthermore, the present invention relates to mesenchymal stem cells for delivering an oncolytic virus with improved tumor targeting ability by introducing the GRP78 gene into the mesenchymal stem cells as a viral vector.

[85] The GRP78 gene is a glucose-regulated protein 78 gene whose role in enhancing cell infiltration by increasing cell motility is already known, and the sequence of this gene is shown under GenBank NM 005347 SEQ NO: 1 (human). According to the present invention, a tumor homing marker serves to improve tumor targeting ability.

[86] [SEQ ID NO: 1]

[88] According to the present invention, it was confirmed that the GRP78 gene improved tumor tropism, and in particular, the virus was delivered to tumor tissue sites without adsorption to organs other than tumors.

[89] Although continued passage in the case of human mesenchymal stem cells to facilitate the introduction of such a specific gene is difficult, the telomerase reverse transcriptase (TERT) gene, which induces cell proliferation, can be additionally introduced to overcome this difficulty.

[90] The TERT gene is the telomerase reverse transcriptase gene, and the sequence of this gene is shown under GenBank NM 198253, SEQ ID NO: 2 (human).

[91] [SEQ ID NO: 2]

[93] According to the present invention, the MSC-TERT-GRP78 cell line was obtained and the improvement of tumor targeting of mesenchymal stem cells for the proliferation of viral vectors and viruses was confirmed. Therefore, mesenchymal stem cells can be used for both diagnosis and treatment of tumors.

[94] Furthermore, the present invention relates to mesenchymal stem cells for delivery of an oncolytic virus, into which the E1B55K gene is introduced in such a way that its expression is induced by an expression inducer.

[95] The E1B55K gene is a viral gene encoding the E1B55K protein, which is expressed early in the life of the adenovirus. The gene sequence is shown under GenBank accession number AC_000008, SEQ ID NO: 3. (The underlined "a" in the first line was originally a "g", but the KpnI site was removed by substitution. The underlined "t" in the 14th line represents where the "a" was originally replaced with a "t" to remove the HindIII site.)

[96] [SEQ ID NO: 3]

[98] The present inventors obtained a human MSC-CAR-E1B55K cell line from human bone marrow-derived MSCs which had already been applied for (Patent Document 1), and confirmed the increase in virus production, but confirmed that the continuation of passages was not easy, the condition of the cell lines deteriorated with the increase of the passage number, and confirmed that animal experiments and the establishment of a master cell bank were therefore impossible, and it took a long time to find out the reason. In particular, a new function of E1B55K was confirmed. This is because E1B55K not only induces the enhancement of virus proliferation, but also induces a decrease in the viability of cell lines at the same time.

[99] Increasing the oncogenic potential via the TERT gene, which had to be introduced for mass production of MSC cell lines, could provide a solution due to the fact that it was first determined that E1B55K induces not only an increase in viral replication but also a general decrease in MSC cell viability. That is, inducing a temporal shift in the timing of E1B55K gene expression allowed continued passage of MSCs, thereby allowing mass production through cell banking while essentially blocking MSC tumorigenesis.

[100] In the examples, the Tet-on system was used to induce a time-shifted expression point of the E1B55K gene, i.e., to ensure expression after the introduction of mesenchymal stem cells into the body, but any other time-shifting induction system is also possible. In addition, the expression inducer is preferably doxycycline, tetracycline, or a similar compound.

[101] According to the present invention, an MSC-TERT-tetoneE1B55K cell line was obtained and E1B55K (oncolysis) was expressed at desired time points via a time shift to substantially block the massive viral production and tumorigenesis of MSCs.

[102] Furthermore, the present invention relates to mesenchymal stem cells for delivering an oncolytic virus, comprising a glucose-regulated protein 78 (GRP78) gene and an E1B55K gene, wherein the E1B55K gene is expressed by an expression inducer.

[103] According to the present invention, the MSC-TERT-tetoneE1B55K-GRP78 cell line was obtained, and it was confirmed that the virus production and release time could be controlled so that tumor targeting could be improved and viruses could be mass-produced.

[104] The mesenchymal stem cell line used according to the present invention is preferably of human origin. The mesenchymal stem cell line is preferably derived from bone marrow, umbilical cord blood, or fat.

[105] The oncolytic virus used according to the present invention may be an oncolytic adenovirus, an oncolytic adeno-associated virus (AAV), an oncolytic retrovirus, an oncolytic lentivirus, an oncolytic herpes simplex virus, or an oncolytic vaccinia virus.

[106] In addition, the oncolytic adenovirus may be the oncolytic adenovirus described in Korean Patent Application No. 2016-0166171 developed by the inventors of the present invention.

[107] The present invention also relates to a composition for delivering an anti-cancer gene comprising mesenchymal stem cells for delivering an oncolytic virus and an oncolytic virus.

[108] The gene delivery composition of the present invention may be intended for systemic administration.

[109] The stem cells (Ad-MSC) loaded with the virus according to the present invention, in addition to having cancer-specific characteristics, are characterized by low or no immune response, and therefore can be administered systemically, and are well targeted to tumor cells, are not characterized by an effect causing toxicity, and therefore can significantly improve the efficiency of gene delivery.

[110] Mesenchymal stem cells can be isolated from bone marrow, umbilical cord blood, fat and similar organs and tissues, and allogeneic mesenchymal stem cells can be used using a patient's own cells or blood bank databases. Therefore, when using the adenovirus-loaded stem cells of the present invention as a gene carrier, not only self-treatment but also allogeneic treatment is possible, so that the unit cost of gene therapy can be further reduced.

[111] Methods for introducing an adenovirus comprising a gene according to the present invention into mesenchymal stem cells can be carried out by various methods known in the art. In particular, the gene can be introduced by a method of infection with a virus known in the art.

[112] As used herein, the term “anti-cancer pharmaceutical composition” refers to “a pharmaceutical composition for use in the treatment of cancer.”

[113] Since the mesenchymal stem cells included as an active ingredient in the anticancer pharmaceutical composition of the present invention are the same as the above-described mesenchymal stem cells of the present invention, the detailed description of the mesenchymal stem cells is equally applicable to the compositions of the present invention. Therefore, the description of the general issues is omitted in order to avoid excessive complexity due to unnecessary repetition of the present disclosure.

[114] Since the oncolytic adenovirus included in the compositions of the present invention exhibits effective killing against various tumor cells as described above, the pharmaceutical composition of the present invention can be used to treat cancer such as gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, biliary tract cancer, bladder cancer, colorectal cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, skin cancer, kidney cancer, polyploid carcinoma, thyroid cancer, parathyroid cancer or ureteral cancer. As used herein, the term "treatment" includes: (i) preventing the formation of tumor cells; (ii) inhibiting tumor-associated diseases or disorders after removal of tumor cells; and (iii) alleviation of tumor-related diseases or disorders following removal of tumor cells. Therefore, as used herein, the term "therapeutically effective amount" refers to an amount sufficient to achieve the aforementioned pharmacological effect.

[115] The pharmaceutically acceptable carrier included in the composition according to the present invention is a pharmaceutically acceptable carrier commonly used in the formulation and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil and the like, without limitation.

[116] The pharmaceutical composition according to the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative and the like in addition to the above-mentioned ingredients.

[117] The pharmaceutical composition of the present invention is preferably administered parenterally, and can be administered, for example, using intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration or local administration. The pharmaceutical composition of the present invention is administered intraperitoneally for the treatment of ovarian cancer and administered through the hepatic portal vein for the treatment of liver cancer by an injection method, in the case of liver cancer, melanoma and breast cancer, the pharmaceutical composition can be administered by direct injection into the tumor mass, in the case of colon cancer, the pharmaceutical composition can be administered by direct injection into an enema, and in the case of bladder cancer, the pharmaceutical composition can be administered by direct injection into a catheter.

[118] A suitable amount for administration of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, the administration route, the age, body weight, sex or disease state of the patient, diet, administration time, route of administration, excretion rate and response sensitivity, and a physician with ordinary skill can easily determine and prescribe an effective dose for the desired treatment. Typically, the pharmaceutical composition of the present invention comprises 1×10 ⁵ to 1×10 ¹⁵ PFU/ml of the oncolytic virus, and usually the amount of virus that infects MSCs (per 1×10 ⁶ cells) in one injection is 1×10 ⁷ to 1×10 ⁸ PFU.

[119] The pharmaceutical composition according to the present invention may be prepared in the form of a unit dose or may be contained in a multi-dose container, being formulated using a pharmaceutically acceptable carrier and/or an excipient according to a method that can be easily used by a person of ordinary skill in the art to which the present invention pertains. In this case, the composition may also be in the form of a solution in an oily or aqueous medium, a suspension, or in the form of an emulsion, an extract, a powder, a granule, a tablet, or a capsule, and the pharmaceutical composition according to the present invention may further include a dispersant or a stabilizer.

[120] The pharmaceutical composition of the present invention can be used as a monotherapy, but can also be used in combination with a conventional type of chemotherapy or radiotherapy, and by using such a combination therapy, cancer can be treated more effectively. The chemotherapeutic agent that can be used together with the composition of the present invention includes gemcitabine, sorafenib, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol, transplatin, 5-fluorouracil, vincristine, vinblastine, methotrexate and the like. Radiation therapy that can be used in conjunction with the composition of the present invention includes x-rays, γ-rays and the like.

[121] The mesenchymal stem cells according to the present invention can exhibit an anti-cancer effect against gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, biliary tract cancer, bladder cancer, colorectal cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, skin cancer, kidney cancer, polyploid carcinoma, thyroid cancer, parathyroid cancer or ureter cancer.

[122] Furthermore, the present invention may relate to a composition for diagnosing cancer comprising a virus/stem cell complex comprising mesenchymal stem cells and an oncolytic virus.

[123] As used herein, the term "diagnosis" means confirmation of the presence or characteristics of a pathological condition. Diagnosis according to the present invention consists of confirmation of the presence or absence of cancer and its progression.

[124] The virus/stem cell complex refers to stem cells infected or loaded with a virus.

[125] The virus/stem cell complex can be combined with a luminescent material, fluorophore or isotope for imaging-based cancer diagnosis.

[126] Luminescent material refers to any material that emits light.

[127] In the examples of the present invention, it was confirmed that after MSCs were infected with a luciferase-expressing adenovirus, luminescence was recorded in the form of images upon intraperitoneal administration of luciferin to mice.

[128] The fluorophore is preferably, but is not limited to, a phosphor, fluorescent protein, or other imaging materials capable of binding to a peptide that specifically binds to NRP1.

[129] The fluorophore is preferably, but is not limited to, fluorescein, BODYPY, tetramethylrhodamine, Alexa, cyanine, allopycocyanin, or derivatives thereof.

[130] The fluorescent protein is preferably, but not limited to, Dronpa protein, gene fluorescent protein (EGFP), red fluorescent protein (DsRFP), Cy5.5, which is a cyanine fluorophore exhibiting fluorescence in the near infrared region, or other fluorescent proteins. Other imaging materials are preferably, but not limited to, iron oxide, radioactive isotopes, and the like, and can be used in imaging equipment such as MP and PET.

