Gene therapeutic agent for cancer

Abstract

The present disclosure provides a cancer gene therapy technique having an enhanced cancer inhibition effect. The inventors have found the possibility that an excellent cancer inhibition effect can be obtained using an oncolytic adenoviral vector comprising a nucleic acid, which contains an artificial gene obtained by fusing a CD44 extracellular portion, a Notch core region, and HIF-3α4, and an E1A gene and an E1B gene that are arranged under the control of a COX-2 promoter, and in which the artificial gene, the E1A gene, and the E1B gene are arranged in the same orientation, and thereby completed the present disclosure.

Description

Cancer gene therapy drugs

[0001] The present disclosure relates to, for example, novel gene therapy drugs or novel gene therapy methods for cancer, and their active ingredients, etc. The contents of all documents described in this specification (including Non-Patent Documents 1 to 12 listed below as prior art documents) are incorporated herein by reference.

[0002] In recent years, viral vector-based gene therapy has attracted attention as a new cancer treatment. Taking advantage of the properties of various viruses, viruses are used as gene carriers and viral vectors. One method of gene transfer is "in vivo gene therapy," in which a recombinant viral vector incorporating a therapeutic gene is directly administered into the body to treat the disease. Cancer gene therapy has a different mechanism of action from conventional treatments such as chemotherapy. It involves introducing genes into cancer cells to directly suppress genes involved in cancer development and proliferation, or introducing tumor suppressor genes into cells to directly induce cell death. Therefore, cancer gene therapy is expected to be an effective treatment for cancers that are difficult to treat with conventional chemotherapy or radiation therapy.

[0003] International Publication No. 2021 / 131944

[0004] Kosaki R., Watanabe K., Yamaguchi Y. (1999). Overproduction of hyaluronan by expression of the hyaluronan synthase Has2 enhances anchorage-independent growth and tumorigenicity. Cancer Res. 1999 Mar 1;59(5):1141-5.Bourguignon LY, Zhu H, Shao L, Chen YW (2001). CD44 interaction with c-Src kinase promotes cortactin-mediated cytoskeleton function and hyaluronic acid-dependent ovarian tumor cell migration. J Biol Chem. 2001 Mar 9;276(10):7327-36. Epub 2000 Nov 17.Fujita Y., Kitagawa M., Nakamura S., Azuma K., Ishii G., Higashi M., Kishi H., Hiwasa T., Koda K., Nakajima N., Harigaya K.(2002). CD44 signaling through focal adhesion kinase and its anti-apoptotic effect. FEBS Lett. 2002 Sep 25;528(1-3):101-8.Bazil V., Horejsi V.(1992). Shedding of the CD44 adhesion molecule from leukocytes induced by anti-CD44 monoclonal antibody simulating the effect of a natural receptor ligand. J Immunol. 1992 Aug 1;149(3):747-53.Murakami D., Okamoto I., Nagano O., Kawano Y., Tomita T., Iwatsubo T., De Strooper B., Yumoto E., Saya H.(2003). Presenilin-dependent gamma-secretase activity mediates the intramembranous cleavage of CD44. Oncogene. 2003 Mar 13;22(10):1511-6.Ueda M., Saji H.(2014). Radiolabeled Probes Targeting Hypoxia-Inducible Factor-1-Active Tumor Microenvironments. ScientificWorldJournal. 2014;2014:165461. doi: 10.1155 / 2014 / 165461. Epub 2014 Aug 18.Gao N., Shen L., Zhang Z., Leonard SS., He H., Zhang XG., Shi X., Jiang BH.(2004). Arsenite induces HIF-1alpha and VEGF through PI3K, Akt and reactive oxygen species in DU145 human prostate carcinomacells. Mol Cell Biochem. 2004 Jan;255(1-2):33-45.Kopan R., Ilagan MX.(2009). The canonical Notch signaling pathway: unfolding the activation mechanism. Cell. 2009 Apr 17;137(2):216-33. doi: 10.1016 / j.cell.2009.03.045.Shona, T., Graeme, J.(2009). A Cancer Gene Therapy Approach that Targets Tumor-associated Hyaluronan. Cancer Growth and Metastasis 2009(2)November 2009 with 31 Reads.Morsut L., Roybal KT., Xiong X., Gordley RM., Coyle SM., Thomson M., Lim WA. (2016). Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell. 2016 Feb 11;164(4):780-91. doi: 10.1016 / j.cell.2016.01.012. Epub 2016 Jan 28. Makino Y., Cao R., Svensson K., Bertilsson G., Asman M., Tanaka H., Cao Y., Berkenstam A., Poellinger L. (2001). Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression. Nature. 2001 Nov 29;414(6863):550-4. Maynard MA., Evans AJ., Hosomi T., Hara S., Jewett MA., Ohh M. (2005). Human HIF-3alpha4 is a dominant-negative regulator of HIF-1 and is down-regulated in renal cell carcinoma. FASEB J. 2005 Sep;19(11):1396-406.(2).

[0005] The present inventors have previously found that an artificial gene fusing CD44 (particularly the extracellular portion), Notch (particularly the core region), and HIF-3α4 can exert a cancer-suppressing effect (Patent Document 1). The present inventors have made further improvements, with the main objective of providing a cancer gene therapy technology with an even stronger cancer-suppressing effect.

[0006] The present inventors have found that the above-mentioned problems can be solved by an oncolytic adenoviral vector comprising an artificial gene in which the extracellular portion of CD44, the Notch core region, and HIF-3α4 are fused, and a nucleic acid in which the artificial gene and the E1A gene and the E1B gene are arranged in the same orientation under the control of the COX-2 promoter, and have made further improvements, which has led to the completion of the present disclosure.

