A method for producing an adenovirus

Abstract

This application belongs to the field of cell engineering technology, and specifically discloses a method for producing adenovirus, which includes the following steps: adding a JAK inhibitor during the process of adenovirus infecting host cells, and the JAK inhibitor is selected from one or more of a JAK1 specific inhibitor, a Fedratinib inhibitor, a Ruxolitinib inhibitor, a SAR20347 inhibitor, and a PF-06700841 inhibitor. This application has at least one of the following beneficial effects: The method for producing adenovirus provided by this application can significantly increase the yield of single-cell adenovirus by adding a JAK inhibitor during the process of adenovirus infecting host cells, thereby reducing the production cost of adenovirus-related drugs.

Description

Technical Field

[0001] This application belongs to the technical field of cell engineering. More specifically, it relates to a production method for improving the single-cell yield of adenovirus. Background Art

[0002] Cancer is the second most common disease in terms of morbidity and mortality after cardiovascular diseases. With the improvement of the economic development level, the mortality rate of cardiovascular patients has decreased relatively, and cancer will become the main disease hindering the increase of life expectancy. China has a large population base, and the number of cancer patients ranks first in the world. Due to the generally late diagnosis and poor subsequent treatment effects, the cancer mortality rate in China is higher than the global average level. Improving cancer prevention, diagnosis, and treatment can reduce the cancer incidence and mortality rate and improve the quality of life of cancer patients.

[0003] Traditional cancer therapies include surgical treatment, radiotherapy, and chemotherapy, each with its own limitations, such as low efficacy, high mortality rate, and poor prognosis. With the continuous in-depth research on life medicine, cancer treatment has entered the era of precision targeted biotherapy, including tumor immunotherapy, gene therapy, and oncolytic virus therapy, etc. In particular, oncolytic virus therapy has various advantages such as safety, effectiveness, and specificity. Regarding the specific selection problem of tumor cells, researchers have modified the adenovirus vector. One is to delete the genes that are necessary for virus replication in normal cells but not in tumor cells, and the other is to introduce a tumor-specific promoter to control the genes necessary for virus replication. To enhance the anti-tumor effect, various therapeutic gene expression cassettes can also be introduced. The new anti-cancer strategy combining gene therapy and virus therapy is more targeted, safe, and efficient.

[0004] Interferon is widely used in the treatment of cancer patients, such as metastatic melanoma and malignant hematological tumors, etc. Research shows that IFN-α can directly inhibit the proliferation of tumor cells both in vivo and in vitro. In addition, it can indirectly act on tumor cells, including downregulating the expression of oncogenes, upregulating the expression of tumor suppressor genes, and increasing the expression of major histocompatibility complex. Type I interferon has a regulatory function on various host immune cells and plays a core role in the overall anti-tumor immunity. Therefore, the targeted gene-virus drug obtained by inserting the recombinant interferon expression cassette into the oncolytic adenovirus has a stronger tumor-killing effect. Summary of the Invention

[0005] However, the inventors of this application found that when using cells to produce oncolytic adenovirus containing a recombinant interferon expression cassette, its single-cell yield significantly decreased (compared with the empty virus without the recombinant interferon expression cassette, the single-cell yield of the oncolytic adenovirus containing the recombinant interferon expression cassette decreased by more than 60%), significantly increasing the production cost of related drugs.

[0006] Based on this, in order to improve the single-cell yield of adenovirus, the present application provides a method for producing adenovirus, which can significantly improve the single-cell yield of adenovirus without affecting the activity of adenovirus, especially improve the single-cell yield of recombinant oncolytic adenovirus inserted with a recombinant interferon expression cassette, thereby reducing the production cost of adenovirus-related drugs.

[0007] The present application is achieved through the following solutions:

[0008] The present application provides a production method for improving the single-cell yield of adenovirus, which includes the following steps: adding a JAK inhibitor during the process of adenovirus infecting host cells, and the JAK inhibitor is selected from one or more of a JAK1 specific inhibitor, a Fedratinib inhibitor, a Ruxolitinib inhibitor, a SAR20347 inhibitor, and a PF-06700841 inhibitor.