[131] The present invention also relates to a method of treating cancer, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising mesenchymal stem cells and an oncolytic virus to a subject.

[132] All of the above-described contents regarding the method for treating cancer can be applied to the above-described mesenchymal stem cells according to the present invention and the composition including them as they are or applied accordingly.

[133] Cancer may specifically be, but is not limited to, stomach cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, biliary tract cancer, bladder cancer, colorectal cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, skin cancer, kidney cancer, polyploid carcinoma, thyroid cancer, parathyroid cancer, or ureteral cancer.

[134] As used herein, the term "subject" refers to a mammal who is the subject of treatment, observation, or experiment, preferably a human.

[135] As used herein, the term "therapeutically effective amount" refers to the amount of an active ingredient or pharmaceutical composition that produces a biological or medical response in a tissue system, animal, or human under consideration by a researcher, veterinarian, physician, or other clinician, and includes an amount that produces an alleviation of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective amount and frequency of administration of the active ingredient of the present invention will vary depending on the desired effect. Therefore, the optimal dosage for administration can be readily determined by those skilled in the art, and its range varies depending on the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of formulation, body weight, age, sex, health condition and diet of the patient, the time of administration, the route of administration, the rate of excretion, and the like. The method of treatment according to the present invention provides from 1×10 ⁵ to 1×10 ¹⁵ PFU/ml of oncolytic virus, and usually the amount of virus that is infected into MSC (calculated per 1×10 ⁶ cells) in one injection is from 1×10 ⁷ to 1×10 ⁸ PFU.

[136] In the treatment method according to the present invention, the pharmaceutical composition according to the present invention can be administered orally or parenterally (for example, administered intravenously, subcutaneously, intraperitoneally or locally) according to the desired route.

[137]

Embodiment of the invention

[138] The present invention will now be described in more detail using examples according to the present invention, but the scope of the present invention is not limited to the examples provided below.

[139]

[140]

Examples

[141] Reference Example 1: Construction of human oncolytic adenovirus YSC-02

[142] YSC-02 was constructed as described in Acquired Example 19 of Korean Patent Application No. 2016-0166171.

[143]

[144] Reference Example 2: Construction of human oncolytic adenovirus dl324-3484-H1-shHSP27-U6-shTGFβ1

[145] dl324-3484-H1-shHSP27-U6-shTGFβ1 was constructed as described in Example 9 of Korean Patent Application No. 2016-0166171.

[146]

[147] Example of production 1: Introduction of TERT gene for mass production of MSC cell line

[148] 1) Confirmation of expression of a single clone of human adipose tissue MSC-TERT

[149] The TERT gene was introduced to induce the expression of a specific gene that compensates for the deficiency and short lifespan of MSCs. In the case of human MSCs, continued passage is not easy. To overcome this problem, the telomerase reverse transcriptase (TERT) gene, which induces cell proliferation, was inserted and clones with high TERT expression were selected.

[150] The method of introducing the TERT gene is as follows.

[151] The pBABE-hygro-hTERT retroviral vector was purchased from Addgene, and cell clones resistant to 200 μg/ml hygromycin B Gold were generated by infecting human adipose tissue-derived MSCs with filtered medium obtained by transfecting Platinum-A packaging cells (Cell Biolabs) with 1 μg of the pBABE-hygro-hTERT retroviral vector, and then screened and selected as in Fig. 1. Finally, clone 22 of the MSCs was selected, and clone 22 was used as the human adipose tissue-derived MSC-TERT used in subsequent experiments.

[152] 2) Confirmation of continuation of passages

[153] Figure 2 shows the confirmation of continued passage of human MSCs with proliferation up to passage 30. It was confirmed that the increased expression level of TERT in MSCs subjected to prolonged passage up to passage 30 remained clearly elevated, and the expression of other MSC-related core markers was also maintained or increased compared to wild type.

[154] 3) Confirmation of the ability to be infected with adenovirus

[155] TERT-expressing MSC clone (No. 22) was infected with replication-incompetent adenovirus expressing red fluorescent protein (RFP) at MOI 20 and MOI 100, and 48 hours later, the fluorescently stained cells were observed under a fluorescence microscope. As a result of comparison with wild-type MSC, which is a control, it was confirmed that the infection rate of human MSC-TERT was improved compared with the control, and at least a two-fold increase in the infection rate at MOI 20 and an infection rate of 90% or more at MOI 100 were achieved (Fig. 3).

[156]

[157] Example 1: Confirmation of a new function of the adenoviral E1B55K gene

[158] If MSCs are to be used as a cell line for clinical mass production, it is necessary to establish a master cell bank. For this purpose, it is important, first of all, that the continuation of MSC passages be possible. However, on the other hand, it is also necessary to adhere to the principle that the induction of continuation should not contribute to an increase in tumorigenic potential. Although the dilemma of simultaneously satisfying these two conditions was initially considered insoluble, a solution was first proposed in the laboratory of the present inventor by constructing a stable MSC cell line in which CAR and E1B55K genes were introduced into MSCs. A human MSC-CAR-E1B55K cell line was obtained from human bone marrow-derived MSCs, which has already been applied for, and an increase in virus production was confirmed (Patent Document 1). However, it was confirmed several times that the continuation of passages was not easy, and the condition of the cell line worsened as the number of passages increased. Accordingly, it was confirmed that in the case of animal experiments, the establishment of a master cell bank was impossible, and as a result of elucidating the reason, it was confirmed that E1B55K induced the enhancement of viral proliferation and the decrease in the viability of cell lines at the same time (Fig. 4). That is, it was confirmed that not only p53, which is well known as a tumor suppressor gene and cytostatic factor, was clearly accumulated, but also the expression of cell survival-related factors (HSP27, HSP70, Daxx, phospho-p65, NF-κB, c-Me) was decreased by E1B55K.

[159] The experimental process in this case is as follows.

[160] E1B55K was first cloned into pcDNA3.1 hygro(+). To extract the E1B55K gene from pBSK-3484 containing the E1B55K gene, after PCR (initial denaturation: 95°C for 2 min, denaturation: 95°C for 30 sec, annealing: 58°C for 30 sec, extension: 72°C for 2 min; 30 cycles) and BamHI/NotI digestion with primers each containing BamHI and NotI (forward, 5'-GCGGATCCATGGAGCGAAGAAACCCATCT-3': SEQ ID NO: 4, reverse, 5'-GAG CGGCCGCTCAATCTGTATCTTCATCGCT-3': SEQ ID NO: 5), a plasmid was constructed by introducing the E1B55K gene into pcDNA3.1 hygro (+) (Invitrogen), which had previously been digested with BamHI/NotI.

[161] It was confirmed by transfection of a plasmid expressing E1B55K into bone marrow-derived MSCs and adipose-derived MSCs that prolonged expression of E1B55K in the cell line reduced the viability of the cell line. That is, accumulation of p53 and reduction of various survival-related signals were observed.

[162] In order to clarify how E1B55K expression causes p53 accumulation, decreased cell survival signals, and induction of cell death, the following hypothesis was made. That is, it was known in the art that E1B55K maintains cell survival through the well-known degradation of p53 only by the complex with the E4orf6 protein. Therefore, it was hypothesized that when E1B55K alone is induced to express in MSC cells, (stoichiometrically) free E1B55K itself acts as a transcriptional activator that promotes p53 transcription to decrease cell viability, due to the comparative quantitative predominance of E1B55K over E4orf6, and the experiments were carried out as follows.

[163] After co-transfection of the MSC-TERT-tetoneE1B55K cell line with 1 μg pFlag-CMV2-E4orf6 subcloned with the pGL2-p53 promoter (pGL2-356bp, Addgene) engineered such that the constant portion (-344 to +12) of the p53 promoter was located upstream of the site into which the luciferase-encoding gene and the E4orf6 gene were inserted, or with 1 μg pFlag-CMV2 (Sigma) as a control, luciferase activity with and without doxycycline (1.25 μg/ml for 48 h) was confirmed by luminescence intensity.

[164] In this case, the analytical method used was the Dual-Luciferase Reporter assay system from Promega (catalog number #E1910, Fig. 5).

[165] E1B55K, the expression of which was induced in the MSC-TERT-tetoneE1B55K cell line by doxycycline treatment, was directly or indirectly associated with the p53 promoter to activate transcription, resulting in a 4-fold increase in luminescence intensity due to luciferase activity (Fig. 5). On the other hand, it was confirmed that when E4orf6 was expressed, as in E1B55K-expressing MSCs, the increase in luminescence intensity was no more than 1.5-fold. These results indicate that E4orf6 interferes with the action of E1B55K itself by forming a complex with E1B55K and at the same time more actively performs its already known role in promoting p53 protein degradation (Fig. 6).

[166] Subcloning of the E4orf6 gene (GenBank AC_000008; also called E434K) was then performed to transfer the E4orf6 gene from the dl324-BstBI adenoviral backbone into the pFlag-CMV2 vector. For this purpose, the region of the gene encoding E4orf6 was amplified by PCR (initial denaturation: 95°C for 2 min, denaturation: 95°C for 30 sec, annealing: 58°C for 30 sec, extension: 72°C for 1 min and 30 sec; 30 cycles) in dl324-BstBI using the E4orf6 PCR primer (forward 5′-GTACAAGCTTATGACTACGTCCGGCGTTCC-3′, incorporating HindIII: SEQ ID NO: 6, and reverse 5′-CACCTCTAGACTACATGGGGGTAGAGTCAT-3′, incorporating XbaI: SEQ ID NO: 7) and then double-digested with HindIII/XbaI and ligated with pFlag-CMV2 digested with HindIII and XbaI. 100% identity was confirmed by sequencing using CMV30, one of the universal primers from Cosmogenetech Co. Ltd., to confirm the correct subcloning (Fig. 7).

[167] Based on the above experimental results, the induction of E1B55K overexpression acts directly or indirectly on the p53 promoter, which is contrary to the main action of E1B55K in wild-type adenovirus (forming a complex with E4orf6 to induce p53 protein degradation), so that a new action was discovered that induces p53 accumulation through transcriptional induction. Furthermore, in order to examine how the increase in MSC cell death and decrease in viability signal dependent on the increase in E1B55K expression affects the virus producing ability of MSCs, we decided to further examine whether the virus producing ability of MSCs is enhanced by externally supplying E1B55K using an adenoviral vector. For this purpose, a replication-incompetent adenovirus expressing E1B55K was constructed as follows.