[0007] The present disclosure includes, for example, the subject matter described in the following sections: Section 1. An oncolytic adenoviral vector comprising a nucleic acid comprising: a base sequence (I): a base sequence having a structure in which (A) a base sequence encoding a protein having CD44 extracellular function, (B) a base sequence encoding a protein having Notch core region function, and (C) a base sequence encoding a protein having HIF-3α4 function are linked in the order of (A)-(B)-(C), and a base sequence (II): a base sequence comprising: (D) a base sequence comprising a COX-2 promoter, (E) a base sequence encoding an E1A protein placed under the control of the COX-2 promoter, and (F) a base sequence encoding an E1B protein placed under the control of the COX-2 promoter, wherein the base sequence (I) and the base sequence (II) are arranged in the same orientation.Item 2. An oncolytic adenoviral vector comprising: a base sequence (I): a base sequence having a structure in which the base sequence (A), the base sequence (B), and the base sequence (C) are linked in the order of (A)-(B)-(C); and a base sequence (II): a base sequence comprising: (D) a base sequence comprising a COX-2 promoter, (E) a base sequence encoding an E1A protein placed under the control of the COX-2 promoter, and (F) a base sequence encoding an E1B protein placed under the control of the COX-2 promoter, wherein the base sequence (I) and the base sequence (II) are arranged in the same direction, and wherein the base sequence (A) is: (a-1): a base sequence consisting of the base sequence of SEQ ID NO: 1, or (a-2): a base sequence consisting of a base sequence having 85% or more sequence identity with the base sequence of (a-1) and encoding a protein capable of binding to hyaluronic acid, and the base sequence (B) is: (b-1): a base sequence consisting of the base sequence of SEQ ID NO: 2, or (b-2): a base sequence having a sequence identity of 85% or more with the base sequence of (b-1), and encoding a protein that can be cleaved by a protease, wherein the base sequence (C) is: (c-1): a base sequence consisting of the base sequence of SEQ ID NO: 3, or (c-2): a base sequence having a sequence identity of 85% or more with the base sequence of (c-1), and encoding a protein that can bind to HIF-1α, wherein the base sequence (D) is: (d-1): a base sequence consisting of the base sequence of SEQ ID NO: 4, or (d-2): a base sequence having a sequence identity of 85% or more with the base sequence of (d-1), and encoding a promoter whose expression is induced under COX-2 expression-inducing conditions, wherein the base sequence (E) is: (e-1): a base sequence consisting of the base sequence of SEQ ID NO: 5, or (e-2): a base sequence having 85% or more sequence identity with the base sequence of (e-1) and encoding a protein having E1A function, and the base sequence (F) is: (f-1): a base sequence consisting of the base sequence of SEQ ID NO: 6, or (f-2): a base sequence having 85% or more sequence identity with the base sequence of (f-1) and encoding a protein having E1B function.Item 3. The base sequence (A) is (a-1): a base sequence consisting of the base sequence of SEQ ID NO: 1, or (a-2): a base sequence consisting of a base sequence having 85% or more sequence identity with the base sequence of (a-1), and encoding a protein capable of binding to hyaluronic acid, the base sequence (B) is (b-1): a base sequence consisting of the base sequence of SEQ ID NO: 2, or (b-2): a base sequence having 85% or more sequence identity with the base sequence of (b-1), and encoding a protein that can be cleaved by a protease, in a protein encoded by a nucleic acid having a structure in which (A)-(B)-(C) are linked in this order, the base sequence (C) is (c-1): a nucleic acid consisting of the base sequence of SEQ ID NO: 3, or (c-2): a nucleic acid consisting of a base sequence having 85% or more sequence identity with the base sequence of (c-1), and encoding a protein capable of binding to HIF-1α, and the base sequence (D) is Item 2. The oncolytic adenoviral vector according to Item 1, wherein the base sequence (E) is: (d-1): a base sequence consisting of the base sequence of SEQ ID NO: 4; or (d-2): a base sequence having 85% or more sequence identity with the base sequence of (d-1), and encoding a promoter whose expression is induced under COX-2 expression-inducing conditions; the base sequence (E) is: (e-1): a base sequence consisting of the base sequence of SEQ ID NO: 5; or (e-2): a base sequence having 85% or more sequence identity with the base sequence of (e-1), and encoding a protein having E1A function; and the base sequence (F) is: (f-1): a base sequence consisting of the base sequence of SEQ ID NO: 6; or (f-2): a base sequence having 85% or more sequence identity with the base sequence of (f-1), and encoding a protein having E1B function.Item 4. An oncolytic adenoviral vector comprising: a base sequence (III): (g-1) consisting of the base sequence of SEQ ID NO: 7; or (g-2) consisting of a base sequence having 85% or more sequence identity with the base sequence of SEQ ID NO: 7, and having a structure in which a portion encoding a protein capable of binding to hyaluronic acid, a portion encoding a protein that can be cleaved by a protease, and a portion encoding a protein capable of binding to HIF-1α are linked in this order; or (g-3) consisting of a base sequence having 85% or more sequence identity with the base sequence of SEQ ID NO: 7, and encoding a protein having anti-cancer activity; and a base sequence (II): (D) a base sequence comprising a COX-2 promoter, (E) a base sequence encoding an E1A protein placed under the control of the COX-2 promoter, and (F) a base sequence encoding an E1B protein placed under the control of the COX-2 promoter, wherein the base sequence (III) and the base sequence (II) are arranged in the same direction, and wherein the base sequence (D) is Item 4. An oncolytic adenoviral vector comprising a nucleic acid, wherein the base sequence (E) is: (d-1): a base sequence consisting of the base sequence of SEQ ID NO: 4, or (d-2): a base sequence having 85% or more sequence identity with the base sequence of (d-1), and encoding a promoter whose expression is induced under COX-2 expression-inducing conditions, and the base sequence (E) is: (e-1): a base sequence consisting of the base sequence of SEQ ID NO: 5, or (e-2): a base sequence having 85% or more sequence identity with the base sequence of (e-1), and encoding a protein having E1A function, and the base sequence (F) is: (f-1): a base sequence consisting of the base sequence of SEQ ID NO: 6, or (f-2): a base sequence having 85% or more sequence identity with the base sequence of (f-1), and encoding a protein having E1B function. Item 5. The oncolytic adenoviral vector according to any one of Items 1 to 4, further comprising: (H) a nucleotide sequence encoding an Ad5 / 35 chimeric fiber protein.Item 6. The oncolytic adenoviral vector according to Item 5, wherein the base sequence (H) consists of: (h-1): a base sequence of SEQ ID NO: 8, or (h-2): a base sequence having 85% or more sequence identity with the base sequence of (h-1), and encoding a protein capable of binding to CD46. Item 7. The oncolytic adenoviral vector according to any of Items 2 to 6, wherein the protease is ADAM protease or γ-secretase. Item 8. An anti-cancer composition comprising the oncolytic adenoviral vector according to any of Items 1 to 7. Item 9. The anti-cancer composition according to Item 8, which is in the form of an injection. Item 10. The anti-cancer composition according to Item 8 or 9, which is used to treat COX-2-positive cancer. Item 11. The anti-cancer composition according to any of Items 8 to 10, which is used to treat at least one cancer selected from the group consisting of bladder cancer, breast cancer, and prostate cancer. Item 12. A method for treating cancer, comprising administering a therapeutically effective amount of the oncolytic adenoviral vector of any of Items 1 to 7 to a subject in need thereof. Item 13. The method of Item 12, wherein the administration is by injection. Item 14. The method of Item 12 or 13, wherein the cancer is a COX-2 positive cancer. Item 15. The method of any of Items 12 to 14, wherein the cancer is at least one type of cancer selected from the group consisting of bladder cancer, breast cancer, and prostate cancer. Item 16. The oncolytic adenoviral vector of any of Items 1 to 7, for use in cancer treatment. Item 17. The oncolytic adenoviral vector of Item 16, wherein the vector is administered by injection. Item 18. The oncolytic adenoviral vector of Item 16 or 17, wherein the cancer is a COX-2 positive cancer. Item 19. The oncolytic adenoviral vector of any of Items 16 to 18, wherein the cancer is at least one type of cancer selected from the group consisting of bladder cancer, breast cancer, and prostate cancer. Item 20. Application of the oncolytic adenoviral vector according to any one of Items 1 to 7 for the manufacture of a cancer therapeutic agent. Item 21. The application according to Item 20, wherein the therapeutic agent is an injection. Item 22. The application according to Item 20 or 21, wherein the cancer is a COX-2 positive cancer.Item 23. The application according to any one of Items 20 to 22, wherein the cancer is at least one cancer selected from the group consisting of bladder cancer, breast cancer, and prostate cancer.

