WO2022092795A1 - 종양 타겟팅 향상 및 바이러스 대량 생산을 가능한 중간엽줄기세포 - Google Patents
종양 타겟팅 향상 및 바이러스 대량 생산을 가능한 중간엽줄기세포 Download PDFInfo
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Definitions
- the present invention relates to the production of mesenchymal stem cells capable of controlling virus production and release timing to improve tumor targeting of mesenchymal stem cells for virus delivery and virus propagation and to enable mass production of viruses.
- Non-Patent Document 1 In the case of vesicular stomatitis virus, compared to the intravenous virus (input virus), the rate of reaching the tumor target was reported to be 0.01% within 24 hours [Non-Patent Document 1]. The very low delivery efficiency to the target during intravenous injection of the virus was the biggest constraint.
- Non-Patent Document 3 confirms that intravascular delivery of MSCs for oncolytic virus delivery has access to treatment for multiple brain tumors, but has problems such as the possibility of MSC tumor growth.
- Non-Patent Document 4 This is a carrier loaded with a gene that induces apoptosis when MSC is used as a viral carrier
- MSCs multi-cells
- Patent Document 1 Domestic Registered Patent No. 10-2169798 (2020.10.26)
- 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
- 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
- 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
- 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
- the present inventors have improved the tumor targeting of mesenchymal stem cells for virus delivery and virus propagation and mesenchymal stem cells capable of controlling virus production and release timing to enable mass production of viruses By developing the present invention was completed.
- an object of the present invention is to provide a mesenchymal stem cell for delivery of an oncolytic virus having an improved tumor targeting ability, into which the GRP78 (glucose regulated protein 78) gene is introduced.
- Another object of the present invention is to provide mesenchymal stem cells for oncolytic virus delivery into which the E1B55K gene is introduced so that expression is induced by an expression inducer.
- the present invention includes a GRP78 (glucose regulated protein 78) gene and an E1B55K gene, wherein the E1B55K gene is expressed by an expression inducer
- GRP78 glycose regulated protein 78
- E1B55K gene is expressed by an expression inducer
- Another object is to provide mesenchymal stem cells for oncolytic virus delivery.
- Another object of the present invention is to provide a composition for anticancer gene delivery comprising the mesenchymal stem cells and oncolytic virus.
- Another object of the present invention is to provide a pharmaceutical composition for anticancer comprising the mesenchymal stem cells and oncolytic virus.
- Another object of the present invention is to provide a composition for diagnosing cancer comprising the mesenchymal stem cells and oncolytic virus.
- Another object of the present invention is to provide a method for treating cancer comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising the mesenchymal stem cells and the oncolytic virus.
- virus-only tumor treatment The three major challenges posed by virus-only tumor treatment are:
- mesenchymal stem cells were used as a cell carrier to facilitate systemic blood transport of oncolytic viruses, and in addition to transporting viruses, they moved to the target tumor site.
- a genetically modified mesenchymal stem cell line (MSC) was developed for effective anti-tumor action by a large amount of virus produced in the middle.
- the present invention relates to the production of mesenchymal stem cells capable of controlling virus production and release timing to improve tumor targeting of mesenchymal stem cells for virus delivery and virus propagation and to enable mass production of viruses.
- the MSC according to the present invention significantly improves tumor targeting and at the same time uses the MSC as a virus production plant with an efficiency comparable to that of an existing virus-producing cell line. It was possible to dramatically improve safety and anticancer efficacy. In other words, it is possible to simultaneously maximize the effectiveness and minimize toxicity of oncolytic virus-loaded MSCs by solving the biggest challenges of virus-based anticancer drugs, such as tumorigenicity, tumor tropism, tumor mobility, and virus production/release control at an appropriate time. there is.
- Figure 2 confirms by Western blotting whether the passage growth of human adipose MSC-TERT is effective even at passage 30.
- Figure 4 confirms by Western blotting that p53 accumulation and a decrease in survival-related signals occur upon induction of E1B55K expression in human bone marrow and adipose MSCs.
- 5 is a view showing the increase in p53 promoter activity by expression of E1B55K in the presence of doxycycline in the Tet-One induced expression system as a result of luminescence intensity.
- Figure 7 shows that the E4orf6 gene in the adenovirus backbone, d1324-BstBI, was amplified by PCR, transferred to the pFlag-CMV2 vector, and sequenced to confirm 100% agreement.
- Figure 9 confirms the E1B55K gene sequence of pCA14-E1B55K.
- 10B shows that the normal expression of the purified E1B55K-expressing replication-incompetent virus was confirmed as a result of detection by MOI.
- 11 is a transfection of the E1B55K-expressing plasmid into MSC-TERT and confirms by virus titration that virus production is improved upon infection with oncolytic adenovirus.
- Figure 14 shows the PCR conditions when producing pRetro-x-tetone-puro-E1B55K.
- 16 is a cleavage map of the pRetro-x-Tetone-puro-E1B55K vector used for construction.
- 17 is a sequence analysis of the E1B55K plasmid inserted into pRetro-x-Tetone to confirm the final introduction.