[168] 1) Construction of a replication-incompetent adenovirus (pCA14-E1B55K) expressing E1B55K

[169] The E1B55K gene was cloned into pCA14, which is an adenoviral shuttle vector. E1B55K was incorporated into pBSK-3484, and a primer incorporating XbaI and HindIII at both its ends (sense 5'-TTCATCTAGAATGGAGCGAAGAAACCCATC-3' (XbaI): SEQ ID NO: 9, antisense 5'-GACGAAGCTTTCAATCTGTATCTTCATCGC-3 (HindIII): SEQ ID NO: 10) was designed and amplified by PCR (initial denaturation: 95°C for 2 min, denaturation: 95°C for 30 sec, annealing: 58°C for 30 sec, extension: 72°C for 2 min; 30 cycles) and ligated with pCA14 (Microbix, Canada) previously digested with XbaLHindIII. As a result, it was confirmed that all the E1B55K PCR fragments were inserted into PCA14 (Fig. 8). The confirmed adenovirus shuttle vector pCA14-E1B55K was linearized again by XmnI digestion after sequence confirmation (Fig. 9), the adenovirus vector dl324-BstBI was linearized by Bsp119I digestion, both of them were co-transformed into E. coli BJ5183 to induce homologous recombination to construct an adenovirus vector expressing E1B55K, then the vector was transfected into 293A, then the virus production was induced to increase the production, and the titer was calculated after isolation and purification.

[170] It was confirmed that 1, 2, and 3 of the plasmid clones pc14-E1B55K 1, 2, 3, and 4, read in the forward and reverse directions using a primer designed for pCA14 sequence analysis (self-designed forward (hCMV): 5'-GGGAGGTCTATATAAGCAGAGCTCG-3': SEQ ID NO: 11, reverse (pCA14 SV40): 5'-CATGATCGATGCTAGACGATCCAGA-3': SEQ ID NO: 12), were matched, and the results of the sequence analysis of the plasmid DNA of sample clone #3 are shown in Fig. 9 herein. In the two forward mismatched (red) sequences shown in Fig. 9, the first base of E1B55K of existing wild type 5 adenovirus, “a”, is the 2019th base of AC_000008 in GenBank, the 2048th base “g” in the forward primer was intentionally replaced with “a” to remove the KpnI site (GGTACC→AGTACC), and the 2084th base “a” in the forward primer (overlapping with red in the reverse primer) was replaced with “t” to intentionally remove the HindIII site (AAGCTT→TAGCTT) (homology 100%).

[171] 2) Confirmation of E1B55K expression in MSC lysate infected with E1B55K-expressing virus

[172] After it was confirmed that the detection of E1B55K by the polyclonal antibody obtained by using the E1B55K specific antibody construct was not abnormal in terms of E1B55K expression (Fig. 10A), and by detecting the obtained and purified virus at each MOI, the normal expression was confirmed by Western blotting (Fig. 10B). The image on the right side of Fig. 10A is attached as raw data for the image on the left side, in order to confirm the correct size and band of the E1B55K polyclonal antibody. In lane 3 of Fig. 10A 3 μg of adenoviral vector DNA (dl324-E1B55K) expressing E1B55K was digested with PacI and then transfected into 293A, and after virus production was confirmed, human adipocyte-derived MSC-TERT were infected with the pooled mixture, in lane 4 the final mixture was infected by freezing and thawing the mixture with cells prepared in the same manner as in lane 3, and in lane 5 the initial amount of DNA during the first transfection is 2.5 μg. In Fig. 10B, it was confirmed that the anti-E1B55K polyclonal antibody works normally by confirming that after amplification and purification of the viral mixture obtained under the conditions of lane 5, the intensity of the band corresponding to E1B55K also increases as the MOI increases when MSC-TERT are infected with the virus expressing replication-incompetent E1B55K at each MOI by counting viral infectious particles.

[173] The method for constructing a polyclonal antibody specific to E1B55K is as follows.

[174] First, the peptide sequence located in the N-terminal region of E1B55K (MERRNPSERGVPAGFSGHASVESGC: SEQ ID NO: 13) was synthesized, bovine serum albumin (BSA) was conjugated to the immunogenic carrier, and then New Zealand white rabbits were immunized and the serum was purified to obtain a polyclonal antibody (GW Vitek, Korea).

[175] 3) Comparison of virus production according to E1B55K expression level in human adipose tissue-derived MSC-TERT → confirmation of innovative enhancement of MSC virus production capacity according to gene transfer

[176] First, human adipocyte-derived MSC-TERT were transfected with 1 μg of E1B55K-expressing plasmid (pcDNA3.1-E1B55K) (Fig. 11) or infected with replication-incompetent adenovirus expressing E1B55K at each MOI (Fig. 12), and then the medium was replaced after 4 hours. And after infection with oncolytic adenovirus (YSC-02) at MOI 100, all the medium and cells were collected after 48 hours, then frozen and thawed to excite the virus as much as possible, and then the results of titration of infectious virus in the supernatant obtained by centrifugation are shown. As a result, it was confirmed that virus production was obviously increased compared with the control after infection with oncolytic adenovirus in E1B55K-expressing MSC-TERT (Fig. 12).

[177]

[178] Example 2: Innovative induction of increased virus production, regulation of the expression timing of the loaded gene, and a method for internal control of tumorigenesis can be realized simultaneously by introducing a time-shifted E1B55K expression regulation system.

[179] Increasing the manipulation of tumorigenic potential by the TERT gene, which had to be introduced for mass production of MSC cell lines, could provide a solution due to the fact that it was first determined that E1B55K induces not only an increase in viral replication but also a general decrease in MSC cell viability. That is, a manipulation method was developed in which inducing a temporal shift in the timing of E1B55K gene expression allowed continued passage of MSCs, thereby enabling mass production through cell banking while essentially blocking MSC tumorigenesis. Importantly, this marked an innovative turning point for virus/MSC therapeutics.

[180] The protocol in Fig. 13 refers to the Retro-X™Tet-One™ induced expression system (TAKARA cat# 634307).

[181] 1) Design of pRetro-X-Tet-One-puro-E1B55K

[182] In order to insert the E1B55K gene into the Retro-X-Tet-One-purovector plasmids, both ends must contain EcoRI and BamHI recognition sites, respectively. pCA14-E1B55K was used as a template to construct E1B55K containing the recognition sites, the E1B55K In-Fusion EcoRI sense primer is 5′-CCCTCGTAAAGAATTCATGGAGCGAAGAAACCCATCTGAG-3′ (SEQ ID NO: 14), and the E1B55K In-Fusion BamHI antisense primer is 5′-GAGGTGGTCTGGATCCTCAATCTGTATCTTCATCGCTAGA-3′ (SEQ ID NO: 15).

[183] The PCR conditions were as follows (Fig. 14).

[184] After an initial denaturation at 95°C for 2 min, 30 cycles of denaturation at 95°C for 40 s, annealing at 61°C for 40 s, and extension at 72°C for 1 min and 50 s were performed.

[185] Promega product (#M750 B) was used for PCR.

[186] After obtaining the plasmid construct (pRetro-X-Tetone-E1B55K sample #13 in Fig. 15), in which E1B55K was properly inserted into pRetro-X-Tetone using the In-Fusion exogenous gene introduction method, the introduction was finally confirmed by performing sequence analysis (Fig. 17). Then, it was checked whether the expression of the E1B55K protein, which is actually the inserted gene, was induced by doxycycline (Fig. 18). As in lane 5 in Fig. 18, it was confirmed that the expression of E1B55K was induced when A549 was transfected with the pRetro-X-Tetone-E1B55K plasmid only in the presence of doxycycline (2.5 μg/ml), and the expression of E1B55K was not induced when A549 was transfected with the same plasmid in the absence of doxycycline. That is, there was no leakage phenomenon. The far right lane represents the positive condition in which E1B55K was constitutively expressed, and thus the expression of E1B55K was confirmed.

[187] The pRetro-X-tetone-puro-ElB55K vector used for construction is shown in Fig. 16.

[188] 2) Confirmation of pRetro-X-tetone-puro-ElB55K expression

[189] After transfection with pRetro-X-Tetone-puro-ElB55K plasmid, the expression of E1B55K was confirmed in pRetro-X-tetone-puro-E1B55K according to the amount of doxycycline during doxycycline treatment. When the Tet One system was introduced to investigate whether the Tet One system could control the regulation of E1B55K expression in a time-shifting manner, it was confirmed that the expression of E1B55K did not occur at all before doxycycline treatment (Fig. 19). That is, it was confirmed that no leakage occurred. On the other hand, even treatment with a very low concentration of 0.05 μg/mL doxycycline immediately induced the expression of E1B55K. This means that cell viability can be maintained before doxycycline treatment, and the expression can be induced immediately after doxycycline treatment after reaching tumors.

[190] Time-shifted virus release was re-tested as follows.

[191] Following transfection of human adipose tissue MSC-TERT with pRetro-tetone-E1B55K plasmid (4 h) and then infection with oncolytic adenovirus expressing red fluorescent protein (RFP) (4 h), virus production was compared with and without doxycycline treatment after 48 h.

[192] As a result, it was confirmed that virus production was clearly increased by 10 or more times after doxycycline treatment (Fig. 20).

[193] 3) Obtaining the MSC-TERT-tetoneE1B55K cell line

[194] After the gene encoding E1B55K, which is the early region of adenovirus 1B, 55 kDa, was introduced into the Retro-X-Tet-One inducible expression system (TAKARA Bio Inc.), MSC-TERT were infected with this system to generate clones expressing E1B55K in the presence of doxycycline. For this purpose, GP2-293 packaging cells were co-transfected with pRetroX-TetOne-ElB55K and the pAmpho vector encoding a membrane protein (included in the TAKRA kit). After 48 hours, MSC-TERT, which are the target cells, were infected with the filtered retroviral supernatant. All clones selected with puromycin during culture were again subjected to Western blotting to confirm that the induction of E1B55K expression (Fig. 21) and p53 accumulation (Fig. 22) occurred simultaneously in the presence of doxycycline (2.5 μg/ml).

[195] These results showed that the induction of virus production could be increased at desired time points by adjusting the timing of expression of E1B55K, which is the loaded gene, depending on doxycycline treatment. (Since E1B55K gene expression needs to be induced, the amount of virus appears to start increasing at least 24 hours after doxycycline treatment.)

[196] 4) Implementation of the method of internal control of tumor formation

[197] It was tested whether clones 2 or 10 in Fig. 21 could drive tumorigenesis. For this purpose, long-term (approximately 14 days) culturing was performed after doxycycline treatment of 6 wells into which the cloned cell lines were aliquoted.

[198] As a result, it was confirmed that after 2 weeks of culture with doxycycline treatment (1.25 μg/mL), most of the cells lost their viability (1.25 μg/mL) (Fig. 23). Accordingly, even for uninfected MSCs, not those that were lysed due to infection with an oncolytic virus, the cell lines used, infected and selected with an E1B55K-expressing retrovirus, eventually undergo cell death, thereby essentially eliminating the possibility of tumorigenesis.

[199]

[200] Example 3: Establishing an Innovative Tumor Targeting Enhancement - Discovery of a Tumor Homing Enhancement Factor

[201] By inducing additional expression of GRP78 in human adipose tissue-derived MSC-TERT, the expression of tumor tropism-associated markers was confirmed.