[0008] According to the present disclosure, a novel cancer gene therapy drug with extremely high cancer suppression effect can be provided.

[0009] Figure 2 shows a schematic diagram of the adenoviral vector CRAd-synNotch (forward orientation) prepared in an example of the present disclosure. Figure 3 shows an excerpt of the CD44 / Notch / HIF-3α4 fusion gene, Ad5 / 35 chimeric fiber gene, COX-2 promoter region, E1A gene, and E1B gene from the schematic diagram shown in Figure 2. Figure 4 shows a schematic diagram of the adenoviral vector CRAd-GFP prepared in an example of the present disclosure. Test Example 2.1.1 shows the results of expression analysis of CD46, CAR, and CD44 in BT474 cells (n=3, average ±SE bars, **p<0.01). Test Example 2.1.2 shows the results of expression analysis of CD46, CAR, and CD44 in T24 cells (n=3, average ±SE bars, **p<0.01). Test Example 2.1.3: Results of expression analysis of CD46, CAR, and CD44 in MDA-MB-231 cells (n=3, average ±SE bars, **p<0.01). Test Example 2.1.4: Results of expression analysis of CD46, CAR, and CD44 in DU145 cells (n=3, average ±SE bars, **p<0.01). Test Example 2.2: Results of quantification of COX-2 gene expression in T24 cells, MDA-MB-231 cells, and DU145 cells (n=3, average ±SE bars, **p<0.01). Test Example 2.3.1: Results of quantitative expression of HAS-1, HAS-2, and HAS-3 genes in T24 cells (n=3, average ±SE bars, **p<0.01, *p<0.05). Test Example 2.3.2: Results of quantitative expression of HAS-1, HAS-2, and HAS-3 genes in MDA-MB-231 cells (n=3, average ±SE bars, **p<0.01). Test Example 2.3.3: Results of quantitative expression of HAS-1, HAS-2, and HAS-3 genes in DU145 cells (n=3, average ±SE bars, **p<0.01). Test Example 2.5.1: Results of CD44 expression analysis by Western blotting.Test Example 2.5.2: Results of quantification of HIF-3α4 gene expression by real-time RT-PCR (n=3, average ±SE bars, **p<0.01). Test Example 2.6.1.1: Results of CD44 expression analysis by Western blotting in CRAd-synNotch (forward) clone 1 are shown. Test Example 2.6.1.2: Results of CD44 expression analysis by Western blotting in CRAd-synNotch (forward) clones 2 and 3 are shown. Test Example 2.6.2: Results of quantification of CD44 gene and HIF-3α4 gene expression by real-time RT-PCR are shown (n=3, average ±SE bars, **p<0.01). Test Example 2.7.1: This shows the results of CD44 expression analysis by Western blotting in MDA-MB-231 cells and T24 cells infected with CRAd-synNotch (forward) clone 3 after mass culture. Test Example 2.7.2: This shows the results of quantifying CD44 gene and HIF-3α4 gene expression by real-time RT-PCR in T24 cells infected with CRAd-synNotch (forward) clone 3 after mass culture (n=3, average ±SE bars, **p<0.01). Test Example 2.8: This shows the results of evaluating the proliferation ability of adenovirus vectors (n=3, average ±SE bars, **p<0.01). The vertical axis shows virus copy numbers (real numbers). A: wtAd, B: ADX730, C: CRAd-GFP, D: CRAd-synNotch. Test Example 2.9.1: Results of evaluation of the inhibitory effect of CRAd-synNotch on BT474 cell proliferation (n=3, average ±SE bars). The vertical axis indicates absorbance measured at a wavelength of 492 nm. Test Example 2.9.2: Results of evaluation of the inhibitory effect of CRAd-synNotch on T24 cell proliferation (n=3, average ±SE bars, **p<0.01). The vertical axis indicates absorbance measured at a wavelength of 492 nm. Test Example 2.9.3: Results of evaluation of the inhibitory effect of CRAd-synNotch on MDA-MB-231 cell proliferation (n=3, average ±SE bars). The vertical axis represents the absorbance measured at a wavelength of 492 nm.Test Example 2.9.4: Shows the results of evaluating the inhibitory effect of CRAd-synNotch on DU145 cell proliferation (n=3, average ±SE bars, **p<0.01, *p<0.05). The vertical axis shows absorbance measured at a wavelength of 492 nm. Test Example 2.10.1: Shows the results of evaluating the inhibitory effect of CRAd-synNotch on VGEF gene expression (n=3, average ±SE bars, **p<0.01). Test Example 2.10.2: Shows the results of evaluating the inhibitory effect of CRAd-synNotch on SOX-2 gene expression (n=3, average ±SE bars, **p<0.01). Test Example 2.10.3: Results of evaluation of the CCL2 gene suppression effect of CRAd-synNotch are shown (n=3, average ±SE bars, **p<0.01). Test Example 2.11: Results of evaluation of the in vivo bladder cancer therapeutic effect of CRAd-synNotch are shown. In this figure, the vertical axis represents tumor volume, and the horizontal axis represents the number of days from the start of administration of PBS, ADX730, CRAd-GFP, or CRAd-synNotch (n=5, average ±SE bars, **p<0.01). Test Example 2.11: Results of evaluation of the in vivo bladder cancer therapeutic effect of CRAd-synNotch are shown. In this figure, the vertical axis represents survival rate, and the horizontal axis represents the number of days from the start of administration of PBS, ADX730, CRAd-GFP, or CRAd-synNotch (n=5). The nucleotide sequence of SEQ ID NO: 1 is shown. The nucleotide sequence of SEQ ID NO: 2 is shown. The nucleotide sequence of SEQ ID NO: 3 is shown. The nucleotide sequence of SEQ ID NO: 4 is shown. The nucleotide sequence of SEQ ID NO: 5 is shown. The nucleotide sequence of SEQ ID NO: 6 is shown. The nucleotide sequence of SEQ ID NO: 7 is shown. The nucleotide sequence of SEQ ID NO: 8 is shown. The nucleotide sequence of SEQ ID NO: 9 is shown. The amino acid sequences of SEQ ID NOs: 10 to 12 are shown. The amino acid sequence of SEQ ID NO: 13 is shown. The amino acid sequence of SEQ ID NO: 14 is shown. The amino acid sequence of SEQ ID NO: 15 is shown. Test 2.12: T24 cells were infected with 50 MOI of CRAd-synNotch using the method described in 1.7.2 and cultured for 24 or 72 hours. After culture, Western blotting was performed using a CD44 antibody as the primary antibody, and the results are shown on the left. The results of Western blotting using HIF3α4 antibody as the primary antibody are shown on the right.Experiment 2.12: DU145 cells were infected with CRAd-synNotch at 25 MOI or 50 MOI using the method described in 1.7.2 and cultured for 24, 48, or 96 hours. After culture, the results of Western blotting using CD44 antibody as the primary antibody are shown on the left. The results of Western blotting using HIF3α4 antibody as the primary antibody are shown on the right. Experiment 2.13: Results of analysis of the expression levels of Cortactin, OCT4, and Nanog in T24 cells are shown. n=3, average±SE bars, **p<0.01. Experiment 2.13: Results of analysis of the expression levels of PHD3, GLUT1, and Cyclin G2 in T24 cells are shown. n=3, average±SE bars, **p<0.01, *p<0.05. Experiment 2.13: Results of analyzing the expression levels of Cortactin, OCT4, Nanog, and SOX-2 in DU145 cells. n=3, average±SE bars, **p<0.01. Experiment 2.13: Results of analyzing the expression levels of VEGF, GLUT1, Cyclin G2, and PHD3 in DU145 cells. n=3, average±SE bars, **p<0.01, *p<0.05. Experiment 2.14: Results of immunohistochemical staining of CD44 in a T24 mouse model. Bar=100 μm.