- FIG. 18 shows whether E1B55K, a gene inserted into pRetro-x-Tetone, is induced as a protein by doxycycline.
- Figure 24a confirms that the expression of all factors acting on each step of MSC homing is increased by the increase of GRP78.
- Figure 24b confirms the increase in the mRNA expression of GRP78 by introducing pcDNA3.1-GRP78 into human MSC-TERT to confirm that GRP78 expression is clear.
- 25 is a result of confirming whether the GRP78 gene is inserted into the pcDNA3.1-hygro vector [C: pcDNA3.1-hygro].
- 26 is a view illustrating gene introduction by confirming the increase in GRP78 protein expression by Western blotting after transfection of the plasmid obtained from the pcDNA3.1-GRP78 clone into a liver cancer cell line.
- 29 is a result of screening plasmid clones in which the GRP78 gene is inserted into LNCXneo by restriction enzyme digestion.
- 30 is a result of screening clones expressing GRP78 by introducing a retrovirus expressing the GRP78 gene from the MSC-TERT cell line.
- Figure 32 is the result of confirming the tumor targeting validation (quick and accurate to tumor) of the final type of MSC-TERT-tetoneE1B55K-GRP78 introgression completed in lung cancer (a), liver cancer (b), and pancreatic cancer (c) cell lines.
- Fig. 34 shows the expression of adenovirus-related proteins in tumor tissues in the presence of doxycycline when the final type MSC-TERT-tetoneE1B55K-GRP78 was infected with an oncolytic virus.
- FIG. 37 shows the amount of virus production according to the presence of doxycycline or expression of E4orf6 when the final type MSC-TERT-tetoneE1B55K-GRP78 was infected with an oncolytic virus.
- Figure 38 confirms the expression of tumor homing markers according to the increase in passage of MSC-TERT.
- Figure 39 is a graph confirming the tumor size change in mice on the antitumor effect of the oncolytic adenovirus infected with MSC-TERT-tetoneE1B55K-GRP78.
- Figure 40a confirms the antitumor effect of the oncolytic adenovirus infected with MSC-TERT-tetoneE1B55K-GRP78 by changing the size of the mouse after excision of the tumor.
- Figure 40b confirms the antitumor effect of the oncolytic adenovirus infected with MSC-TERT-tetoneE1B55K-GRP78, the change in the size of the tumor in the mouse mouse photographic photograph.
- Figure 41 shows the rate of virus adsorption to tumors (a) and other organs (liver (b), lung (c), spleen (d), kidney (e) and heart (f)).
- the present invention includes human GRP78 (Glucose regulated protein 78) gene (GenBank NM_005347) introduced, mesenchymal stem cells for oncolytic virus delivery.
- GRP78 Glucose regulated protein 78 gene
- mesenchymal stem cells for oncolytic virus delivery with improved tumor targeting ability by introducing the GRP78 gene into mesenchymal stem cells used as virus carriers.
- the GRP78 gene is a glucose regulatory protein 78 gene, which is already known for its role in increasing cell invasion by increasing cell motility, and this gene sequence is as shown in GenBank NM_005347 SEQ ID NO: 1 (human). In the present invention, it serves to improve tumor targeting ability as a tumor homing marker.
- the tumor tropism was improved due to the GRP78 gene, and in particular, the virus was delivered to the tumor tissue site without adsorption to organs other than the tumor.
- telomerase reverse transcriptase (TERT) gene that induces cell proliferation can be additionally introduced.
- the TERT gene is a telomerase reverse transcriptase gene, and the gene sequence is as shown in GenBank NM_198253, SEQ ID NO: 2 (human).
- MSC-TERT-GRP78 in the present invention Cell lines were established, and the improvement of tumor targeting of mesenchymal stem cells for viral transport and virus propagation was confirmed. Therefore, the mesenchymal stem cells can be used for both diagnosis and treatment of tumors.
- the present invention provides a mesenchymal stem cell for delivery of an oncolytic virus virus into which the E1B55K gene is introduced so that expression is induced by an expression inducer.
- the E1B55K gene is a viral gene encoding an E1B55K protein expressed early in the lifespan of an adenovirus. This gene sequence is as shown in GenBank AC_000008, SEQ ID NO:3. (The underlined a in the first line was originally g, but the KpnI site was removed by substitution. The underlined t in the fourteenth line was originally a replaced with t and the HindIII site was removed.)
- the present inventors established a human MSC-CAR-E1B55K cell line from bone marrow-derived human MSC previously applied (Patent Document 1), and confirmed an increase in virus production, but it was not easy to propagate by passage, and it was confirmed that the cell line condition deteriorated as passage passed. , it took a lot of time to confirm that animal testing and master cell banking were impossible and to understand the cause. In particular, the new function of E1B55K was confirmed. This is the fact that, in addition to E1B55K's ability to induce increased viral proliferation, it simultaneously induces a decrease in cell line viability.
- the Tet-on system was applied in the Example, but any other time difference induction system is possible.
- the expression inducer is preferably doxycycline or tetracycline.
- the MSC-TERT-tetoneE1B55K cell line was established, and E1B55K was expressed at a desired time through time difference (oncolytic) to fundamentally block virus mass production and MSC tumorigenesis.