[202] It was confirmed that the expression of all factors acting at each stage of MSC homing was increased by increasing the level of GRP78 (Fig. 24A).

[203] To confirm that GRP78 was accurately expressed, the increase in GRP78 mRNA expression was confirmed by transfecting human MSC-TERT with pcDNA3.1-GRP78 (2 μg) (Fig. 24B).

[204] 1) Cloning of GRP78 in pcDNA3.1 hygro (+)

[205] RNA was extracted from SNU449, a liver cancer cell line with high levels of GRP78 expression, using Trizol. The extracted RNA was reverse transcribed using the Superscript™ First Strand Synthesis System for Invirogen RT-PCR Kit. The primers used for PCR were then sequenced by adding an XhoI site in the forward direction and an XbaI site in the reverse direction. Cloning was performed by PCR (initial denaturation: 95°C for 2 min; denaturation: 95°C for 40 sec; annealing: 58°C for 40 sec; extension: 72°C for 2 min and 30 sec; 30 cycles) using forward (5'-GATTCTCGAGATGAAGCTCTCCCTGG-3': SEQ ID NO: 16) and reverse (5'-GGCCTCTAGACTACAACTCATCTTTT-3': SEQ ID NO: 17) primers. After PCR and XhoI/XbaI digestion, the pcDNA3.1-hygro vector backbone was also digested with XhoI/XbaI and ligated. Colonies obtained after transformation into bacteria were cultured. Each of the thus obtained plasmids was simultaneously digested with XhoI/XbaI to confirm the insert of the appropriate size only in plasmid 5 by insert confirmation (Fig. 25). After transfection of liver cancer cell line SNU449 with #5 pcDNA3.1-GRP78, the increase in GRP78 protein expression was confirmed by Western blotting (Fig. 26). In addition, sequence analysis of plasmid sample 5 showed 100% homology with NCBINM_005347 (Fig. 27).

[206] 2) Cloning GRP78 into PLNCX neo

[207] To introduce the GRP78 gene into MSC-TERT or MSC-TERT-tetonE1B55K, the following procedure was performed.

[208] To introduce GRP78 into the retroviral vector pLNCXneo, the pcDNA3.1-GRP78 vector was digested with XhoI/PmeI to extract the GRP78 region, and the GRP78 region was ligated and inserted into PLNCXneo previously digested with XhoI/PmeI.

[209] pLNCX neo was initially constructed by expanding the restriction enzyme recognition site from HindIII-HpaI-ClaI, which is the cloning site in the existing LNCX, to HindIII-Pm1I-BstXI-NotI-XhoI-Sa1I-ApaI-PmeI-HpaI-ClaI (Fig. 28).

[210] In this case, the sequence of threads looks like this.

[211] Upper 5'-AGCTTCACGTGCCAGCACAGTGGCGGCCGCTCGAGTCGACGGGCCCGTTTAAACGTTAACAT-3', (SEQ ID NO: 18)

[212] Bottom 5'-CGATGTTAACGTTTAAAACGGGCCCGTCGACTCGAGCGGCCGCCACTGTGCTGGCACGTGA-3 (SEQ ID NO: 19).

[213] In order to check whether LNCXneo-GRP78 was inserted, pLNCXneo (control) and pcDNA3.1-GRP78 (GRP78 gene-carrying plasmid) as a control were digested with HindIII/PmeI, respectively, and the GRP78 fragments obtained from the linearized PLNCXneo and pcDNA3.1-GRP78 were ligated and transformed into DH5a competent cells, and then it was confirmed that the pLNCXneo-GRP78 candidate constructs (samples 1 to 24) were digested at the same time and inserted into samples 2, 5, 9, 11, 12, 20, and 21, thereby obtaining plasmids (Fig. 29).

[214] 3) Construction of MSC-TERT-GRP78 cell line [215] Platinum-A packaging cells (Cell Biolabs) were transfected with the retroviral vector pLNCXneo-GRP78, and after 48 hours, the medium was filtered. Cell clones resistant to G418 (500 μg to 600 μg/ml) were obtained by infecting MSC-TERT with the retrovirus-containing medium solution obtained by filtration, and then screened to select clones 2 and 4 clearly expressing GRP78 and MMP2 (Fig. 30).

[216] 4) Targeting MSCs to tumor with gene transfer: Confirmation of conditions for specifically reaching only the tumor site within a very short period of time

[217] Validation of tumor targeting of MSC-TERT-GRP78

[218] 2× ¹⁰⁶ SNU398 cells (a liver cancer cell line) were subcutaneously transplanted into the shoulders of 6-week-old BALB/c nude mice, and 7 days later, 1× ¹⁰⁶ MSC-TERT-GRP78 cells infected with a replication-incompetent adenovirus expressing firefly luciferase were injected into the tail veins. 150 mg D-luciferin/kg mouse was injected intraperitoneally into the mice to use an in vivo imaging system (IVIS) device that detects luminescence. The in vivo biodistribution of MSC-TERT-GRP78 infected with the luciferase-expressing adenovirus was monitored 6 hours after tail vein injection by imaging using an IVIS Spectrum system (PerkinElmer). The special feature of this experiment is that MSC-TERT-GRP78 with GRP78 gene transfer can form tumors in the shoulder, which can distinguish them from tumors in internal organs such as the liver and lung, to accurately determine how great the tumor migration ability of MSC-TERT-GRP78 is, which clearly confirms that the MSC-TERT-GRP78 entry sites accumulated in the tumor tissue sites within a very short time without adsorption to other organs (Fig. 31).

[219] Validation of targeting of the final tumor type MSC-TERT-tetoneE1B55K-GRP78 with gene transfer

[220] 8× ¹⁰⁶ A549 cells (lung cancer cell line), 2× ¹⁰⁶ SNU398 cells (liver cancer cell line) or 2× ¹⁰⁶ MiaPaCa-2 cells (pancreatic cancer cell line) were transplanted subcutaneously into the shoulders of 6-week-old BALB/c nude mice, and 7 days later, 1× ¹⁰⁶ MSC-TERT-tetoneE1B55K-GRP78 cells infected with replication-incompetent adenovirus expressing firefly luciferase were injected into the tail veins. 150 mg D-luciferin/kg mouse was injected intraperitoneally into the mice to use the IVIS device, which detects luminescence. The in vivo biodistribution of MSCs infected with luciferase-expressing adenovirus was monitored after 6 h or 24 h using the IVIS Spectrum system (PerkinElmer).

[221] As a result, similar to the previous targeting experiment (T), tumors were formed on the shoulder side, which can distinguish them from tumors in internal organs such as the liver and lungs, thereby clearly confirming that the MSC entry sites accumulated in the tumor tissue sites in a very short time without adsorption to other organs (Fig. 32). As can be seen in this case, it can be observed that the final type MSC-TERT-tetoneE1B55K-GRP78 containing GRP78 migrated to the tumor site in a very short time compared with MSCs without GRP78 regardless of the cancer type, and it can be confirmed that there is a certain difference in intensity when comparing the luciferase activity intensity values (minimum 10 times, maximum up to 100 times depending on the luminescence intensity in the tumors). This shows that the tumor targeting is very fast and accurate. In this case, E1B55K is not expressed because the diet does not include doxycycline.

[222] Such remarkably rapid and precise migration of luciferase-expressing MSC-GRP78 into tumors and targeting of different tumor types greatly expands the potential of MSC-GRP78 for tumor diagnosis. In the primary mouse model, solid cancer was artificially transplanted subcutaneously and the tumor site was confirmed to allow MSC-GRP78 to migrate, but when a tumor similar to that in the primary mouse model is present in the body, even in the metastatic model, MSC-GRP78 can be delivered to the tumor site to emit light, or tumors can be detected by isotope labeling of tumors with higher resolution. This can be applied to tumor diagnosis, and eventually it will be possible to construct an OV/MSC complex that can both diagnose and treat tumors.

[223] Confirmation of MSC-TERT-tetoneE1B55K-GRP78 final type infiltration with gene transfer into tumors

[224] It was confirmed that the final type MSC-TERT-tetoneE1B55K-GRP78 reached the tumor sites, and the following experiment was conducted to confirm that the MSCs that actually reached the tumor sites infiltrated the tumor tissue.

[225] First, A549 cells were transplanted into the subcutaneous tissue of mice to form tumors. Then, the final type MSCs (MSC-TERT-tetoneE1B55K-GRP78) were labeled with a fluorescent cell tracking probe (Invitrogen, C34565) and then injected into the tail vein of mice. At 24 hours, 48 hours, and 72 hours after the injection, the tumor tissues were removed and tissue sections were made, and then the fluorescent region was confirmed under a fluorescence microscope. As shown in Fig. 33, it could be confirmed that an excessive number of MSCs with GRP78 were present in the tumor. In the case of MSCs without GRP78, it was observed that the MSCs mainly remained on the tumor surface with low fluorescence intensity.

[226] Confirmation of expression of adenovirus-associated proteins in tumor tissue

[227] Viruses were released from MSC-TERT-tetoneE1B55K-GRP78 that infiltrated into tumor tissue to infect tumor cells, and the following experiments were performed to confirm that the virus replicated and persisted for a certain period of time after virus production.

[228] 8× ¹⁰⁶ A549 cells (lung cancer cell line) were subcutaneously transplanted into the iliac fossa of 6-week-old BALB/c nude mice, and 1× ¹⁰⁶ MSC-TERT-tetoneE1B55K-GRP78 cells infected with oncolytic adenovirus expressing shHSP27-shTGFβ were injected into the tail veins of each of every 2 mice, in which the tumor size reached an average of 150 ^mm3 . Then, 3 days later, an additional 1× ¹⁰⁶ MSC-TERT-tetoneE1B55K-GRP78 cells infected with oncolytic adenovirus expressing shHSP27-shTGFβ1 were injected. Then, 7 days after the second injection, tumors were removed from mice in each group and tumor section samples were reacted with adenovirus type 5 specific antibody (Abeam, Cambridge, UK) to perform DAB staining reaction with secondary antibody, and control staining for immunohistochemistry was performed with the nuclear stain hematoxylin. In this case, all groups were fed a diet containing doxycycline (625 mg/kg) as chow.

[229] As a result, adenovirus-specific proteins were confirmed in most of the confirmed regions (Fig. 34). Adenovirus was released from MSCs in tumor tissue for at least 7 days after tail vein injection, indicating that replication, proliferation, and dissemination occurred repeatedly, and supporting the possibility that the virus remained until approximately 20 days when the tumor size had decreased to almost zero.