[0010] Each embodiment of the present disclosure will be described in more detail below. The present disclosure preferably includes, for example, an oncolytic adenoviral vector incorporating a specific nucleic acid, an anticancer composition containing the oncolytic adenoviral vector, and a method for treating cancer using the composition, but is not limited thereto. The present disclosure includes all of the disclosures herein that would be recognized by a person skilled in the art.

[0011] The oncolytic adenoviral vector encompassed by the present disclosure is preferably an oncolytic adenoviral vector comprising: a base sequence (I): (A) a base sequence encoding a protein having CD44 extracellular function, (B) a base sequence encoding a protein having Notch core region function, and (C) a base sequence encoding a protein having HIF-3α4 function, linked in the order of (A)-(B)-(C); and a base sequence (II): (D) a base sequence comprising a COX-2 promoter, (E) a base sequence encoding an E1A protein placed under the control of the COX-2 promoter, and (F) a base sequence encoding an E1B protein placed under the control of the COX-2 promoter, wherein the base sequence (I) and the base sequence (II) are oriented in the same direction. This oncolytic adenoviral vector may be referred to as the "oncolytic adenoviral vector of the present disclosure." Furthermore, the above-described nucleic acid may be referred to as the "nucleic acid of the present disclosure."

[0012] 1. Oncolytic Adenovirus Vectors Oncolytic viruses are restricted replication viruses that can replicate specifically in targeted cancer cells. Oncolytic viruses generally cannot infect normal cells. They adsorb to specific receptors highly expressed on cancer cells and infect the cancer cells. Once incorporated into cancer cells, the oncolytic virus replicates within the cells to produce viral proteins, causing the host cancer cells to lyse and die. The oncolytic virus that has replicated within the cells is released from the dead cancer cells into the tumor microenvironment. Oncolytic adenovirus is a typical type of oncolytic virus.

[0013] The oncolytic adenoviral vector used in the technology of the present disclosure is not particularly limited as long as the adenovirus derived from the vector can infect and grow in cancer cells, but is preferably an oncolytic adenoviral vector capable of infecting and growing in cancer cells expressing the CD46 receptor. Examples of such oncolytic adenoviral vectors include oncolytic adenoviral vectors based on human type 35 adenovirus, and more specifically, oncolytic adenoviral vectors derived from pAd1129-06 (O.D.260).

[0014] A preferred embodiment of the oncolytic adenoviral vector of the present disclosure is one in which the nucleic acid of the present disclosure is contained in an oncolytic adenoviral vector based on human adenovirus type 35. A more preferred embodiment of the oncolytic adenoviral vector of the present disclosure is one in which the nucleic acid of the present disclosure is contained in an oncolytic adenoviral vector derived from pAd1129-06.

[0015] The oncolytic adenoviral vector of the present disclosure can be produced by known methods or methods that can be easily derived from known methods, such as genetic engineering techniques such as nucleic acid synthesis techniques, PCR, restriction enzyme cleavage, DNA ligation techniques, and in vitro transcription techniques.

[0016] 2. Nucleic Acid of the Present Disclosure As described above, the nucleic acid of the present disclosure is a nucleic acid comprising: base sequence (I): a base sequence having a structure in which (A) a base sequence encoding a protein having CD44 extracellular function, (B) a base sequence encoding a protein having Notch core region function, and (C) a base sequence encoding a protein having HIF-3α4 function are linked in the order of (A)-(B)-(C); and base sequence (II): (D) a base sequence including a Cox-2 promoter, (E) a base sequence encoding an E1A protein placed under the control of the Cox-2 promoter, and (F) a base sequence encoding an E1B protein placed under the control of the Cox-2 promoter, wherein the base sequences (I) and (II) are arranged in the same orientation.