- the present invention includes a GRP78 (glucose regulated protein 78) gene and an E1B55K gene, wherein the E1B55K gene is expressed by an expression inducer and mesenchymal stem cells for oncolytic virus delivery.
- GRP78 glycose regulated protein 78
- E1B55K gene is expressed by an expression inducer and mesenchymal stem cells for oncolytic virus delivery.
- MSC-TERT-tetoneE1B55K-GRP78 in the present invention A cell line was established, and it was confirmed that the virus production and release timing could be controlled to improve the tumor targeting of mesenchymal stem cells for virus delivery and virus propagation and to enable mass production of viruses.
- the mesenchymal stem cell line used herein is preferably human-derived.
- the mesenchymal stem cell line is preferably derived from bone marrow, cord blood or adipose.
- Oncolytic virus as used herein is 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.
- AAV oncolytic adeno-associated virus
- AAV oncolytic retrovirus
- an oncolytic lentivirus an oncolytic lentivirus
- an oncolytic herpes simplex virus or an oncolytic vaccinia virus.
- the oncolytic adenovirus may be an oncolytic adenovirus described in Korean Patent Application No. 2016-0166171 developed by the present inventors.
- the present invention also includes a composition for delivering an anticancer gene comprising the mesenchymal stem cells for delivery of the oncolytic virus and the oncolytic virus.
- composition for gene delivery of the present invention may be for systemic administration.
- stem cells (Ad-MSC) loaded with the virus of the present invention
- Ad-MSC adoptive multi-mediastinum
- it in addition to cancer-specific characteristics, it has a low or no immune response, so systemic administration is possible, and it has excellent targeting to tumor cells and has an effect that does not induce toxicity. Therefore, gene transfer efficiency can be significantly increased.
- the mesenchymal stem cells can be isolated from bone marrow, cord blood, adipose, and the like, and allogeneic mesenchymal stem cells can be used by using the database of the patient himself or a blood bank. Therefore, when the adenovirus-loaded stem cell of the present invention is used as a gene delivery agent, not only self-treatment but also the same type of treatment is possible, thereby further lowering the unit cost of gene therapy.
- the method of introducing the adenovirus containing the gene of the present invention into mesenchymal stem cells can be carried out through various methods known in the art. Specifically, the gene introduction may be performed according to a viral infection method known in the art.
- anticancer pharmaceutical composition means "a pharmaceutical composition for use in cancer treatment”.
- mesenchymal stem cells included as an active ingredient in the anticancer pharmaceutical composition of the present invention are the same as the mesenchymal stem cells of the present invention described above, the detailed description of the mesenchymal stem cells is also applied to the composition of the present invention as it is. Accordingly, in order to avoid excessive complexity due to unnecessary repetition of descriptions of the present specification, descriptions of common matters are omitted.
- the oncolytic adenovirus included in the composition of the present invention exhibits killing efficacy against various tumor cells, so the pharmaceutical composition of the present invention is gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer Cancers such as head 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 can be used for treatment.
- gastric cancer such as head 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.
- the term “treatment” refers to (i) prevention of tumor cell formation; (ii) inhibition of a disease or disorder associated with a tumor upon removal of tumor cells; and (iii) alleviation of a disease or condition associated with the tumor upon removal of the tumor cells.
- the term “therapeutically effective amount” refers to an amount sufficient to achieve the aforementioned pharmacological effect.
- compositions of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate. , microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, and the like. not.
- the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.
- the pharmaceutical composition of the present invention is preferably administered parenterally, for example, can be administered using intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration or local administration.
- intraperitoneal administration in ovarian cancer and portal vein administration in liver cancer it may be administered by an injection method. It can be administered by direct injection, and in the case of bladder cancer, it can be administered by direct injection into the catheter.
- a suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration mode, age, weight, sex, disease severity, food, administration time, administration route, excretion rate, and reaction sensitivity of the patient. and an ordinary skilled physician can easily determine and prescribe an effective dosage for the desired treatment.
- the pharmaceutical composition of the present invention contains 1 ⁇ 10 5 to 1 ⁇ 10 15 pfu/ml of an oncolytic virus, and usually contains a virus that infects MSCs (based on the number of 1 ⁇ 10 6 cells) at one injection. The amount is 1 ⁇ 10 7 to 1 ⁇ 10 8 pfu.
- the pharmaceutical composition of the present invention is prepared in a unit dosage form by being formulated using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or it may be prepared by incorporation into a multi-dose container.
- the formulation may be in the form of a solution, suspension, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.
- the pharmaceutical composition of the present invention may be used as a single therapy, but may also be used in combination with other conventional chemotherapy or radiation therapy, and when such a combination therapy is performed, cancer treatment can be more effective.
- Chemotherapeutic agents that can be used together with the composition of the present invention include gemcitabine, sorafenib, cisplatin, carboplatin, procarbazine, mechlorethamine ( mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, diactinomycin, Daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol, translatinum, 5-fluorouracil, vincristin, vinblastin, and methotrexate.