[230] 5) Generation of human adipose tissue MSC-TERT-tetoneE1B55K-GRP78 cell line - Confirmation of E1B55K expression after treatment with 1.25 μg/ml doxycycline for 48 hours

[231] Following infection with a retroviral mixture obtained by co-transfection of GP2-293 packaging cells with pRetroX-TetOne-E1B55K and the pAmpho vector encoding the membrane protein, selection was performed with puromycin (0.8 μg/ml to 1.0 μg/ml), following infection with a retroviral mixture obtained by re-transfection of Platinum-A packaging cells with the pLNCXneo-GRP78 retroviral vector, selection was performed with G418 (500 μg/ml to 600 μg/ml), and the resulting clones with clear expression of E1B55K and MMP2 proteins were selected in the presence of doxycycline (1.25 μg/ml) (Fig. 35, resulting clones 21, 24, 25 and 26).

[232] It was shown that in the final clone 21, the E1B55K induction concentration by doxycycline was efficiently and sufficiently induced without any leakage even at a very low concentration, less than 1/10 of the 1.25 μg/ml used (Fig. 36). This suggests that it can be reliably turned on and off in vivo and thus virus production can be induced at desired time points since it is not greatly affected by the amount of doxycycline, which is an inducer of in vivo expression, during treatment and is very sensitive to the presence or absence of doxycycline.

[233] Regarding the ability of human adipose tissue-derived MSC-TERT-tetoneE1B55K-GRP78 (clone 21) to produce viruses after infection with an oncolytic adenovirus expressing shHSP27/shTGFβ1, increased virus production was confirmed after 48 h in MSCs treated with doxycycline (1.25 μg/ml), and in this case, a difference in virus production depending on E4orf6 gene expression was also confirmed. For this experiment, MSC clone 21 were transfected with 1 μg of pFlag-CMV2-E4orf6 into which the E4orf6 gene was subcloned or 1 μg of pFlag-CMV2 as a control, and after additional infection with an oncolytic adenovirus expressing shHSP27/shTGFβ1, the cells were treated with doxycycline (1.25 μg/ml for 48 h), and then the total amount of virus in the pooled mixture and the remaining cells was determined by titration. As a result, in the final clone 21, an almost 10-fold increase in virus production was confirmed upon doxycycline treatment, and a difference in virus production was observed depending on the presence or absence of transfection with the E4orf6 gene (Fig. 37). That is, the amount of virus production was reduced in accordance with the external induction of E4orf6 expression. It appears that the increase in virus production by E1B55K alone acts as an inhibitory factor for virus production, which is caused by the formation of the E40RF6 complex. (It has been confirmed that the induction of MSC cell death by E1B55K alone promotes virus production. The E1B55K/E4orf6 complex prevents MSC cell death, which results in the omission of the optimal time for virus production.)

[234] 6) Comparison of maintenance of human adipose tissue MSC-TERT efficacy at low and high passage numbers

[235] It is important to confirm that the efficacy of MSCs is maintained in mass production of 200 or more vials of cell lines from a master cell bank. That is, it was confirmed by Western blotting on samples in which each cell was lysed from low passage number (p13) and high passage number (p23) whether biomarkers known as tumor homing-associated markers, which is a characteristic of MSCs due to the difference in passage number of the resulting MSC-TERT-tetoneE1B55K-GRP78, were retained.

[236] As a result, it was confirmed that even when the passage number was increased to 20, there was no difference in the expression of proteins known as tumor homing marker (Fig. 38).

[237]

[238] Example 4: Anticancer efficacy of MSC-TERT-tetoneE1B55K-GRP78 infected with oncolytic adenovirus

[239] 8× ¹⁰⁶ A549 cells (lung cancer cell line) were subcutaneously transplanted into the flanks of 6-week-old BALB/c nude mice, and mice bearing tumors that averaged 150 ^mm3 in size were divided into groups of 10 mice each. 1× ¹⁰⁶ MSC-TERT-tetoneE1B55K-GRP78 cells infected with an oncolytic adenovirus expressing shHSP27-shTGFβ1 were injected into the tail veins of mice. Then, 3 days later, 1× ¹⁰⁶ MSC-TERT-tetoneE1B55K-GRP78 cells infected with an oncolytic adenovirus expressing shHSP27-shTGFβ1 were further injected intravenously. A group that was transplanted with tumor cells only (no treatment) and a group that was injected with the final MSC-TERT-tetoneE1B55K-GRP78 (MSC only) were used as controls. In this case, all groups were fed a diet containing doxycycline (625 mg/kg) as food.

[240] As a result, it was found that when comparing the antitumor effect obtained by direct intratumor injection of oncolytic virus (Ad-3484-shHSP27-shTGFβ1) (a condition in which the growth rate of tumor size decreased compared with the control, but the tumor size itself decreased and therefore did not regress), the tumor size was significantly reduced and the tumor almost disappeared within a few days when the oncolytic virus (Ad-3484-shHSP27-shTGFβ1) was injected into the tail veins in the final MSC-TERT-tetoneE1B55K-GRP78 compared to the untreated tumors or the control when only the final MSC was injected into the tail vein, as shown in Fig. 39, and it can be seen that within 20 days after the injection, the tumors almost disappeared and then the tumors grew slightly again in some individuals. Using individual photographs of mice in each group and photographs of tumors taken for each group after tumor removal one month after the first OV/MSC injection, the antitumor effect of the oncolytic virus (Ad-3484-shHSP27-shTGFβ1) was more reliably confirmed in the resulting MSC-TERT-tetoneE1B55K-GRP78 (Fig. 40).

[241]

[242] Example 5: Confirmation of adsorption to organs other than tumors

[243] After tumor targeting was confirmed to be highly efficient, the adenoviral gene copy number was measured at each time point for the amount of adenovirus in each major organ, including tumors, to further confirm the amount captured in major organs other than tumors (lung, liver, spleen, and similar organs), migration to the tumor site, elapsed time, and similar parameters. Viral genomic plasmid DNA was defined as the standard known DNA and confirmed by constructing a standard curve (in the case of the viral DNA of the currently used oncolytic adenovirus, 1 copy equals 10 ³ pg).

[244] For this purpose, after infection with the replication-incompetent defective virus with E1B55K, one dose of MSC-TERT-GRP78 (1× ¹⁰⁶ ) infected with the oncolytic virus YSC-02 of Reference Example 1 was administered intravenously through the tail once, and the samples were collected by date (day 0, 1, 2, 3, 4, 5, 10, and 15). The collected tissues (tumors, lungs, liver, spleen, kidneys, heart) were immediately stored in liquid nitrogen, and on the day when the tissue collection was completed, genomic DNA was extracted from each tissue under the same conditions. To confirm gene expression, real-time PCR was performed using the extracted genomic DNA using primers for the E40RF gene. The primers for E40RF screening used this time are as follows.

[245] Forward 5'-CGTGGTCAAACTCTACAGCC-3': SEQ ID NO: 20

[246] Reverse 5'-GCATGAGCATGACTACGATG-3': SEQ ID NO: 21

[247] As can be seen in Fig. 41, although the differences between individuals are significant, it can be seen that the range in individual tissues when the oncolytic virus enters tumor tissue is from 10 ⁴ to 10 ¹⁰ copies, and almost all of the viruses are adsorbed and absorbed into tumor tissue compared to a few to 10 copies found in all other organs.

--->

<110> DAYS BIO CO., LTD.