[0017] In the present disclosure, the term "nucleic acid" includes DNA, RNA, PNA, and the like. The nucleic acid in the present disclosure is preferably DNA or RNA, and particularly preferably DNA. Furthermore, the nucleic acid may be subjected to known chemical modifications, as exemplified below. In order to prevent degradation by hydrolases such as nucleases, the phosphoric acid residue (phosphate) of each nucleotide can be substituted with a chemically modified phosphoric acid residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate. Furthermore, the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide can be substituted with -OR (where R is, for example, CH ３ (2'-O-Me), CH ２ CH ２ OCH ３ (2'-O-MOE), CH ２ CH ２ NHC (NH) NH ２ , C.H. ２ CONHCH ３ , C.H. ２ CH ２ CN, etc.). Furthermore, the base moiety (pyrimidine, purine) may be chemically modified. Specific examples include the introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or the substitution of the carbonyl group at the 2-position with a thiocarbonyl. Further examples include those in which the phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc., but are not limited to these. Furthermore, BNA (LNA), in which the conformation of the sugar moiety is fixed to N-type by bridging the 2' oxygen and 4' carbon of the sugar moiety of the nucleotide, may also be preferably used.

[0018] 2-1. Nucleotide Sequence (A) The nucleotide sequence (A) encodes a protein with CD44 extracellular function. CD44 is a receptor for hyaluronic acid and other receptors. Upon binding to a ligand (e.g., hyaluronic acid), it clusters and transmits signals. This is known to result in intracellular phenomena such as the activation of various kinases involved in cell proliferation and migration, such as c-Src, FAK, and MAPK. It is also known that after signal transduction, the intracellular domain is cleaved by proteases, translocates to the nucleus, and the detached extracellular domain is released as soluble CD44. Due to these properties, CD44 is highly expressed in many types of cancer cells, including colon cancer, breast cancer, gastric cancer, pancreatic cancer, and prostate cancer, and is also being studied as a marker for cancer stem cells.

[0019] In the present disclosure, the extracellular function of CD44 refers to the receptor function for a ligand, and this function preferably includes, for example, the ability to bind to hyaluronic acid.

[0020] The protein having the CD44 extracellular function may be, for example, a protein of the entire extracellular portion of CD44, or may include the cell membrane portion of CD44, or may be a protein of a portion of the extracellular portion of CD44, so long as it does not impair the receptor function for a ligand. Furthermore, such a protein may have one or more amino acids deleted, substituted, or added, so long as it retains the function. A preferred example of a protein having the CD44 extracellular function is a protein comprising the amino acid sequence of SEQ ID NO: 10, and a more preferred example is a protein consisting of the amino acid sequence of SEQ ID NO: 10. The base sequence (A) is not particularly limited, as long as it is a base sequence that encodes such a polypeptide.

[0021] More specifically, examples of the base sequence (A) include the following (a-1) and (a-2): (a-1): a base sequence consisting of the base sequence of SEQ ID NO: 1 (a-2): a base sequence having 85% or more sequence identity with the base sequence of (a-1) and encoding a protein capable of binding to hyaluronic acid

[0022] In the present disclosure, "identity" of a base sequence refers to the degree of match between the base sequences of two or more comparable base sequences. Thus, the higher the match between two base sequences, the higher the identity or similarity between those sequences. The level of identity of a base sequence can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. 87:2264-2268 (1990); Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc Natl Acad Sci USA. 90:5873-7 (1993)). Specific techniques for these analysis methods are known, and can be found on the National Center for Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of amino acid sequences is also defined in the same manner as above. The identity of amino acid sequences can be analyzed using a program called BLASTX.

[0023] An example of a base sequence X' that does not have 100% sequence identity to a certain base sequence X is a base sequence in which one or more bases have been substituted, deleted, added, or inserted (preferably substituted) relative to the base sequence X. Here, "multiple" means, for example, 2 to 100, preferably 2 to 80, more preferably 2 to 50, and even more preferably 2 to 10. The upper or lower limit of the range is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, It may be 2, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0024] Furthermore, in the present disclosure, when a certain base sequence X is a base sequence encoding a polypeptide, a base sequence X' that does not have 100% sequence identity thereto preferably does not cause a frameshift compared to the base sequence X. When the amino acid sequence encoded by the base sequence X is referred to as P and the amino acid sequence encoded by the base sequence X' is referred to as P', it is particularly preferred that P' is identical to P.

[0025] When the sequence identity of amino acid sequence P' to amino acid sequence P is not 100%, the sequence identity may be 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, and particularly preferably 95% or more. Examples of such P' include amino acid sequences in which one or more amino acids have been substituted, deleted, added, or inserted (preferably by substitution, more preferably by conservative substitution) with respect to P. Here, "plurality" refers to, for example, 2 to 20, preferably 2 to 10, more preferably 2 to 5, and even more preferably 2 or 3. The upper or lower limit of the range may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0026] "Conservative substitution" means that an amino acid residue is substituted with an amino acid residue having a similar side chain. For example, substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine are conservative substitutions. Other conservative substitutions include substitutions between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0027] As described above, the base sequence (a-2) is a base sequence that has a sequence identity of 85% or more with the base sequence of (a-1) and encodes a protein capable of binding to hyaluronic acid. The sequence identity may be 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more.

[0028] Furthermore, in the present disclosure, the term "a protein capable of binding to another protein" refers to the ability of the protein to specifically interact with the other protein. Whether a protein can bind to another protein can be confirmed by methods known in the art. Such methods include, for example, co-immunoprecipitation (Co-IP), pull-down assay, Western blotting, cross-linking, label transfer reaction, interaction mapping, surface plasmon resonance, and FRET (fluorescence resonance energy transfer). Furthermore, whether a protein can bind to another protein can also be predicted by computer simulation based on the amino acid sequences of the protein and the other protein.