- Radiation therapy that can be used together with the composition of the present invention is X-ray irradi
- the mesenchymal stem cells according to the present invention are 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, It can show anticancer effect on head and neck cancer, skin cancer, kidney cancer, polyploid carcinoma, thyroid cancer, parathyroid cancer or ureter cancer.
- the present invention may include a composition for diagnosing cancer comprising the virus/stem cell complex including the mesenchymal stem cells and the oncolytic virus.
- diagnosis refers to ascertaining the presence or characteristics of pathophysiology. Diagnosis in the present invention is to confirm the onset and progress of cancer.
- the virus/stem cell complex refers to a stem cell infected with or loaded with a virus.
- the virus/stem cell complex may bind to a luminescent material, a fluorescent substance, or an isotope to diagnose cancer through imaging.
- the light-emitting material refers to any material that emits light.
- MSCs were infected with an adenovirus expressing luciferase, and then luciferin was administered to the abdominal cavity of the mouse to capture the luminescence as an image.
- the phosphor is preferably a phosphor, a fluorescent protein, or other imaging material capable of binding to the peptide that specifically binds to NRP1, but is not limited thereto.
- the phosphor is preferably fluorescein, BODYPY, tetramethylrhodamine, Alexa, cyanine, allophicocyanine, or a derivative thereof, but limited thereto doesn't happen
- the fluorescent protein is preferably, but not limited to, Dronpa protein, a fluorescent gene (EGFP), a red fluorescent protein (DsRFP), Cy5.5, which is a cyanine fluorescent substance exhibiting near-infrared fluorescence, or other fluorescent proteins.
- Other imaging materials are preferably iron oxide, radioactive isotopes, etc., but are not limited thereto, and may be applied to imaging equipment such as MR and PET.
- the present invention also includes a method of treating cancer comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising said mesenchymal stem cells and an oncolytic virus.
- the cancer is specifically 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 ureter cancer.
- the term “subject” refers to a mammal that is the subject of treatment, observation or experimentation, and preferably refers to a human.
- the term "therapeutically effective amount” refers to an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human as thought by a researcher, veterinarian, physician or other clinician, This includes amounts that result in amelioration of the symptoms of the disease or disorder being treated. It is apparent to those skilled in the art that the therapeutically effective amount and frequency of administration for the active ingredient of the present invention will vary depending on the desired effect.
- the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of active ingredients and other components contained in the composition, the type of formulation, the patient's weight, age, sex, and health condition , the range varies depending on the diet, administration time, administration method, excretion rate, etc.
- 1 ⁇ 10 5 to 1 ⁇ 10 15 pfu/ml of oncolytic virus is included, and the amount of virus that infects MSCs (based on the number of 1 ⁇ 10 6 cells) is usually 1 ⁇ 10 7 to 1 ⁇ 10 8 pfu.
- the pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally or locally applied) according to a desired method.
- TERT telomerase reverse transcriptase
- the TERT gene introduction method is as follows.
- Platinum-A packaging cells were transfected with 1 ⁇ g of a retroviral vector, pBABE-hygro-hTERT, purchased from Addgene, and the filtered medium obtained was infected with human adipose MSC and hygromycin (200 ⁇ g of hygromycin B Gold). /ml) after obtaining cell clones resistant to screening and selection as shown in FIG. MSC clone #22 was finally selected, and clone #22 was used for human fat MSC-TERT used in subsequent experiments.
- Figure 2 confirms the passage proliferation of human MSC, and it was expanded up to passage 30. It was confirmed that the increase in the amount of TERT expression was still very clearly increased from the MSCs passaged up to 30 passages, and the expression of other MSC-related major markers was maintained or increased compared to the wild type.
- TERT-expressing MSC clone No. 22 was infected with replication-defective adenovirus expressing RFP (red fluorescence protein) at 20 MOI and 100 MOI, and after 48 hours, fluorescently stained cells were observed under a fluorescence microscope.
- RFP red fluorescence protein
- Example 1 Confirmation of new functions of the adenovirus gene E1B55K
- MSCs are to be used as a cell line for mass production for clinical use, master cell banking is required. For this, it is essential that the passage extension of the MSC be possible above all else. On the one hand, however, one must also maintain the principle that extension induction must never contribute to an increase in tumorigenicity. The dilemma of simultaneously satisfying these two problems was initially considered impossible, but a solution was presented for the first time in our laboratory by constructing a stable MSC cell line in which CAR and E1B55K genes were introduced into MSC. A human MSC-CAR-E1B55K cell line was established from the previously applied bone marrow-derived human MSC, and an increase in virus production was confirmed [Patent Document 1].
- E1B55K was cloned into pcDNA3.1 hygro(+).
- primers having BamHI and NotI respectively (forward, 5'-GCGGATCCATGGAGCGAAGAAACCCATCT-3': SEQ ID NO: 4, reverse, 5'-GAG CGGCCGCTCAATCTGTATCTTCATCGCT-3': sequence No.