[0009] The inventors of the present application selected a variety of inhibitors that can improve the single-cell yield of adenovirus by testing different JAK inhibitors.

[0010] In a specific embodiment of the present application, the JAK1 specific inhibitor includes a Solcitinib inhibitor and an Upadacitinib inhibitor. The JAK1 specific inhibitor described in the present application includes an inhibitor that only inhibits JAK1, and also includes an inhibitor that mainly inhibits JAK1.

[0011] In a specific embodiment of the present application, the concentration of the Solcitinib inhibitor added is 1.0 uM to 20.0 uM. For example, the concentration of the Solcitinib inhibitor added is 1.0 uM, 1.5 uM, 1.8 uM, 2.0 uM, 2.5 uM, 2.8 uM, 3.0 uM, 4.0 uM, 5.0 uM, 6.0 uM, 7.0 uM, 7.5 uM, 8.0 uM, 9.0 uM, 10.0 uM, 11.0 uM, 12.0 uM, 12.5 uM, 13.0 uM, 14.0 uM, 15.0 uM, 16.0 uM, 17.0 uM, 18.0 uM, 19.0 uM or 20.0 uM, etc.

[0012] In a specific embodiment of the present application, the concentration of the Solcitinib inhibitor added is 1.5 uM to 3.0 uM.

[0013] In a specific embodiment of the present application, the concentration of the Solcitinib inhibitor added is 1.5 uM or 3.0 uM.

[0014] In a specific embodiment of the present application, the added concentration of the Upadacitinib inhibitor is 1.0 uM to 20.0 uM, such as 1.0 uM, 2.0 uM, 3.0 uM, 4.0 uM, 5.0 uM, 6.0 uM, 7.0 uM, 7.5 uM, 8.0 uM, 9.0 uM, 10.0 uM, 11.0 uM, 12.0 uM, 12.5 uM, 13.0 uM, 14.0 uM, 15.0 uM, 16.0 uM, 17.0 uM, 18.0 uM, 19.0 uM or 20.0 uM, etc.

[0015] In a specific embodiment of the present application, the added concentration of the Upadacitinib inhibitor is 3.0 uM.

[0016] In a specific embodiment of the present application, the added concentration of the Fedratinib inhibitor is 0.5 uM to 10.0 uM, such as 0.5 uM, 1.0 uM, 2.0 uM, 3.0 uM, 4.0 uM, 5.0 uM, 6.0 uM, 7.0 uM, 8.0 uM, 9.0 uM or 10.0 uM, etc.

[0017] In a specific embodiment of the present application, the added concentration of the Fedratinib inhibitor is 3.0 uM.

[0018] In a specific embodiment of the present application, the added concentration of the Ruxolitinib inhibitor is 0.1 uM to 10.0 uM. For example, the added concentration of the Ruxolitinib inhibitor is 0.1 uM, 0.5 uM, 1.0 uM, 1.5 uM, 1.8 uM, 2.0 uM, 2.5 uM, 3.0 uM, 3.5 uM, 4.0 uM, 4.5 uM, 5.0 uM, 5.5 uM, 6.0 uM, 6.5 uM, 7.0 uM, 7.5 uM, 8.0 uM, 8.5 uM, 9.0 uM, 9.5 uM or 10.0 uM, etc.

[0019] In a specific embodiment of the present application, the added concentration of the Ruxolitinib inhibitor is 0.5 uM to 10.0 uM.

[0020] In a specific embodiment of the present application, the added concentration of the Ruxolitinib inhibitor is 0.5 uM to 5.0 uM.

[0021] In a specific embodiment of the present application, the added concentration of the Ruxolitinib inhibitor is 0.5 to 3.0 uM.

[0022] In a specific embodiment of the present application, the concentration of the Ruxolitinib inhibitor added is 1.0 - 3.0 uM.

[0023] In a specific embodiment of the present application, the concentration of the Ruxolitinib inhibitor added is 3.0 uM.