[0029] 2-2. Nucleotide Sequence (B) Nucleotide sequence (B) is a nucleotide sequence encoding a protein with Notch core region function. Notch is a receptor expressed on the cell surface, and the Notch signaling system is one of the major signaling systems responsible for intercellular signaling. In many cases, the mechanism of intercellular signaling is such that a signal-sending cell produces and releases a soluble ligand, which then binds to a receptor on the surface of the signal-receiving cell. Furthermore, in the receiving cell, intracellular signaling pathways, including downstream phosphorylation cascades, are activated, and gene expression is regulated by changes in the activity of specific transcription factors. On the other hand, the Notch signaling system is characterized by signal transmission through direct interaction between adjacent cells. Notch, which acts as a receptor, binds to its ligand Delta or Serrate (called Jagged in mammals) on the cell surface, and when physical force is applied, a conformational change occurs, and the intracellular domain of Notch is separated by the action of protein-cleaving enzymes (proteases) such as ADAM protease or γ-secretase. The intracellular domain of Notch thus released from the cell membrane migrates to the nucleus, where it interacts with transcription factors and coactivators upstream of target genes to regulate their transcription.

[0030] In the present disclosure, the Notch core region is a region that includes a site that is cleaved by the action of a protease, and the Notch core region function is the function of being able to be cleaved (preferably being able to be separated in a limited manner) by a protease that can cleave the Notch core region.

[0031] A protein having Notch core region function may be, for example, a protein containing only the portion of the Notch core region necessary for protease cleavage, or may be a protein containing one or more (e.g., 1 to 30, 1 to 20, 1 to 10, or 1, 2, 3, 4, or 5) amino acids before and after the portion necessary for protease cleavage, as long as it is cleaved by the action of a protease. Furthermore, as long as it is cleaved by the action of a protease, such a protein may have one or more (e.g., 1 to 30, 1 to 20, 1 to 10, or 1, 2, 3, 4, or 5) amino acids deleted, substituted, or added. A preferred example of a protein having Notch core region function is a protein comprising the amino acid sequence of SEQ ID NO: 11, and a more preferred example is a protein consisting of the amino acid sequence of SEQ ID NO: 11. The base sequence (B) is not particularly limited, as long as it is a base sequence that encodes such a protein.

[0032] As described above, preferred examples of proteases include ADAM protease and γ-secretase, and it is more preferred to encode a polypeptide that is cleaved by either or both of these proteases. ADAM protease is a proteolytic enzyme that belongs to the A Disintegrin and Metalloprotease family.

[0033] Whether a certain protein can be cleaved by a protease can be confirmed by treating the protein with a protease and then subjecting the protein to electrophoresis (for example, SDS-PAGE).

[0034] More specifically, examples of the base sequence (B) include the following (b-1) and (b-2): (b-1): a base sequence consisting of the base sequence of SEQ ID NO: 2 (b-2): a base sequence consisting of a base sequence having 85% or more sequence identity with the base sequence of (b-1), and encoding a protein that can be cleaved by a protease Furthermore, (b-2) is more preferably (b-2'): a base sequence consisting of a base sequence having 85% or more sequence identity with the base sequence of (b-1), and encoding a protein that can be cleaved by an intracellular protease when a ligand binds to the protein encoded by the base sequence (A), in a protein encoded by a base sequence having a structure in which (A)-(B)-(C) are linked in that order.

[0035] The sequence identity may be 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more.

[0036] 2-3. Base Sequence (C) Base sequence (C) is a base sequence encoding a protein with HIF-3α4 function. HIF (Hypoxia Inducible Factor) is a transcription factor activated when cells experience hypoxia. It is a heterodimer consisting of HIF-1α and HIF-1β. It has been revealed that HIF-1α is not only degraded by PHDs under normal oxygen concentrations, but also suppressed by a transcription factor called IPAS (Inhibitory PAS domain protein) discovered in mice. IPAS was identified as a splicing variant of HIF-3α, a HIF. While IPAS itself does not exhibit transcriptional activity, it inhibits DNA binding through its interaction with HIF-1α, thereby suppressing the function of HIF-1α. In humans, HIF-3α4, which was identified as a splicing variant of HIF-3α, has been shown to perform a function similar to that of IPAS.

[0037] In the present disclosure, the function of HIF-3α4 is the ability to suppress HIF-1α, and more specifically, the function of interacting with (binding to) HIF-1α.

[0038] The protein having HIF-3α4 function may be, for example, HIF-3α4 itself, or may be a protein in which one or two or more (e.g., 1 to 30, 1 to 20, 1 to 10, or 1, 2, 3, 4, or 5) amino acids have been added to HIF-3α4, so long as it is capable of inhibiting HIF-1α. Furthermore, such a protein may have one or two or more (e.g., 1 to 30, 1 to 20, 1 to 10, or 1, 2, 3, 4, or 5) amino acids deleted, substituted, or added, so long as it is capable of inhibiting HIF-1α. A preferred example of a protein having HIF-3α4 function is a protein comprising the amino acid sequence of SEQ ID NO: 12, and a more preferred example is a protein consisting of the amino acid sequence of SEQ ID NO: 12. The base sequence (C) is not particularly limited, as long as it is a base sequence that encodes such a protein.

[0039] More specifically, examples of the base sequence (C) include the following (c-1) and (c-2): (c-1): a base sequence consisting of the base sequence of SEQ ID NO: 3 (c-2): a base sequence consisting of a base sequence that has 85% or more sequence identity with the base sequence of (c-1) and encodes a protein that can bind to HIF-1α The sequence identity may be 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more.

[0040] 2-4. Base Sequence (I) The base sequence (I) is a base sequence having a structure in which the above-described base sequence (A), base sequence (B), and base sequence (C) are linked in the order (A)-(B)-(C). In the base sequence (I), the base sequence (A) may be at the 3'-end or the 5'-end, but is preferably at the 5'-end. The base sequences (A), (B), and (C) may be linked directly or via a linker, but are preferably linked directly. The linker is not particularly limited as long as it does not impair the effects of the present disclosure, but may be, for example, a linker consisting of one or more bases. When the base sequence (I) includes one or more linkers, the base length of each linker may be 1 to 1000 bp or 1 to 500 bp, preferably 1 to 100 bp or 1 to 50 bp, and particularly preferably 1 to 10 bp. The upper or lower limit of the range is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, It may be 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 bp.

[0041] The base length of the base sequence (I) may be, for example, 8000 bp or less, preferably 7500, 7000, 6500, or 6000 bp or less, more preferably 5500, 5000, or 4500 bp or less, and particularly preferably 4000, 3500, or 3100 bp or less.