- p53 promoter In MSC-TERT-tetoneE1B55K cell line, a certain part of the p53 promoter (-344 to +12) is located in front of the luciferase coding gene insertion site pGL2-p53 promoter (pGL2-356bp, Addgene) and E4orf6 gene subcloned pFlag-CMV2-E4orf6 or pFlag-CMV2 (Sigma) as a control, 1 ⁇ g each, after cotransfection, luciferase activity with or without doxycycline (1.25 ⁇ g/ml for 48 h) was measured by luminescence intensity. was confirmed with
- the analysis method used at this time was Promega's dual-luciferase reporter assay system (catalog number #E1910, FIG. 5).
- E1B55K expression induced in MSC-TERT-tetoneE1B55K cell line by doxycycline treatment directly or indirectly binds to the p53 promoter to activate transcription, thereby increasing luminescence intensity by luciferase activity by more than 4 times (FIG. 5).
- the increase in luminescence intensity did not exceed 1.5 times when E4orf6 expression occurred in MSC expressing E1B55K.
- E4orf6 GeneBank AC_000008; also called E4 34K
- E4 34K the subcloning of the E4orf6 gene was carried out by transferring the E4orf6 gene from the adenovirus backbone d1324-BstBI to the pFlag-CMV2 vector.
- E1B55K directly or indirectly acts on the p53 promoter, which is opposite to the main action of E1B55K in the adenovirus wild type (inducing degradation of p53 protein by forming a complex with E4orf6) through transcriptional induction.
- a novel action inducing p53 accumulation was discovered.
- E1B55K was supplied from the outside using an adenovirus carrier to determine whether the virus production capacity was increased in MSCs. I decided to check it out again.
- a replication-defective adenovirus expressing E1B55K was prepared as follows.
- E1B55K gene was cloned into an adenovirus shuttle vector, pCA14.
- E1B55K is contained in pBSK-3484, and primers (sense 5'-TTCATCTAGAATGGAGCGAAGAAACCCATC-3'(XbaI): SEQ ID NO: 9, antisense 5'-GACGAAGCTTTCAATCTGTATCTTCATCGC-3 (HindIII): Sequence No.
- adenoviral vector dl324-BstBI was linearized by Bsp119I digestion, and the two were cotransformed into E. coli BJ5183. ) to induce homologous recombination to construct an adenovirus vector expressing E1B55K, and then transfect 293A to induce virus production, expand production, and separate and purify titers.
- FIG. 10a After confirming that there is no abnormality in E1B55K detection by the polyclonal antibody obtained by requesting production of a specific antibody for E1B55K expression for E1B55K expression (Fig. It was confirmed by Western blotting (Fig. 10b).
- the right figure of Figure 10a is the raw data of the left figure, and was attached to confirm the exact size and band of the polyclonal antibody E1B55K.
- 3 ⁇ g of E1B55K-expressing adenovirus vector (dl324-E1B55K) DNA was transfected into 293A after PacI digestion, and the soup harvested after confirming virus production was infected with human adipocyte-derived MSC-TERT.
- the 4th lane is the final soup by freezing and thawing the soup and plus cells obtained in the same way as the 3rd lane, and the 5th lane is 2.5 ⁇ g of DNA at the time of first transfection.
- Figure 10b shows the infectivity particles of the virus after amplifying and purifying the viral soup obtained under the conditions of the 5th lane, and infecting the virus expressing replication-incompetent E1B55K for each MOI with MSC-TERT. As the MOI increases, E1B55K It was confirmed that the polyclonal antibody against E1B55K was operating normally by confirming that the intensity of the band corresponding to was also increased.
- the production process of the polyclonal antibody which is a specific antibody against E1B55K, is as follows.
- a peptide sequence located at the N-terminal site of E1B55K (MERRNPSERGVPAGFSGHASVESGC: SEQ ID NO: 13) was synthesized, and BSA (bovine serum albumin) was conjugated to an immunogen carrier, and then New Zealand White rabbits were immunized to purify the serum. obtained (GW Vitek, Korea).
- E1B55K-expressing plasmid (pcDNA3.1-E1B55K) was transfected into human adipocyte-derived MSC-TERT (Fig. 11) or E1B55K-expressing replication-defective adenovirus was infected by MOI (Fig. 12) after 4 hours. After changing the medium, and 48 hours after infection with oncolytic adenovirus (YSC-02) at 100 MOI, all medium and cells were collected, and then frozen and thawed to maximize virus excitation, followed by centrifugation. It shows the results of titration of the infective virus in the supernatant obtained by doing so. As a result, it was confirmed that the virus production was significantly increased in MSC-TERT expressing E1B55K compared to the control group after oncolytic adenovirus infection ( FIG. 12 ).
- Example 2 The simultaneous realization of innovative virus production increase induction, payload gene expression time control, and original tumorigenicity control technology is made possible by the introduction of the E1B55K time difference expression control system.
- Retro-XTM Tet-OneTM inducing expression system (Takara cat# 634307) was referenced in the protocol of FIG. 13 .
- E1B55K In-fusion EcoRI sense primer was 5'-CCCTCGTAAAGAATTCATGGAGCGAAGAAACCCATCTGAG-3' (SEQ ID NO: 14)
- E1B55K In-fusion BamHI antisense primer was 5'-TAGAGG-3'TGGTCTGGATCC (SEQ ID NO: 15).