[0024] In a specific embodiment of the present application, the concentration of the SAR20347 inhibitor added is 3.0 uM - 20.0 uM. For example, the concentration of the SAR20347 inhibitor added is 3.0 uM, 4.0 uM, 5.0 uM, 6.0 uM, 7.0 uM, 8.0 uM, 9.0 uM, 10.0 uM, 11.0 uM, 12.0 uM, 13.0 uM, 14.0 uM, 15.0 uM, 16.0 uM, 17.0 uM, 18.0 uM, 19.0 uM or 20.0 uM, etc.

[0025] In a specific embodiment of the present application, the concentration of the SAR20347 inhibitor added is 10.0 uM.

[0026] In a specific embodiment of the present application, the concentration of the PF - 06700841 inhibitor added is 0.5 uM - 15.0 uM. For example, the concentration of the PF - 06700841 inhibitor added is 0.5 uM, 1.0 uM, 1.5 uM, 1.8 uM, 2.0 uM, 2.5 uM, 3.0 uM, 4.0 uM, 5.0 uM, 6.0 uM, 7.0 uM, 8.0 uM, 9.0 uM, 10.0 uM, 11.0 uM, 12.0 uM, 13.0 uM, 14.0 uM or 15.0 uM, etc.

[0027] In a specific embodiment of the present application, the concentration of the PF - 06700841 inhibitor added is 3.0 uM.

[0028] In a specific embodiment of the present application, the adenovirus includes a recombinant adenovirus or a wild - type adenovirus.

[0029] In a specific embodiment of the present application, the recombinant adenovirus includes a recombinant adenovirus capable of expressing an exogenous therapeutic gene.

[0030] In a specific embodiment of the present application, the exogenous therapeutic gene is a gene capable of expressing interferon, IL - 2, IL - 12, IL - 15, IL - 24, GM - CFS, TRAIL, PD - 1 binding protein, CTLA4 binding protein or CD47 binding protein, etc.

[0031] In a specific embodiment of the present application, the exogenous gene is a gene capable of expressing interferon.

[0032] In a specific embodiment of the present application, the adenovirus is an oncolytic adenovirus.

[0033] In a specific embodiment of the present application, the adenovirus is wild-type adenovirus Ad-WT, empty virus OncoMul-V2 or oncolytic adenovirus YSCH-01.

[0034] In a specific embodiment of the present application, the adenovirus has no special requirements for host cells.

[0035] In a specific embodiment of the present application, the host cell is an engineered cell for adenovirus production.

[0036] In a specific embodiment of the present application, the host cell is Hela-S3, HeLa, HEK293 or A549.

[0037] On the other hand, the present application provides the use of the above JAK inhibitor in increasing the yield of single-cell adenovirus.

[0038] The production method of the adenovirus provided by the present application has at least one of the following beneficial effects:

[0039] The production method of the adenovirus provided by the present application, by adding a JAK inhibitor, such as a JAK1-specific inhibitor, Fedratinib inhibitor, Ruxolitinib inhibitor, SAR20347 inhibitor or PF-06700841 inhibitor, etc., during the process of adenovirus infecting host cells, can significantly increase the yield of single-cell adenovirus without affecting the activity of the adenovirus, thereby reducing the production cost of adenovirus-related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a result graph comparing the single-cell yields of YSCH-01 virus and empty control virus provided in the examples of the present application.

[0041] Figure 2 It is an experimental result graph of different broad-spectrum JAK inhibitors on the single-cell yield of YSCH-01 virus provided in the examples of the present application.

[0042] Figure 3 It is an experimental result graph of different specific JAK inhibitors on the single-cell yield of YSCH-01 virus provided in the examples of the present application.

[0043] Figure 4 It is an experimental result graph of the effect of different concentrations of Ruxolitinib inhibitor on the single-cell yield of YSCH-01 virus provided in the examples of the present application.

[0044] Figure 5 This is a graph showing the experimental results of the effect of the Ruxolitinib inhibitor provided in the embodiments of the present application on the single-cell yield of the YSCH-01 virus produced by HEK293 cells.