[0042] Preferred embodiments of the base sequence (I) include, for example, the following (g-1), (g-2), and (g-3). Note that (g-1), (g-2), and (g-3) may be collectively referred to as the base sequence (III). (g-1) A base sequence consisting of the base sequence of SEQ ID NO: 7. (g-2) A base sequence consisting of a base sequence that has a sequence identity of 85% or more with the base sequence of SEQ ID NO: 7 and has a structure in which a portion encoding a protein capable of binding to hyaluronic acid, a portion encoding a protein that can be cleaved by protease, and a portion encoding a protein capable of binding to HIF-1α are linked in this order. (g-3) A base sequence consisting of a base sequence that has a sequence identity of 85% or more with the base sequence of SEQ ID NO: 7 and encoding a protein having anti-cancer activity. The sequence identity may be 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more.

[0043] A preferred example of the protein encoded by the base sequence (I) is a protein comprising the amino acid sequence of SEQ ID NO:13, and a more preferred example is a protein comprising the amino acid sequence of SEQ ID NO:13.

[0044] In the present disclosure, the term "anticancer effect" includes effects such as suppressing the proliferation of cancer cells, killing cancer cells, reducing the number of cancer cells, and reducing tumor volume.

[0045] 2-5. Base Sequence (D) Base sequence (D) is a base sequence containing the COX-2 (cyclooxygenase-2) promoter. COX-2 is involved in the conversion of arachidonic acid to prostaglandin H2. COX-2 is not expressed in most cells under normal conditions, but its expression is known to increase in inflammatory conditions. COX-2 expression is also known to increase in many cancer cells. In other words, the expression of a gene or protein encoded by a base sequence placed under the control of the COX-2 promoter can increase in cells under inflammatory conditions or cancer cells.

[0046] A promoter typically includes a transcription start point, a sequence upstream (5' side), and, if necessary, a sequence downstream (3' side). Examples of COX-2 promoters include any DNA region containing a transcription start point within a DNA region ranging from -10,000 to +500, preferably from -5,000 to +200, and more preferably from -2,000 to +150, where +1 is the base at the transcription start point of a COX-2 gene in humans, +1 is the base downstream (3' side), and 0 or a negative value is the base upstream (5' side). When multiple transcription start points exist, the transcription start point with the highest transcription amount can be selected. The base length of the base sequence (D) can be, for example, 500 to 10,000 bp, preferably 8,000 to 5,000 bp, and more preferably 1,000 to 3,000 bp.

[0047] The COX-2 promoter contained in base sequence (D) may have a mutation (e.g., substitution, deletion, insertion, addition, etc.) in its base sequence relative to the endogenous COX-2 promoter of an organism (preferably human), so long as the expression of a gene or protein under its control can be increased in cells in an inflammatory state or cancer cells. In this case, the COX-2 promoter contained in base sequence (D) has, for example, 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 97% or more, and particularly preferably 99% or more identity with the corresponding base sequence of the endogenous promoter. The mutation site is preferably, for example, a site other than known expression control elements (e.g., inflammatory response regions, general transcription factor binding regions, various activator binding regions, etc.).

[0048] More specifically, examples of the base sequence (D) include the following (d-1) and (d-2): (d-1): a base sequence consisting of the base sequence of SEQ ID NO: 4 (d-2): a base sequence consisting of a base sequence that has 85% or more sequence identity with the base sequence of (d-1), and that encodes a promoter whose expression is induced under COX-2 expression-inducing conditions. Note that "COX-2 expression-inducing conditions" refer to, for example, conditions in cells in an inflammatory state or in cancer cells. Furthermore, "expression is induced under COX-2 expression-inducing conditions" means that the expression level of a gene or protein under the control of the promoter is increased in cells in an inflammatory state or in cancer cells compared to the expression level in normal cells that are not in an inflammatory state.

[0049] In the present disclosure, the term "expression level" encompasses both the amount of transcription product and the amount of translation product. Expression levels can be analyzed by conventionally known methods or methods that can be easily derived from conventionally known methods. Examples of such methods include reverse transcription quantitative PCR (RT-qPCR), RNA-seq, Western blotting, and immunohistochemical staining.

[0050] The sequence identity may be 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more.

[0051] 2-6. Nucleotide Sequence (E) The nucleotide sequence (E) is a nucleotide sequence encoding the E1A protein, and is placed under the control of the COX-2 promoter. Therefore, the nucleotide sequence (E) is placed on the 3'-terminal side of the nucleotide sequence (D). The nucleotide sequence (E) may be directly linked to the 3'-terminal of the nucleotide sequence (D), or another nucleotide sequence (e.g., a linker, a nucleotide sequence (F), etc.) may be present between the nucleotide sequence (E) and the nucleotide sequence (D). In the latter case, the length of the nucleotide sequence present between the nucleotide sequence (E) and the nucleotide sequence (D) is not limited as long as the nucleotide sequence (E) is under the control of the COX-2 promoter.

[0052] The E1A protein, together with the E1B protein described below, is an essential factor for the proliferation of adenovirus. More specifically, the E1A protein is a nonstructural protein that is transcribed and translated in the early stage of viral replication, thereby initiating viral replication.

[0053] In the present disclosure, the E1A function refers to the function of initiating viral replication by being transcribed and translated at the early stage of viral replication, similar to the E1A protein. A protein having the E1A function may be, for example, the E1A protein itself, or a protein to which one or more (e.g., 1 to 30, 1 to 20, 1 to 10, or 1, 2, 3, 4, or 5) amino acids have been added to the E1A protein, as long as it is capable of initiating viral replication by being transcribed and translated at the early stage of viral replication, similar to the E1A protein. Furthermore, as long as it is capable of initiating viral replication by being transcribed and translated at the early stage of viral replication, similar to the E1A protein, one or more (e.g., 1 to 30, 1 to 20, 1 to 10, or 1, 2, 3, 4, or 5) amino acids may be deleted, substituted, or added to such a protein. The nucleotide sequence (E) is not particularly limited, as long as it is a nucleotide sequence that encodes such a protein.

[0054] More specifically, examples of the base sequence (E) include the following (e-1) and (e-2): (e-1): a base sequence consisting of the base sequence of SEQ ID NO: 5 (e-2): a base sequence having 85% or more sequence identity with the base sequence of (e-1) and encoding a protein having E1A function

[0055] The sequence identity may be 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, and particularly preferably 95% or more.