- the pRetro-X-tetone-puro-E1B55K vector used for construction is shown in FIG. 16 .
- E1B55K After transfection with pRetro-X-Tetone-puro-E1B55K plasmid, expression of E1B55K was confirmed in pRetro-X-tetone-puro-E1B55K according to the amount of doxycycline during doxycycline treatment. In order to examine whether the tet one system can control the time difference expression of E1B55K, it was confirmed that the expression of E1B55K did not occur at all before doxycycline treatment when the tet one system was introduced (FIG. 19). That is, it was confirmed that leakage did not occur.
- E1B55K expression was induced immediately. This means that cell survival can be maintained before doxycycline treatment, and expression can be induced immediately after doxycycline treatment after reaching a tumor.
- Time difference virus release verification was secondarily verified as follows.
- E1B55K which is early region 1B-55kDa of adenovirus
- Retro-X-Tet-One inducible expression system TAKARA Bio Inc.
- MSC-TERT Retro-X-Tet-One inducible expression system
- Clones expressing E1B55K in the presence of doxycycline were obtained.
- GP2-293 packaging cells were transfected with pRetroX-TetOne-E1B55K and the pAmpho vector encoding the membrane protein (included in the Takara kit). After 48 hours, the filtered retroviral supernatant was infected with the target cell, MSC-TERT.
- Example 3 Establishment of a breakthrough in tumor targeting - Discovering factors to improve tumor homing
- tumor tropism-related markers was confirmed by inducing additional expression of GRP78 in human adipose MSC-TERT.
- human MSC-TERT was transfected with pcDNA3.1-GRP78 (2 ⁇ g) to confirm the increase in GRP78 mRNA expression ( FIG. 24B ).
- the extracted RNA was reverse transcribed using invirogen's SuperScriptTM First-Strand Synthesis System for RT-PCR kit.
- the primers used for PCR were sequenced by adding an XhoI site in the forward direction and an XbaI site in the reverse direction.
- Forward (5'-GATTCTCGAGATGAAGCTCTCCCTGG-3': SEQ ID NO: 16
- Reverse 5'-GGCCTCTAGACTACAACTCATCTTTT-3': SEQ ID NO: 17
- primers PCR initial denaturing after 95 ° C. 2 minutes denaturing: 95 ° C.
- GRP78 In order to introduce GRP78 into the retroviral vector pLNCXneo, it was cut with XhoI/PmeI from pcDNA3.1-GRP78 vector to extract the GRP78 site, which was then ligated to pLNCXneo previously cut with XhoI/PmeI and inserted.
- pLNCX neo was produced by increasing the restriction enzyme recognition site from HindIII-HpaI-ClaI, which is a cloning site in the existing LNCX, to HindIII-PmlI-BstXI-NotI-XhoI-SalI-ApaI-PmeI-HpaI-ClaI (FIG. 28).
- the strand sequence is as follows.
- pLNCXneo-GRP78 control and pcDNA3.1-GRP78 (GRP78 gene providing plasmid) as a control are cut with HindIII/PmeI, respectively, and linearized by linking pLNCXneo and GRP78 fragment derived from pcDNA3.1-GRP78.
- pLNCXneo-GRP78 candidate constructs samples 2,5,9,11,12,20,21 (FIG. 29)
- Platinum-A packaging cells were transfected with pLNCXneo-GRP78, a retroviral vector, and after 48 hours, the medium was filtered.
- the medium solution containing retrovirus obtained by filtration was infected with MSC-TERT to obtain cell clones resistant to G418 (500 ⁇ g ⁇ 600 ⁇ g/ml), and then screened to obtain clones 2 and 4 with clear GRP78 and MMP2 expression. was selected (FIG. 30).
- the in vivo distribution of MSC-TERT-GRP78 infected with adenovirus expressing luciferase was observed by imaging using an IVIS Spectrum System (PerkinElmer) 6 hours after tail vein injection.
- the characteristic of this experiment is that the GRP78 transgenic MSC-TERT-GRP78 reaches the target site of MSC-TERT-GRP78 by allowing it to form a tumor on the shoulder to distinguish it from internal organs such as liver and lungs in order to accurately distinguish how much tumor migrating ability is. It was clearly confirmed that the tumors gathered into the tumor tissue site within a very short time without adsorption to other organs (FIG. 31).
- the tumor site was identified by artificially implanting a solid cancer subcutaneously and allowing MSC-GRP78 to migrate. By labeling this good isotope, the tumor can be detected. This can be applied to tumor diagnosis, and in the long term, it becomes possible to manufacture an OV/MSC complex that can diagnose and treat tumors at the same time.
- A549 cells were transplanted into the subcutaneous tissue of a mouse to form a tumor.
- the final type of MSC cells (MSC-TERT-tetoneE1B55K-GRP78) were labeled with a fluorescent cell tracker probe (Invitrogen, C34565) and injected into the tail vein of the mouse.
- a fluorescent cell tracker probe Invitrogen, C34565
- the tumor tissue was excised, and the fluorescence site was confirmed with a fluorescence microscope after tissue section.