[0045] Figure 6 This is a graph showing the experimental results of the effect of the Ruxolitinib inhibitor provided in the embodiments of the present application on the single-cell yield of the YSCH-01 virus produced by A549 cells.

[0046] Figure 7 This is a graph showing the experimental results of the effect of the Ruxolitinib inhibitor provided in the embodiments of the present application on the single-cell yield of the wild-type adenovirus Ad-WT.

[0047] Figure 8 This is a graph showing the experimental results of the effect of the Ruxolitinib inhibitor provided in the embodiments of the present application on the single-cell yield of the empty adenovirus OncoMul-V2. Detailed implementation manners

[0048] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application pertains.

[0049] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For those reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0050] The sources of some reagents or materials in this application are as follows:

[0051] Name Source Ruxolitinib MCE, CAS No.: 941678-49-5 Cerdulatinib MCE, CAS No.: 1198300-79-6 Gandotinib MCE, CAS No.: 1229236-86-5 SAR20347 TOPSCIENCE, CAS No.: 1450881-55-6 PF-06700841 TOPSCIENCE, CAS No.: 2140301-96-6 Solcitinib MCE, CAS No.: 1206163-45-2 XL019 MCE, CAS No.: 945755-56-6 Upadacitinib MCE, CAS No.: 1310726-60-3 Fedratinib MCE, CAS No.: 936091-26-8 PF-06651600 MCE, CAS No.: 1792180-81-4 Tyk2-IN-2 MCE, CAS No.: 2098466-94-3 Hela-S3 <![CDATA[ATCC, CCL-2.2 TM > HEK293 ATCC, CRL-1573 A549 <![CDATA[ATCC, CRM-CCL-185 TM >

[0052] In this application, the oncolytic adenovirus YSCH-01 (CCTCC NO: V201871) is used as the experimental object. The oncolytic adenovirus YSCH-01 carries a recombinant interferon expression cassette. Under the same conditions, compared with the control empty virus OncoMul-V2 (i.e., without the interferon expression cassette), the single-cell yield of YSCH-01 decreased by about 61%. The results are shown in Figure 1 as shown. In order to improve the single-cell yield of YSCH-01, the inventors of this application made the following attempts.

[0053] In the following examples, when DMSO was added (DMAO group), the single-cell yield of adenovirus was taken as 1, and the ratio of the single-cell yield of adenovirus in other experimental groups to that in the DMSO group was used as the result for description.

[0054] Example 1 Screening of Broad-Spectrum JAK Inhibitors for Improving the Single-Cell Yield of Adenovirus

[0055] In this example, the effect of broad-spectrum JAK inhibitors on the single-cell yield of adenovirus YSCH-01 was investigated. The broad-spectrum JAK inhibitors included Ruxolitinib inhibitor, Cerdulatinib inhibitor, Gandotinib inhibitor, SAR20347 inhibitor, and PF-06700841 inhibitor.

[0056] The broad-spectrum JAK inhibitors in this application refer to those with more than one main action site.

[0057] In this example, Hela-S3 was used as the host cell. When the recombinant oncolytic adenovirus YSCH-01 was inoculated into Hela-S3, Ruxolitinib inhibitor, Cerdulatinib inhibitor, Gandotinib inhibitor, SAR20347 inhibitor, or PF-06700841 inhibitor was added respectively, and a solvent blank control group of DMSO was set. The inoculation dose of YSCH-01 was 600 VP / cell. The above inhibitors were added to Hela-S3 together with YSCH-01. After 48 h of virus infection, the culture supernatant and cell samples were collected, frozen and thawed repeatedly 3 times from room temperature to -80 °C, and filtered through a 0.22 μm filter membrane. The number of virus particles was measured using a high-performance liquid chromatograph. The addition amounts of various JAK inhibitors were set at two concentrations according to their conventional dosages, and the experimental results are as Figure 2 shown.