<120> MESENCHYMAL STEM CELLS THAT PROVIDE IMPROVEMENT

TUMOR TARGETING AND MASSIVE VIRUS PRODUCTION

<130> X21U18C0183

<150> KR 10-2020-0139918

<151> 2020-10-27

<160> 31

<170> KoPatentIn 3.0

<210> 1

<211> 1965

<212> DNA

<213> Homo sapiens

<400> 1

atgaagctct ccctggtggc cgcgatgctg ctgctgctca gcgcggcgcg ggccgaggag 60

gaggacaaga aggaggacgt gggcacggtg gtcggcatcg acctggggac cacctactcc 120

tgcgtcggcg tgttcaagaa cggccgcgtg gagatcatcg ccaacgatca gggcaaccgc 180

atcacgccgt cctatgtcgc cttcactcct gaaggggaac gtctgattgg cgatgccgcc 240

aagaaccagc tcacctccaa ccccgagaac acggtctttg acgccaagcg gctcatcggc 300

cgcacgtgga atgacccgtc tgtgcagcag gacatcaagt tcttgccgtt caaggtggtt 360

gaaaagaaaa ctaaaccata cattcaagtt gatattggag gtgggcaaac aaagacattt 420

gctcctgaag aaatttctgc catggttctc actaaaatga aagaaaccgc tgaggcttat 480

ttgggaaaga aggttaccca tgcagttgtt actgtaccag cctattttaa tgatgcccaa 540

cgccaagcaa ccaaagacgc tggaactatt gctggcctaa atgttatgag gatcatcaac 600

gagcctacgg cagctgctat tgcttatggc ctggataaga gggaggggga gaagaacatc 660

ctggtgtttg acctgggtgg cggaaccttc gatgtgtctc ttctcaccat tgacaatggt 720

gtcttcgaag ttgtggccac taatggagat actcatctgg gtggagaaga ctttgaccag 780

cgtgtcatgg aacacttcat caaactgtac aaaaagaaga cgggcaaaga tgtcaggaaa 840

gacaatagag ctgtgcagaa actccggcgc gaggtagaaa aggccaaacg ggccctgtct 900

tctcagcatc aagcaagaat tgaaattgag tccttctatg aaggagaaga cttttctgag 960

accctgactc gggccaaatt tgaagagctc aacatggatc tgttccggtc tactatgaag 1020

cccgtccaga aagtgttgga agattctgat ttgaagaagt ctgatattga tgaaattgtt 1080

cttgttggtg gctcgactcg aattccaaag attcagcaac tggttaaaga gttcttcaat 1140

ggcaaggaac catcccgtgg cataaaccca gatgaagctg tagcgtatgg tgctgctgtc 1200

caggctggtg tgctctctgg tgatcaagat acaggtgacc tggtactgct tgatgtatgt 1260

ccccttacac ttggtattga aactgtggga ggtgtcatga ccaaactgat tccaaggaac 1320

acagtggtgc ctaccaagaa gtctcagatc ttttctacag cttctgataa tcaaccaact 1380

gttacaatca aggtctatga aggtgaaaga cccctgacaa aagacaatca tcttctgggt 1440

acatttgatc tgactggaat tcctcctgct cctcgtgggg tcccacagat tgaagtcacc 1500

tttgagatag atgtgaatgg tattcttcga gtgacagctg aagacaaggg tacagggaac 1560

aaaaataaga tcacaatcac caatgaccag aatcgcctga cacctgaaga aatcgaaagg 1620

atggttaatg atgctgagaa gtttgctgag gaagacaaaa agctcaagga gcgcattgat 1680

actagaaatg agttggaaag ctatgcctat tctctaaaga atcagattgg agataaagaa 1740

aagctgggag gtaaactttc ctctgaagat aaggagacca tggaaaaagc tgtagaagaa 1800

aagattgaat ggctggaaag ccaccaagat gctgacattg aagacttcaa agctaagaag 1860

aaggaactgg aagaaattgt tcaaccaatt atcagcaaac tctatggaag tgcaggccct 1920

cccccaactg gtgaagagga tacagcagaa aaagatgagt tgtag 1965

<210> 2

<211> 3399

<212> DNA

<213> Homo sapiens

<400> 2

atgccgcgcg ctccccgctg ccgagccgtg cgctccctgc tgcgcagcca ctaccgcgag 60

gtgctgccgc tggccacgtt cgtgcggcgc ctggggcccc agggctggcg gctggtgcag 120

cgcggggacc cggcggcttt ccgcgcgctg gtggcccagt gcctggtgtg cgtgccctgg 180

gacgcacggc cgccccccgc cgccccctcc ttccgccagg tgtcctgcct gaaggagctg 240

gtggcccgag tgctgcagag gctgtgcgag cgcggcgcga agaacgtgct ggccttcggc 300

ttcgcgctgc tggacggggc ccgcgggggc ccccccgagg ccttcaccac cagcgtgcgc 360

agctacctgc ccaacacggt gaccgacgca ctgcggggga gcggggcgtg ggggctgctg 420

ctgcgccgcg tgggcgacga cgtgctggtt cacctgctgg cacgctgcgc gctctttgtg 480

ctggtggctc ccagctgcgc ctaccaggtg tgcgggccgc cgctgtacca gctcggcgct 540

gccactcagg cccggccccc gccacacgct agtggacccc gaaggcgtct gggatgcgaa 600

cgggcctgga accatagcgt cagggaggcc ggggtccccc tgggcctgcc agccccgggt 660

gcgaggaggc gcggggggcag tgccagccga agtctgccgt tgcccaagag gcccaggcgt 720

ggcgctgccc ctgagccgga gcggacgccc gttgggcagg ggtcctgggc ccacccgggc 780

aggacgcgtg gaccgagtga ccgtggtttc tgtgtggtgt cacctgccag acccgccgaa 840

gaagccacct ctttggaggg tgcgctctct ggcacgcgcc actcccaccc atccgtgggc 900

cgccagcacc acgcgggccc cccatccaca tcgcggccac cacgtccctg ggacacgcct 960

tgtcccccgg tgtacgccga gaccaagcac ttcctctact cctcaggcga caaggagcag 1020

ctgcggccct ccttcctact cagctctctg aggcccagcc tgactggcgc tcggaggctc 1080

gtggagacca tctttctggg ttccaggccc tggatgccag ggactccccg caggttgccc 1140

cgcctgcccc agcgctactg gcaaatgcgg cccctgtttc tggagctgct tgggaaccac 1200

gcgcagtgcc cctacggggt gctcctcaag acgcactgcc cgctgcgagc tgcggtcacc 1260

ccagcagccg gtgtctgtgc ccgggagaag ccccagggct ctgtggcggc ccccgaggag 1320

gaggacag acccccgtcg cctggtgcag ctgctccgcc agcacagcag cccctggcag 1380

gtgtacggct tcgtgcgggc ctgcctgcgc cggctggtgc ccccaggcct ctggggctcc 1440

aggcacaacg aacgccgctt cctcaggaac accaagaagt tcatctccct ggggaagcat 1500

gccaagctct cgctgcagga gctgacgtgg aagatgagcg tgcgggactg cgcttggctg 1560

cgcaggagcc caggggttgg ctgtgttccg gccgcagagc accgtctgcg tgaggagatc 1620

ctggccaagt tcctgcactg gctgatgagt gtgtacgtcg tcgagctgct caggtctttc 1680

ttttatgtca cggagaccac gtttcaaaag aacaggctct ttttctaccg gaagagtgtc 1740

tggagcaagt tgcaaagcat tggaatcaga cagcacttga agagggtgca gctgcggggag 1800

ctgtcggaag cagaggtcag gcagcatcgg gaagccaggc ccgccctgct gacgtccaga 1860

ctccgcttca tccccaagcc tgacgggctg cggccgattg tgaacatgga ctacgtcgtg 1920

ggagccagaa cgttccgcag agaaaagagg gccgagcgtc tcacctcgag ggtgaaggca 1980

ctgttcagcg tgctcaacta cgagcgggcg cggcgccccg gcctcctggg cgcctctgtg 2040

ctgggcctgg acgatatcca cagggcctgg cgcaccttcg tgctgcgtgt gcgggcccag 2100

gacccgccgc ctgagctgta ctttgtcaag gtggatgtga cgggcgcgta cgacaccatc 2160

ccccaggaca ggctcacgga ggtcatcgcc agcatcatca aaccccagaa cacgtactgc 2220

gtgcgtcggt atgccgtggt ccagaaggcc gcccatgggc acgtccgcaa ggccttcaag 2280

agccacgtct ctaccttgac agacctccag ccgtacatgc gacagttcgt ggctcacctg 2340

caggagacca gcccgctgag ggatgccgtc gtcatcgagc agagctcctc cctgaatgag 2400

gccagcagtg gcctcttcga cgtcttccta cgcttcatgt gccaccacgc cgtgcgcatc 2460

aggggcaagt cctacgtcca gtgccagggg atcccgcagg gctccatcct ctccacgctg 2520

ctctgcagcc tgtgctacgg cgacatggag aacaagctgt ttgcggggat tcggcgggac 2580

gggctgctcc tgcgtttggt ggatgatttc ttgttggtga cacctcacct cacccacgcg 2640

aaaaccttcc tcaggaccct ggtccgaggt gtccctgagt atggctgcgt ggtgaacttg 2700

cggaagacag tggtgaactt ccctgtagaa gacgaggccc tgggtggcac ggcttttgtt 2760

cagatgccgg cccacggcct attcccctgg tgcggcctgc tgctggatac ccggaccctg 2820

gaggtgcaga gcgactactc cagctatgcc cggacctcca tcagagccag tctcaccttc 2880

aaccgcggct tcaaggctgg gaggaacatg cgtcgcaaac tctttggggt cttgcggctg 2940

aagtgtcaca gcctgtttct ggatttgcag gtgaacagcc tccagacggt gtgcaccaac 3000

atctacaaga tcctcctgct gcaggcgtac aggtttcacg catgtgtgct gcagctccca 3060

tttcatcagc aagtttggaa gaaccccaca tttttcctgc gcgtcatctc tgacacggcc 3120

tccctctgct actccatcct gaaagccaag aacgcaggga tgtcgctggg ggccaagggc 3180

gccgccggcc ctctgccctc cgaggccgtg cagtggctgt gccaccaagc attcctgctc 3240

aagctgactc gacaccgtgt cacctacgtg ccactcctgg ggtcactcag gacagcccag 3300

acgcagctga gtcggaagct cccggggacg acgctgactg ccctggaggc cgcagccaac 3360

ccggcactgc cctcagactt caagaccatc ctggactga 3399

<210> 3

<211> 1491

<212> DNA

<213> Human adenovirus type 5

<400> 3

atggagcgaa gaaacccatc tgagcgggga gtacctgctg gattttctgg ccatgcatct 60

gtggagagcg gttgtgagac acaagaatcg cctgctactg ttgtcttccg tccgcccggc 120

gataataccg acggagggagc agcagcagca gcaggaggaa gccaggcggc ggcggcagga 180

gcagagccca tggaacccga gagccggcct ggaccctcgg gaatgaatgt tgtacaggtg 240

gctgaactgt atccagaact gagacgcatt ttgacaatta cagaggatgg gcaggggcta 300

aagggggtaa agagggagcg gggggcttgt gaggctacag aggaggctag gaatctagct 360

tttagcttaa tgaccagaca ccgtcctgag tgtattactt ttcaacagat caaggataat 420

tgcgctaatg agcttgatct gctggcgcag aagtattcca tagagcagct gaccacttac 480

tggctgcagc caggggatga ttttgaggag gctattaggg tatatgcaaa ggtggcactt 540

aggccagatt gcaagtacaa gatcagcaaa cttgtaaata tcaggaattg ttgctacatt 600

tctgggaacg gggccgaggt ggagatagat acggaggata gggtggcctt tagatgtagc 660

atgataaata tgtggccggg ggtgcttggc atggacgggg tggttattat gaatgtaagg 720

tttactggcc ccaattttag cggtacggtt ttcctggcca ataccaacct tatcctacac 780

ggtgttagct tctatgggtt taacaatacc tgtgtggaag cctggaccga tgtaagggtt 840

cggggctgtg ccttttactg ctgctggaag ggggtggtgt gtcgccccaa aagcagggct 900

tcaattaaga aatgcctctt tgaaaggtgt accttgggta tcctgtctga gggtaactcc 960

agggtgcgcc acaatgtggc ctccgactgt ggttgcttca tgctagtgaa aagcgtggct 1020

gtgattaagc ataacatggt atgtggcaac tgcgaggaca gggcctctca gatgctgacc 1080

tgctcggacg gcaactgtca cctgctgaag accattcacg tagccagcca ctctcgcaag 1140

gcctggccag tgtttgagca taacatactg acccgctgtt ccttgcattt gggtaacagg 1200

aggggggtgt tcctacctta ccaatgcaat ttgagtcaca ctaagatatt gcttgagccc 1260

gagagcatgt ccaaggtgaa cctgaacggg gtgtttgaca tgaccatgaa gatctggaag 1320

gtgctgaggt acgatgagac ccgcaccagg tgcagaccct gcgagtgtgg cggtaaacat 1380

attaggaacc agcctgtgat gctggatgtg accgaggagc tgaggcccga tcacttggtg 1440

ctggcctgca cccgcgctga gtttggctct agcgatgaag atacagattg a 1491

<210> 4

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer BamHI/NotI

<400> 4

gcggatccat ggagcgaaga aacccatct 29

<210> 5

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer BamHI/NotI

<400> 5

cggccgctca atctgtatct tcatcgct 28

<210> 6

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer HindIII

<400> 6

gtacaagctt atgactacgt ccggcgttcc 30

<210> 7

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer XbaI

<400> 7

cacctctaga ctacatgggg gtagagtcat 30

<210> 8

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223>CMV30

<400> 8

aatgtcgtaa taaccccgcc ccgttgacgc 30

<210> 9

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Sense primer XbaI

<400> 9

ttcatctaga atggagcgaa gaaacccatc 30

<210> 10

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Antisense primer HindIII

<400> 10

gacgaagctt tcaatctgta tcttcatcgc 30

<210> 11

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> hCMV Forward Primer

<400> 11

gggaggtcta tataagcaga gctcg 25

<210> 12

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer pCA14 SV40

<400> 12

catgatcgat gctagacgat ccaga 25

<210> 13

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> N-terminus of E1B55K

<400> 13

Met Glu Arg Arg Asn Pro Ser Glu Arg Gly Val Pro Ala Gly Phe Ser

1 5 10 15

Gly His Ala Ser Val Glu Ser Gly Cys

20 25

<210> 14

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Sense primer E1B55K In-Fusion EcoRI