- FIG. 33 it was confirmed that MSCs having GRP78 were present much more in the tumor. In the case of MSC without GRP78, it was observed that it mainly stayed at the tumor interface due to low fluorescence intensity.
- the virus was released from MSC-TERT-tetoneE1B55K-GRP78 infiltrating the tumor tissue to infect tumor cells.
- the tumors of each group of mice were removed and the tumor-sectioned samples were reacted with an adenovirus type 5 specific antibody (Abcam, Cambridge, UK), followed by DAB color reaction with a secondary antibody and immunization with hematoxylin nuclear staining. Counterstaining was performed for histochemistry. At this time, all groups were fed with doxycycline (625 mg/kg) as feed.
- Adenovirus-specific proteins were identified in most of the identified areas (FIG. 34).
- Adenovirus was released from the MSCs from the tumor tissue up to 7 days after the minimal tail vein injection, showing that replication, proliferation and spreading are occurring repeatedly, and the virus was not present until around 20 days when the tumor size was reduced to almost zero level. Possibility of remaining was confirmed.
- the virus production ability of human adipose MSC-TERT-tetoneE1B55K-GRP78 (clone No. 21) was confirmed after 48 hours of increase in virus production in MSCs treated with doxycycline (1.25 ⁇ g/ml) after infection with an oncolytic adenovirus expressing shHSP27/shTGF ⁇ 1. , At this time, a difference in virus production according to E4orf6 gene expression was also confirmed. For this experiment, 1 ⁇ g of pFlag-CMV2-E4orf6 subcloned with the E4orf6 gene or pFlag-CMV2 as a control were transfected into MSC of clone 21 for this experiment.
- Example 4 Anticancer efficacy of oncolytic adenovirus infected with MSC-TERT-tetoneE1B55K-GRP78
- mice 8 ⁇ 10 6 A549 in the flank region of 6-week-old BALB/c thymic nude mice
- Cells (lung cancer cell line) were subcutaneously transplanted and the mice having an average tumor size of 150 mm 3 were separated into groups of 10 each.
- Mice were injected with 1 ⁇ 10 6 MSC-TERT-tetoneE1B55K-GRP78 infected with an oncolytic adenovirus expressing shHSP27-shTGF ⁇ 1 into the tail vein. And after 3 days, 1 ⁇ 10 6 MSC-TERT-tetoneE1B55K-GRP78 infected with an oncolytic adenovirus expressing shHSP27-shTGF ⁇ 1 were additionally injected intravenously.
- a group transplanted with only tumor cells (no treat) and a group injected with the final MSC-TERT-tetoneE1B55K-GRP78 (MSC only) were used.
- all groups were fed with doxycycline (625 mg/kg) as feed.
- the antitumor effect obtained when the oncolytic virus (Ad-3484-shHSP27-shTGF ⁇ 1) was injected directly into the tumor (the tumor size growth rate was delayed compared to the control group, but the tumor size itself decreased and did not return)
- viruses by major organs including tumors can be identified in more detail, such as the amount captured in major organs (lung, liver, spleen, etc.)
- the amount of adenovirus gene copy number was measured over time.
- the viral genomic plasmid DNA was determined as the standard DNA and a standard curve was drawn and confirmed (in the case of the currently used viral DNA of the oncolytic adenovirus, 1 copy is 10 3 pg).
- MSC-TERT-GRP78 (1 ⁇ 10 6 ) infected with the YSC-02 oncolytic virus of Reference Example 1 was administered as a single IV administration by tail, and each day (0, 1, 2, 3, 4, 5, 10, 15).
- the collected tissues tumor, lung, liver, spleen, kidney, heart
- genomic DNA for each tissue was extracted under the same conditions on the day the tissue sampling was finished.
- Real-time PCR was performed using the primers of the E4ORF gene to check the presence or absence of gene expression using the extracted genomic DNA.
- the primers for the E4 ORF screen used at this time are as follows.
- the range is 10 4 to 10 10 copies in the individual tissue when the oncolytic virus is contained in the tumor tissue, compared to the number of copies found in all other organs ⁇ 10, almost It can be seen that the place where all viruses are adsorbed and absorbed is the tumor tissue.
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Abstract
Description
Claims (18)
- GRP78(Glucose regulated protein 78) 유전자가 도입된, 종양 표적능이 향상된 종양 살상 바이러스 전달용 중간엽 줄기세포.
- 발현 유도 물질에 의해 발현이 유도되도록 E1B55K 유전자가 도입된 종양 살상 바이러스 바이러스 전달용 중간엽 줄기세포.
- GRP78(Glucose regulated protein 78) 유전자 및 E1B55K 유전자를 포함하며, 상기 E1B55K 유전자는 발현유도물질에 의해 발현되는 것인 종양 살상 바이러스 바이러스 전달용 중간엽 줄기세포.
- 제 1 항 내지 제 3 항 중 어느 한 항에 있어서,TERT(telomerase reverse transcriptase) 유전자가 추가로 도입된 중간엽 줄기세포.