[0058] From Figure 2 it can be seen that different broad-spectrum JAK inhibitors have different effects on the single-cell yield of the recombinant oncolytic adenovirus YSCH-01 produced by Hela-S3 cells. Ruxolitinib inhibitor (improvement amount above 130%), SAR20347 inhibitor (improvement amount above 180%), and PF-06700841 inhibitor (improvement amount above 110%) can significantly improve the single-cell yield of YSCH-01. Therefore, in the actual production process, Ruxolitinib inhibitor, SAR20347 inhibitor, or PF-06700841 inhibitor can be added during the process of adenovirus infecting host cells to improve the single-cell yield of adenovirus.

[0059] The broad-spectrum JAK inhibitors screened in this example can be used in combination.

[0060] Example 2: Screening of Specific JAK Inhibitors for Improving the Single-Cell Yield of Adenovirus

[0061] In this example, the effects of JAK inhibitors specifically targeting JAK1, JAK2, JAK3, or Tyk2 on the single-cell yield of adenovirus YSCH-01 were investigated. The specific JAK inhibitors include Solcitinib inhibitor and Upadacitinib inhibitor targeting JAK1; XL019 inhibitor and Fedratinib inhibitor targeting JAK2; PF-06651600 inhibitor targeting JAK3, and Tyk2-IN-2 inhibitor targeting Tyk2.

[0062] The specific JAK inhibitors in this application refer to those with a main action site at one.

[0063] In this example, when Hela-S3 was used as the host cell and the recombinant oncolytic adenovirus YSCH-01 was inoculated into Hela-S3, Solcitinib inhibitor, Upadacitinib inhibitor, XL019 inhibitor, Fedratinib inhibitor, PF-06651600 inhibitor, or Tyk2-IN-2 inhibitor was added respectively, and a solvent blank control group DMSO was set. The inoculation dose of YSCH-01 was 600 VP / cell, and the addition amount of each JAK inhibitor was added at its conventional dose of 3.0 uM. The experimental results are as Figure 3 shown.

[0064] From Figure 3 it can be seen that:

[0065] Among the specific JAK1 inhibitors, Solcitinib inhibitor can significantly improve the single-cell yield of the recombinant oncolytic adenovirus YSCH-01 in Hela-S3 cells, with an increase of about 60%; Upadacitinib inhibitor can also significantly improve the single-cell yield of the recombinant oncolytic adenovirus YSCH-01 in Hela-S3 cells, with an increase of about 140%. Thus, it can be seen that JAK1-specific inhibitors can all significantly improve the single-cell yield of the recombinant oncolytic adenovirus YSCH-01 in Hela-S3 cells.

[0066] Among the specific JAK2 inhibitors, Fedratinib inhibitor can improve the single-cell yield of YSCH-01 by more than 160%, while XL019 inhibitor reduces the single-cell yield of YSCH-01 instead.

[0067] Specific JAK3 inhibitors and specific Tyk2 inhibitors reduce the single-cell yield of YSCH-01.

[0068] Therefore, during the actual production process, JAK1 specific inhibitors and Fedratinib inhibitors can be added during the process of adenovirus infecting host cells to increase the single-cell yield of adenovirus.

[0069] The specific JAK inhibitors screened in this example can be used in combination, or can be used in combination with the broad-spectrum JAK inhibitors screened in Example 1.

[0070] Example 3 Effective dose range of JAK inhibitors

[0071] The inventors of this application tested the effective dose ranges of different JAK inhibitors. The following takes the Ruxolitinib inhibitor as an example for illustration.

[0072] The Ruxolitinib inhibitor was added to Hela-S3 simultaneously with the oncolytic adenovirus YSCH-01 at final concentrations of 1.0 nM, 5.0 nM, 10.0 nM, 50.0 nM, 0.1 μM, 0.5 μM, 1.0 μM, 3.0 μM, 5.0 μM, 10.0 μM, 20.0 μM, or 50.0 μM, respectively. The virus inoculation amount was 600 VP / cell. After 48 h of virus infection, the culture supernatant and cell samples were collected, frozen and thawed repeatedly 3 times from room temperature to -80 °C, and filtered through a 0.22 μm filter membrane. The number of virus particles was measured using a high-performance liquid chromatograph. The experimental results are as Figure 4 shown.