<400> 14

ccctcgtaaa gaattcatgg agcgaagaaa cccatctgag 40

<210> 15

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Antisense Primer E1B55K In-Fusion BamHI

<400> 15

gaggtggtct ggatcctcaa tctgtatctt catcgctaga 40

<210> 16

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer XbaI

<400> 16

gattctcgag atgaagctct ccctgg 26

<210> 17

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer XbaI

<400> 17

ggcctctaga ctacaactca tctttt 26

<210> 18

<211> 62

<212> DNA

<213> Artificial sequence

<220>

<223> Upper thread pLNCX neo

<400> 18

agcttcacgt gccagcacag tggcggccgc tcgagtcgac gggcccgttt aaacgttaac 60

at 62

<210> 19

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Bottom thread pLNCX neo

<400> 19

cgatgttaac gtttaaacgg gcccgtcgac tcgagcggcc gccactgtgc tggcacgtga 60

60

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer E4 ORF

<400> 20

cgtggtcaaa ctctacagcc 20

<210> 21

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer E4 ORF

<400> 21

gcatgagcat gactacgatg 20

<210> 22

<211> 912

<212> DNA

<213> Artificial sequence

<220>

<223> Flag-E4orf6

<400> 22

gactacaaag acgatgacga caagcttatg actacgtccg gcgttccatt tggcatgaca 60

ctacgaccaa cacgatctcg gttgtctcgg cgcactccgt acagtaggga tcgtctacct 120

ccttttgaga cagaaacccg cgctaccata ctggaggatc atccgctgct gcccgaatgt 180

aacactttga caatgcacaa cgtgagttac gtgcgaggtc ttccctgcag tgtgggattt 240

acgctgattc aggaatgggt tgttccctgg gatatggttc taacgcggga ggagcttgta 300

atcctgagga agtgtatgca cgtgtgcctg tgttgtgcca acattgatat catgacgagc 360

atgatgatcc atggttacga gtcctgggct ctccactgtc attgttccag tcccggttcc 420

ctgcagtgta tagccggcgg gcaggttttg gccagctggt ttaggatggt ggtggatggc 480

gccatgttta atcagaggtt tatatggtac cgggaggtgg tgaattacaa catgccaaaa 540

gaggtaatgt ttatgtccag cgtgtttatg aggggtcgcc acttaatcta cctgcgcttg 600

tggtatgatg gccacgtggg ttctgtggtc cccgccatga gctttggata cagcgccttg 660

cactgtggga ttttgaacaa tattgtggtg ctgtgctgca gttactgtgc tgatttaagt 720

gagatcaggg tgcgctgctg tgcccggagg acaaggcgcc ttatgctgcg ggcggtgcga 780

atcatcgctg aggagaccac tgccatgttg tattcctgca ggacggagcg gcggcggcag 840

cagtttattc gcgcgctgct gcagcaccac cgccctatcc tgatgcacga ttatgactct 900

acccccatgt ag 912

<210> 23

<211> 1196

<212> DNA

<213> Artificial sequence

<220>

<223> sample 2 - fig. 7

<400> 23

aaccgtcaga attgatctac catggactac aaagacgatg acgacaagct tatgactacg 60

tccggcgttc catttggcat gacactacga ccaacacgat ctcggttgtc tcggcgcact 120

ccgtacagta gggatcgtct acctcctttt gagacagaaa cccgcgctac catactggag 180

gatcatccgc tgctgcccga atgtaacact ttgacaatgc acaacgtgag ttacgtgcga 240

ggtcttccct gcagtgtggg atttacgctg attcaggaat gggttgttcc ctgggatatg 300

gttctaacgc gggaggagct tgtaatcctg aggaagtgta tgcacgtgtg cctgtgttgt 360

gccaacattg atatcatgac gagcatgatg atccatggtt acgagtcctg ggctctccac 420

tgtcattgtt ccagtcccgg ttccctgcag tgtatagccg gcgggcaggt tttggccagc 480

tggtttagga tggtggtgga tggcgccatg tttaatcaga ggtttatatg gtaccgggag 540

gtggtgaatt acaacatgcc aaaagaggta atgtttatgt ccagcgtgtt tatgaggggt 600

cgccacttaa tctacctgcg cttgtggtat gatggccacg tgggttctgt ggtccccgcc 660

atgagctttg gatacagcgc cttgcactgt gggattttga acaatattgt ggtgctgtgc 720

tgcagttact gtgctgattt aagtgagatc agggtgcgct gctgtgcccg gaggacaagg 780

cgccttatgc tgcgggcggt gcgaatcatc gctgaggaga ccactgccat gttgtattcc 840

tgcaggacgg agcggcggcg gcagcagttt attcgcgcgc tgctgcagca ccaccgccct 900

atcctgatgc acgattatga ctctaccccc atgtagtcta gaggatcccg ggtggcatcc 960

ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag 1020

ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat 1080

tatggggtgg agggggggg tatggaacca aggggcaagt tgggaaaaaa acctgaaggg 1140

cctgcgggtc ttattgggaa ccaaactggg agtgcagtgg accaattttg ggttcc 1196

<210> 24

<211> 960

<212> DNA

<213> Artificial sequence

<220>

<223> pCA14-E1B55K direct

<400> 24

aagacaccgg gaccgatcca gcctggggat cttcgagtcg agggatccct cgagtctaga 60

atggagcgaa gaaacccatc tgagcgggga gtacctgctg gattttctgg ccatgcatct 120

gtggagagcg gttgtgagac acaagaatcg cctgctactg ttgtcttccg tccgcccggc 180

gataataccg acggaggagc agcagcagca gcaggaggaa gccaggcggc ggcggcagga 240

gcagagccca tggaacccga gagccggcct ggaccctcgg gaatgaatgt tgtacaggtg 300

gctgaactgt atccagaact gagacgcatt ttgacaatta cagaggatgg gcaggggcta 360

aagggggtaa agagggagcg gggggcttgt gaggctacag aggaggctag gaatctagct 420

tttagcttaa tgaccagaca ccgtcctgag tgtattactt ttcaacagat caaggataat 480

tgcgctaatg agcttgatct gctggcgcag aagtattcca tagagcagct gaccacttac 540

tggctgcagc caggggatga ttttgaggag gctattaggg tatatgcaaa ggtggcactt 600

aggccagatt gcaagtacaa gatcagcaaa cttgtaaata tcaggaattg ttgctacatt 660

tctgggaacg gggccgaggt ggagatagat acggaggata gggtggcctt tagatgtagc 720

atgataaata tgtggccggg ggtgcttggc atggacgggg tggttattat gaatgtaagg 780

tttactggcc ccaattttag cggtacggtt ttcctggcca ataccaacct tatcctacac 840

ggtgttagct tctatgggtt taacaatacc tgtgtggaag cctggaccga tgtaagggtt 900

cggggctgtg ccttttactg ctgctggaag ggggtggtgt gtcgccccaa aagcagggct 960

960

<210> 25

<211> 1017

<212> DNA

<213> Artificial sequence

<220>

<223> pCA14-E1B55K reverse

<400> 25

tttggtccaa ccggggatga tttgaggggg cttttgggtt tttgcaaagg tgcccttagg 60

ccagattgca attacaaaat cagcaaactt gtaaatttca ggaattgttg ctacatttct 120

gggaacgggg ccgaggtgga gatagatacg gaggataggg tggcctttag atgtagcatg 180

ataaatatgt ggccgggggt gcttggcatg gacggggtgg ttattatgaa tgtaaggttt 240

actggcccca attttagcgg tacggttttc ctggccaata ccaaccttat cctacacggt 300

gttagcttct atgggtttaa caatacctgt gtggaagcct ggaccgatgt aagggttcgg 360

ggctgtgcct tttactgctg ctggaagggg gtggtgtgtc gccccaaaag cagggcttca 420

attaagaaat gcctctttga aaggtgtacc ttgggtatcc tgtctgaggg taactccagg 480

gtgcgccaca atgtggcctc cgactgtggt tgcttcatgc tagtgaaaag cgtggctgtg 540

attaagcata acatggtatg tggcaactgc gaggacagg cctctcagat gctgacctgc 600

tcggacggca actgtcacct gctgaagacc attcacgtag ccagccactc tcgcaaggcc 660

tggccagtgt ttgagcataa catactgacc cgctgttcct tgcatttggg taacaggagg 720

ggggtgttcc taccttacca atgcaatttg agtcacacta agatattgct tgagcccgag 780

agcatgtcca aggtgaacct gaacggggtg tttgacatga ccatgaagat ctggaaggtg 840

ctgaggtacg atgagacccg caccaggtgc agaccctgcg agtgtggcgg taaacatatt 900

aggaaccagc ctgtgatgct ggatgtgacc gaggagctga ggcccgatca cttggtgctg 960

gcctgcaccc gcgctgagtt tggctctagc gatgaagata cagattgaaa gcttgtc 1017

<210> 26

<211> 1172

<212> DNA

<213> Artificial sequence

<220>

<223> primer E1B55K In-Fusion EcoRI sense

<400> 26

ccggggggca gtacctgctg gattctctgg ccatgcatct gtggagagcg gttgtgagac 60

acaagaatcg cctgctactg ttgtcttccg tccgcccggc gataataccg acggagaggagc 120

agcagcagca gcaggaggaa gccaggcggc ggcggcagga gcagagccca tggaacccga 180

gagccggcct ggaccctcgg gaatgaatgt tgtacaggtg gctgaactgt atccagaact 240

gagacgcatt ttgacaatta cagaggatgg gcaggggcta aagggggtaa agagggagcg 300

gggggcttgt gaggctacag aggaggctag gaatctagct tttagcttaa tgaccagaca 360

ccgtcctgag tgtattactt ttcaacagat caaggataat tgcgctaatg agcttgatct 420

gctggcgcag aagtattcca tagagcagct gaccacttac tggctgcagc caggggatga 480

ttttgaggag gctattaggg tatatgcaaa ggtggcactt aggccagatt gcaagtacaa 540

gatcagcaaa cttgtaaata tcaggaattg ttgctacatc tctgggaacg gggccgaggt 600

ggagatagat acggaggata gggtggcctt tagatgtagc atgataaata tgtggccggg 660

ggtgcttggc atggacgggg tggttattat gaatgtaagg tttactggcc ccaattttag 720

cggtacggtt ttcctggcca ataccaacct tatcctacac ggtgttagct tctatgggtt 780

taacaatacc tgtgtggaag cctggaccga tgtaagggtt cggggctgtg ccttttactg 840

ctgctggaag ggggtggtgt gtcgccccaa aagcagggct tcaattaaga aatgcctctt 900

tgaaaggtgt accttgggta tcctgtctga gggtaactcc agggtgcgcc acaatgtggc 960

ctccgactgt ggttgcttca tgctagtgaa aagcgtggct gtgattaagc ataacatggt 1020

atgtggcaac tgcgaggaca gggcctctca gatgctgacc tgctcggacg gcaactgtca 1080

cctgctgaag acattcacgt agccagccct ttcgcaaggc tggccagggt tgagcatacc 1140

tactgacccc ctgttccttg ctttgggaaa aa 1172

<210> 27

<211> 686

<212> DNA

<213> Artificial sequence

<220>

<223> primer: internal primer E1B55K

<400> 27

acctgtaatt tgtttatttg ggcgtaggtc gggcgtgctt ttactgctgc tggaaggggg 60