- 제 1 항 내지 제 3 항 중 어느 한 항에 있어서,상기 종양 살상 바이러스는 종양 살상 아데노바이러스, 종양 살상 아데노-관련 바이러스(Adeno-associated viruses, AAV), 종양 살상 레트로바이러스, 종양 살상 렌티바이러스, 종양 살상 헤르페스 심플렉스 바이러스 또는 종양 살상 백시니아 바이러스인 중간엽 줄기세포.
- 제 1 항 내지 제 3 항 중 어느 한 항에 있어서,상기 종양 살상 바이러스는 종양 살상 아데노바이러스인 중간엽 줄기세포.
- 제 2 항 또는 제 3 항에 있어서,E1B55K 유전자는 중간엽 줄기세포의 체내 도입 후 발현되는 것인 중간엽 줄기세포.
- 제 2 항 또는 제 3 항에 있어서,발현유도물질은 독시사이클린 또는 테트라사이클린인 중간엽 줄기세포.
- 제 1 항 내지 제 3 항 중 어느 한 항에 있어서,인간 유래 중간엽 줄기세포주.
- 제 1 항 내지 제 3 항 중 어느 한 항에 있어서,제대혈, 골수 또는 지방 유래인 중간엽 줄기세포주.
- 청구항 1 내지 청구항 3 중 어느 한 항의 중간엽 줄기세포 및 종양 살상 바이러스를 포함하는 항암 유전자 전달용 조성물.
- 제 11 항에 있어서,상기 암은 위암, 폐암, 유방암, 난소암, 간암, 기관지암, 비인두암, 후두암, 췌장암, 담도암, 방광암, 대장암, 결장암, 자궁경부암, 뇌암, 전립선암, 골암, 두경부암, 피부암, 신장암, 배수성암(polyploid carcinoma), 갑상선암, 부갑상선암 또는 요관암인 유전자 전달용 조성물.
- 청구항 1 내지 청구항 3 중 어느 한 항의 중간엽 줄기세포 및 종양 살상 바이러스를 포함하는 항암용 약제학적 조성물.
- 제 13 항에 있어서,상기 암은 위암, 폐암, 유방암, 난소암, 간암, 기관지암, 비인두암, 후두암, 췌장암, 담도암, 방광암, 대장암, 결장암, 자궁경부암, 뇌암, 전립선암, 골암, 두경부암, 피부암, 신장암, 배수성암(polyploid carcinoma), 갑상선암, 부갑상선암 또는 요관암인 약제학적 조성물.
- 청구항 1 내지 청구항 3 중 어느 한 항에 따른 중간엽 줄기세포 및 종양 살상 바이러스를 포함하는 암 진단용 조성물.
- 제 15 항에 있어서,상기 암은 위암, 폐암, 유방암, 난소암, 간암, 기관지암, 비인두암, 후두암, 췌장암, 담도암, 방광암, 대장암, 결장암, 자궁경부암, 뇌암, 전립선암, 골암, 두경부암, 피부암, 신장암, 배수성암(polyploid carcinoma), 갑상선암, 부갑상선암 또는 요관암인 암 진단용 조성물.
- 치료상의 유효량의, 청구항 1 내지 청구항 3 중 어느 한 항의 중간엽 줄기세포 및 종양 살상 바이러스를 포함하는 약제학적 조성물을 대상체에 투여하는 것을 포함하는 암 치료 방법.
- 제 17 항에 있어서,상기 암은 위암, 폐암, 유방암, 난소암, 간암, 기관지암, 비인두암, 후두암, 췌장암, 담도암, 방광암, 대장암, 결장암, 자궁경부암, 뇌암, 전립선암, 골암, 두경부암, 피부암, 신장암, 배수성암(polyploid carcinoma), 갑상선암, 부갑상선암 또는 요관암인 암 치료 방법.
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KR20170067667A (ko) * | 2015-12-08 | 2017-06-16 | 연세대학교 산학협력단 | GM-CSF 유전자;Flt3L-TRAIL 융합 유전자;TGF-β 발현을 억제하는 shRNA; 및 HSP 발현을 억제하는 shRNA를 포함하는 항종양 조성물 |
KR20190070890A (ko) * | 2017-12-13 | 2019-06-21 | 한양대학교 산학협력단 | 재조합 아데노바이러스 및 이를 포함하는 줄기세포 |
KR20190139763A (ko) * | 2018-06-08 | 2019-12-18 | 연세대학교 산학협력단 | 아데노바이러스의 감염 및 복제가 가능한 중간엽 줄기세포주 |
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WO2019235771A1 (ko) * | 2018-06-08 | 2019-12-12 | 연세대학교 산학협력단 | 아데노바이러스의 감염 및 복제가 가능한 중간엽 줄기세포주 |
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AU2021371806A1 (en) | 2023-03-23 |
EP4239058A4 (en) | 2024-10-16 |
KR20240064602A (ko) | 2024-05-13 |
JP7573898B2 (ja) | 2024-10-28 |
CA3182967A1 (en) | 2022-05-05 |
KR20220056148A (ko) | 2022-05-04 |
JP2023529319A (ja) | 2023-07-10 |
KR102687278B1 (ko) | 2024-07-23 |
EP4239058A1 (en) | 2023-09-06 |
CN116261591A (zh) | 2023-06-13 |
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