[0073] From Figure 4As can be seen, in Hela-S3, when the concentration of the Ruxolitinib inhibitor is below 50.0 nM, it basically has no promoting effect on the single-cell yield of the oncolytic adenovirus YSCH-01; when the concentration reaches above 50.0 nM, with the increase in the concentration of the Ruxolitinib inhibitor, the promoting effect on the single-cell yield of YSCH-01 increases; when the concentration increases to 3.0 μM, the single-cell yield of YSCH-01 reaches the highest value; thereafter, with the increase in concentration, the promoting effect on the single-cell yield of YSCH-01 decreases, and when it reaches 20.0 μM, the single-cell yield of YSCH-01 is significantly inhibited. This indicates that the effective dose range of the Ruxolitinib inhibitor to increase the single-cell yield of the oncolytic adenovirus YSCH-01 is 50.0 nM to 10.0 μM. Preferably, the effective dose range is 0.1 μM to 10.0 μM, more preferably the effective dose range is 0.5 μM to 10.0 μM, further preferably the effective dose range is 0.5 μM to 5.0 μM, even more preferably the effective dose range is 1.0 μM to 3.0 μM, and most preferably the effective dose range is 3.0 μM. When the concentration of the Ruxolitinib inhibitor is 3.0 μM, the single-cell yield of the adenovirus YSCH-01 increases by about 130%.

[0074] Through the same above experiments, the effective dose range of the Solcitinib inhibitor is 1.0 μM to 20.0 μM; the effective dose range of the Upadacitinib inhibitor is 1.0 μM to 20.0 μM; the effective dose range of the Fedratinib inhibitor is 0.5 μM to 10.0 μM; the effective dose range of the SAR20347 inhibitor is 3.0 μM to 20.0 μM; the effective dose range of the PF-06700841 inhibitor is 0.5 μM to 15.0 μM.

[0075] Example 4 Effect of JAK inhibitors on the activity of recombinant adenovirus

[0076] In this example, the effect of JAK inhibitors on the activity of recombinant adenovirus was illustrated by testing the effect of JAK inhibitors on the specific titer of recombinant adenovirus (the specific titer is the ratio of the infectious virus to the number of virus particles, i.e., specific titer = virus titer / number of virus particles). The following JAK inhibitor is exemplified by the Ruxolitinib inhibitor, and the recombinant adenovirus is exemplified by YSCH-01.

[0077] Take the progeny viruses in each group treated with DMSO and 3.0 μM Ruxolitinib inhibitor in Example 3, and use a rapid adenovirus titer assay kit (CELL BIOLABS, VPK-109) to measure their virus titers. Then, calculate the corresponding specific titer based on the number of particles of the progeny viruses measured in Example 3.

[0078] The specific titer of the progeny virus in the DMSO group was 1.62% ifu / vp, and the specific titer of the progeny virus in the Ruxolitinib group was 1.67% ifu / vp. There was no significant difference between the two, indicating that the Ruxolitinib inhibitor did not reduce the specific titer of adenovirus, that is, the Ruxolitinib inhibitor had no effect on the activity of the recombinant adenovirus YSCH-01.

[0079] Example 5 Cell Universality

[0080] In the above examples, human cervical cancer Hela-S3 cells were used as host cells. In actual situations, other engineering cell lines may be used to produce adenoviruses. Therefore, the inventors tested the effect of JAK inhibitors on the production of adenoviruses by other common engineering cell lines.

[0081] The following JAK inhibitor is exemplified by the Ruxolitinib inhibitor, and the engineering cell lines are exemplified by human renal epithelial cells HEK293 and human lung cancer cells A549 for illustration.