tggtgtgtcg ccccaaaagc agggcttcaa ttaagaaatg cctctttgaa aggtgtacct 120

tgggtatcct gtctgagggt aactccaggg tgcgccacaa tgtggcctcc gactgtggtt 180

gcttcatgct agtgaaaagc gtggctgtga ttaagcataa catggtatgt ggcaactgcg 240

aggacagggc ctctcagatg ctgacctgct cggacggcaa ctgtcacctg ctgaagacca 300

ttcacgtagc cagccactct cgcaaggcct ggccagtgtt tgagcataac atactgaccc 360

gctgttcctt gcatttgggt aacaggagg gggtgttcct accttaccaa tgcaatttga 420

gtcacactaa gatattgctt gagcccgaga gcatgtccaa ggtgaacctg aacggggtgt 480

ttgacatgac catgaagatc tggaaggtgc tgaggtacga tgagacccgc accaggtgca 540

gaccctgcga gtgtggcggt aaacatatta ggaaccagcc tgtgatgctg gatgtgaccg 600

aggagctgag gcccgatcac ttggtgctgg cctgcacccg cgctgagttt ggctctagcg 660

atgaagatac agattgagga tccaga 686

<210> 28

<211> 483

<212> DNA

<213> Artificial sequence

<220>

<223> Internal reverse primer near 5' E1B55K

<400> 28

aaagaattca tggagcgaag aaacccatct gagcggggag tacctgctgg attctctggc 60

catgcatctg tggagagcgg ttgtgagaca caagaatcgc ctgctactgt tgtcttccgt 120

ccgcccggcg ataataccga cggaggagca gcagcagcag caggaggaag ccaggcggcg 180

gcggcaggag cagagcccat ggaacccgag agccggcctg gaccctcggg aatgaatgtt 240

gtacaggtgg ctgaactgta tccagaactg agacgcattt tgacaattac agaggatggg 300

caggggctaa agggggtaaa gagggagcgg ggggcttgtg aggctacaga ggaggctagg 360

aatctagctt ttagcttaat gaccagacac cgtcctgagt gtattacttt tcaacagatc 420

aaggataatt gcgctaatga gctgatctgc gcgcagacgc gccaacaaaa caagagtcca 480

gtc 483

<210> 29

<211> 1246

<212> DNA

<213> Artificial sequence

<220>

<223> Primer: E1B55K In-Fusion BamHI antisense

<400> 29

aaaggatggc agggctaaag gggtaaagag gagcggggggc ttggaggcta cagaggaggc 60

taggaatcta gcttttagct taatgaccag acaccgtcct gagtgtatta cttttcaaca 120

gatcaaggat aattgcgcta atgagcttga tctgctggcg cagaagtatt ccatagagca 180

gctgaccact tactggctgc agccagggga tgattttgag gaggctatta gggtatatgc 240

aaaggtggca cttaggccag attgcaagta caagatcagc aaacttgtaa atatcaggaa 300

ttgttgctac atctctggga acggggccga ggtggagata gatacggagg atagggtggc 360

ctttagatgt agctctggga acggggccga ggtggagata gatacggagg atagggtggc 420

ctttagatgt agctctggga acggggccga ggtggagata gatacggagg atagggtggc 480

ctttagatgt agctttactg gccccaattt tagcggtacg gttttcctgg ccaataccaa 540

ccttatccta cacggtgtta gcttctatgg gtttaacaat acctgtgtgg aagcctggac 600

cgatgtaagg gttcggggct gtgcctttta ctgctgctgg aagggggtgg tgtgtcgccc 660

caaaagcagg gcttcaatta agaaatgcct ctttgaaagg tgtaccttgg gtatcctgtc 720

tgagggtaac tccagggtgc gccacaatgt ggcctccgac tgtggttgct tcatgctagt 780

gaaaagcgtg gctgtgatta agcataacat ggtatgtggc aactgcgagg acagggcctc 840

tcagatgctg acctgctcgg acggcaactg tcacctgctg aagaccattc acgtagccag 900

ccactctcgc aaggcctggc cagtgtttga gcataacata ctgacccgct gttccttgca 960

tttgggtaac aggagggggg tgttcctacc ttaccaatgc aatttgagtc acactaagat 1020

attgcttgag cccgagagca tgtccaaggt gaacctgaac ggggtgtttg acatgaccat 1080

gaagatctgg aaggtgctga ggtacgatga gacccgcacc aggtgcagac cctgcgagtg 1140

tggcggtaaa catattagga accagcctgt gatgctggat gtgaccgagg agctgaggcc 1200

cgatcacttg gtgctggcct gcacccgcgc tgagtttggc ccccgt 1246

<210> 30

<211> 995

<212> DNA

<213> Artificial sequence

<220>

<223> pcDNA3.1 GRP78 direct

<400> 30

tggtggaatt ctgcagatat ccagcacagt ggcggccgct cgagatgaag ctctccctgg 60

tggccgcgat gctgctgctg ctcagcgcgg cgcgggccga gggaggaggac aagaaggagg 120

acgtgggcac ggtggtcggc atcgacctgg ggaccaccta ctcctgcgtc ggcgtgttca 180

agaacggccg cgtggagatc atcgccaacg atcagggcaa ccgcatcacg ccgtcctatg 240

tcgccttcac tcctgaaggg gaacgtctga ttggcgatgc cgccaagaac cagctcacct 300

ccaaccccga gaacacggtc tttgacgcca agcggctcat cggccgcacg tggaatgacc 360

cgtctgtgca gcaggacatc aagttcttgc cgttcaaggt ggttgaaaag aaaactaaac 420

catacattca agttgatatt ggaggtgggc aaacaaagac atttgctcct gaagaaattt 480

ctgccatggt tctcactaaa atgaaagaaa ccgctgaggc ttatttggga aagaaggtta 540

cccatgcagt tgttactgta ccagcctatt ttaatgatgc ccaacgccaa gcaaccaaag 600

acgctggaac tattgctggc ctaaatgtta tgaggatcat caacgagcct acggcagctg 660

ctattgctta tggcctggat aagagggagg gggagaagaa catcctggtg tttgacctgg 720

gtggcggaac cttcgatgtg tctcttctca ccattgacaa tggtgtcttc gaagttgtgg 780

ccactaatgg agatactcat ctgggtggag aagactttga ccagcgtgtc atggaacact 840

tcatcaaact gtacaaaaag aagacgggca aagatgtcag gaaagacaat agagctgtgc 900

agaaactccg gcgcgaggta gaaaaggcca aacgggccct gtcttctcag catcaagcaa 960

gaattgaaat tgagtccttc tatgaaggag aagac 995

<210> 31

<211> 1046

<212> DNA

<213> Artificial sequence

<220>

<223> pcDNA3.1 GRP78 reverse

<400> 31

tctatgaagg agaagacttt tctgagaccc tgactcgggc caaatttgaa gagctcaaca 60

tggatctgtt ccggtctact atgaagcccg tccagaaagt gttggaagat tctgatttga 120

agaagtctga tattgatgaa attgttcttg ttggtggctc gactcgaatt ccaaagattc 180

agcaactggt taaagagttc ttcaatggca aggaaccatc ccgtggcata aacccagatg 240

aagctgtagc gtatggtgct gctgtccagg ctggtgtgct ctctggtgat caagatacag 300

gtgacctggt actgcttgat gtatgtcccc ttacacttgg tattgaaact gtgggaggtg 360

tcatgaccaa actgattcca aggaacacag tggtgcctac caagaagtct cagatctttt 420

ctacagcttc tgataatcaa ccaactgtta caatcaaggt ctatgaaggt gaaagacccc 480

tgacaaaaga caatcatctt ctgggtacat ttgatctgac tggaattcct cctgctcctc 540

gtggggtccc acagattgaa gtcacctttg agatagatgt gaatggtatt cttcgagtga 600

cagctgaaga caagggtaca gggaacaaaa ataagatcac aatcaccaat gaccagaatc 660

gcctgacacc tgaagaaatc gaaaggatgg ttaatgatgc tgagaagttt gctgaggaag 720

acaaaaagct caaggagcgc attgatacta gaaatgagtt ggaaagctat gcctattctc 780

taaagaatca gattggagat aaagaaaagc tgggaggtaa actttcctct gaagataagg 840

agaccatgga aaaagctgta gaagaaaaga ttgaatggct ggaaagccac caagatgctg 900

acattgaaga cttcaaagct aagaagaagg aactggaaga aattgttcaa ccaattatca 960

gcaaactcta tggaagtgca ggccctcccc caactggtga agaggataca gcagaaaaag 1020

atgagttgta gtctagagtc ccgtaa 1046

<---

Claims (8)

1. Use of a pharmaceutical composition comprising a mesenchymal stem cell and an oncolytic virus for treating cancer, wherein the cancer is gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, biliary tract cancer, bladder cancer, colorectal cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, skin cancer, kidney cancer, polyploid carcinoma, thyroid cancer, parathyroid cancer or ureteral cancer, wherein the mesenchymal stem cell is introduced with the E1B55K gene, wherein the E1B55K gene is induced by an expression inducer, and said E1B55K gene is expressed after the introduction of the mesenchymal stem cell into the body. 2. Use of a pharmaceutical composition according to claim 1, wherein the mesenchymal stem cell additionally contains the gene of glucose-regulated protein 78 (GRP78). 3. Use of a pharmaceutical composition according to claim 1, wherein the expression inducer is doxycycline or tetracycline. 4. Use of a pharmaceutical composition according to claim 1, wherein the telomerase reverse transcriptase (TERT) gene is additionally introduced into the mesenchymal stem cell. 5. A method for treating cancer, wherein the method comprises administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising a mesenchymal stem cell and an oncolytic virus, wherein the cancer is gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, biliary tract cancer, bladder cancer, colorectal cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, skin cancer, kidney cancer, polyploid carcinoma, thyroid cancer, parathyroid cancer or ureteral cancer, wherein the mesenchymal stem cell is administered with an E1B55K gene, wherein the E1B55K gene is induced by an expression inducer, and said E1B55K gene is expressed after the mesenchymal stem cell is administered to the body. 6. The method for treating cancer according to claim 5, wherein the mesenchymal stem cell additionally contains the gene for glucose-regulated protein 78 (GRP78). 7. A method for treating cancer according to claim 5, wherein the expression inducer is doxycycline or tetracycline. 8. A method for treating cancer according to claim 5, wherein the telomerase reverse transcriptase (TERT) gene is additionally introduced into the mesenchymal stem cell.