[0082] The oncolytic adenovirus YSCH-01 was inoculated into HEK293 cells and A549 cells at an inoculation ratio of 600 VP / cell, and 3.0 μM or 5.0 μM Ruxolitinib was added for treatment respectively. After continuous culture for 48 h, the culture supernatant and cell samples were collected, frozen and thawed repeatedly 3 times from room temperature to -80 °C, and filtered through a 0.22 μm filter membrane. A high-performance liquid chromatograph was used to measure the number of virus particles. The experimental results are as Figure 5 and Figure 6 shown.

[0083] As can be seen from Figure 5 , the inhibitor Ruxolitinib could significantly increase the single-cell yield of YSCH-01 in HEK293 cells, with an increase of more than 50%. As can be seen from Figure 6 , the inhibitor Ruxolitinib could significantly increase the single-cell yield of YSCH-01 in A549 cells, with an increase of more than 190%.

[0084] The above results indicate that the inhibitor Ruxolitinib can significantly improve the single-cell yield of oncolytic adenovirus YSCH-01 in different cells, showing cell universality (i.e., not restricted by cell types), and can be widely applied in actual production.

[0085] Example 6 Adenovirus Universality

[0086] The inventors of this application experimented with the effects of JAK inhibitors on the single-cell yields of different adenoviruses. The following JAK inhibitor is exemplified by Ruxolitinib inhibitor, and the adenoviruses are exemplified by wild-type adenovirus Ad-WT (ATCC, VR-1516) and oncolytic adenovirus OncoMul-V2 (a control empty vector adenovirus without an interferon expression cassette in YSCH-01).

[0087] Both of the above two adenoviruses were inoculated into Hela-S3 cells at an inoculation ratio of 600 VP / cell, and 3.0 uM of Ruxolitinib (Ruxo) was added simultaneously. After continuous culture for 48 h, the culture supernatant and cell samples were collected, frozen and thawed repeatedly 3 times from room temperature to -80 °C, and filtered through a 0.22 um filter membrane. The number of virus particles was measured using a high-performance liquid chromatograph, with solvent DMSO as a control. The experimental results are as Figure 7 and Figure 8 shown.

[0088] As can be seen from Figure 7 , the Ruxolitinib inhibitor can significantly improve the single-cell yield of wild-type adenovirus Ad-WT, and the improvement amount is more than 30%.

[0089] As can be seen from Figure 8 , the Ruxolitinib inhibitor can significantly improve the single-cell yield of empty vector adenovirus OncoMul-V2, and the improvement amount is more than 25%.

[0090] The above results indicate that the inhibitor Ruxolitinib can significantly improve the single-cell yields of wild-type adenovirus Ad-WT and empty vector adenovirus OncoMul-V2, indicating that the Ruxolitinib inhibitor has adenovirus universality in improving the single-cell yield of adenovirus (i.e., not restricted by adenovirus types).

[0091] In summary, the present application has screened out JAK inhibitors that can significantly improve the single-cell yield of adenovirus, namely JAK1-specific inhibitor, Fedratinib inhibitor, Ruxolitinib inhibitor, SAR20347 inhibitor, and PF-06700841 inhibitor. When improving the single-cell yield of adenovirus, JAK inhibitors have no effect on the activity of adenovirus and have universality for adenovirus and cells. The increase in the single-cell yield of adenovirus can significantly reduce the cost of producing adenovirus-related drugs and is more conducive to large-scale industrial production.

[0092] This specific embodiment is only an interpretation of the present application and does not limit the present application. After reading this specification, those skilled in the art can make modifications to this embodiment without creative contributions as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for producing an adenovirus, characterized in that, it comprises the following steps: adding a JAK inhibitor during the process of adenovirus infecting a host cell; the adenovirus includes a recombinant adenovirus or a wild-type adenovirus; the recombinant adenovirus is an oncolytic adenovirus YSCH-01, the oncolytic adenovirus YSCH-01 is an oncolytic virus with a preservation number of CCTCC NO: V201871, the wild-type adenovirus is VR-1516, and the host cell is a Hela-S3 cell, a HEK293 cell and an A549 cell; the JAK inhibitor is Solcitinib; the effective concentration of the Solcitinib is 1.0 uM to 20.0 uM.

Images