[0056] 2-7. Nucleotide Sequence (F) The nucleotide sequence (F) is a nucleotide sequence encoding the E1B protein, and is placed under the control of the COX-2 promoter sequence. Therefore, the nucleotide sequence (F) is placed on the 3'-terminal side of the nucleotide sequence (D). The nucleotide sequence (F) may be directly linked to the 3'-terminal of the nucleotide sequence (D), or another nucleotide sequence (e.g., a linker, a nucleotide sequence (E), etc.) may be present between the nucleotide sequence (F) and the nucleotide sequence (D). In the latter case, the length of the nucleotide sequence present between the nucleotide sequence (F) and the nucleotide sequence (D) is not limited as long as the nucleotide sequence (F) is under the control of the COX-2 promoter.

[0057] The E1B protein, together with the above-mentioned E1A protein, is an essential factor for adenovirus proliferation. More specifically, the E1B protein is a protein required for the formation of a viral replication compartment and controls functions such as replication and transcription of the viral genome and biosynthesis of viral late mRNA.

[0058] In the present disclosure, the E1B function refers to a function that, like the E1B protein, controls functions such as replication and transcription of the viral genome and viral late mRNA biosynthesis, and is involved in the formation of viral replication compartments.

[0059] The protein having E1B function may be, for example, the E1B protein itself, or a protein to which one or more (e.g., 1 to 30, 1 to 20, 1 to 10, or 1, 2, 3, 4, or 5) amino acids have been further added to the E1B protein, as long as it is able to control functions such as replication and transcription of the viral genome and biosynthesis of viral late mRNA, similar to the E1B protein. Furthermore, as long as it is able to control functions such as replication and transcription of the viral genome and biosynthesis of viral late mRNA, similar to the E1B protein, one or more (e.g., 1 to 30, 1 to 20, 1 to 10, or 1, 2, 3, 4, or 5) amino acids may be deleted, substituted, or added to such a protein. The base sequence (F) is not particularly limited as long as it is a base sequence that encodes such a protein.

[0060] More specifically, examples of the base sequence (F) include the following (f-1) and (f-2): (f-1): a base sequence consisting of the base sequence of SEQ ID NO: 6 (f-2): a base sequence having 85% or more sequence identity with the base sequence of (f-1) and encoding a protein having E1B function

[0061] The sequence identity may be 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more.

[0062] 2-8. Base Sequence (II) The base sequence (II) is a base sequence containing the above-described base sequence (D), base sequence (E), and base sequence (F). As described above, the base sequence (E) and base sequence (F) are placed under the control of the COX-2 promoter contained in the base sequence (D), and therefore the base sequence (D) is placed upstream (5'-end) of the base sequence (E) and base sequence (F). Either the base sequence (E) or the base sequence (F) may be placed at the 5'-end. That is, the base sequence (II) may have a structure in which, from the 5'-end, the bases are linked in the order (D)-(E)-(F), or in the order (D)-(F)-(E). The base sequence (D), base sequence (E), and base sequence (F) may be linked directly or via a linker. The linker is not particularly limited as long as it does not impair the effects of the present disclosure, and may be, for example, a linker consisting of one or more bases. When the base sequence (II) contains one or more linkers, the base length of each linker may be 1 to 5000 bp, preferably 1 to 1000 bp, more preferably 1 to 500 bp, and particularly preferably 1 to 300 bp. The upper or lower limit of the range is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, It may be 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 bp.

[0063] In the nucleic acid of the present disclosure, the base sequence (I) or the base sequence (III) and the base sequence (II) are arranged in the same direction. In the present disclosure, the phrase "arranged in the same direction" for multiple base sequences means that the transcription of each base sequence is carried out in the same direction, which can also be said to mean that in double-stranded nucleic acids, transcription is carried out using the same nucleic acid strand as a template (i.e., the sense strands are identical). Either the base sequence (I) or the base sequence (III) or the base sequence (II) may be arranged upstream.

[0064] 2-9. Nucleotide Sequence (H) Although not particularly limited, it is preferred that the nucleic acid of the present disclosure further comprises, in addition to the above-described nucleotide sequences (A) to (F), nucleotide sequence (H): a nucleotide sequence encoding an Ad5 / 35 chimeric fiber protein. In the present disclosure, Ad5 / 35 chimeric fiber protein refers to a chimeric fiber protein comprising a portion or all of the fiber protein derived from adenovirus serotype 35 (Ad35) and a portion or all of the fiber protein derived from adenovirus serotype 5 (Ad5). In the present disclosure, the Ad5 / 35 chimeric fiber protein or the nucleotide sequence encoding the Ad5 / 35 chimeric fiber protein may be referred to simply as "Ad5 / 35 fiber."

[0065] In order to introduce nucleic acid into target cells using the Ad5 vector, which is widely used in gene therapy, it is necessary for the target cells to express an adenovirus receptor (CAR). Therefore, gene therapy using the Ad5 vector is not expected to have a sufficient therapeutic effect on target cells in which CAR is not expressed or in which the amount of CAR expression is low. On the other hand, the Ad35 fiber protein has the ability to bind to CD46, a membrane protein separate from CAR. When the nucleic acid of the present disclosure comprises a nucleotide sequence encoding an Ad5 / 35 chimeric fiber protein, nucleic acid introduction is possible via binding to CD46, even in cancer cells in which CAR is not expressed or in which the amount of CAR expression is low. In other words, from the viewpoint of expecting a high anticancer effect regardless of the presence or absence of CAR expression and the level of CAR expression in target cells, the nucleic acid of the present disclosure preferably comprises the nucleotide sequence (H): a nucleotide sequence encoding an Ad5 / 35 chimeric fiber protein.

[0066] As described above, in the present disclosure, the term "Ad5 / 35 chimeric fiber protein" refers to a chimeric fiber protein comprising a portion or all of the fiber protein derived from Ad35 and a portion or all of the fiber protein derived from Ad5. The specific structure of the chimeric fiber protein is not particularly limited, so long as it has the ability to bind to CD46. For example, it may be a chimeric fiber protein comprising the knob and shaft portions of the fiber protein derived from Ad35 and the tail portion of the fiber protein derived from Ad5. Furthermore, as long as it has the ability to bind to CD46, one or more amino acids may be deleted, substituted, or added in such a protein. A preferred example of an Ad5 / 35 chimeric fiber protein is a protein comprising the amino acid sequence of SEQ ID NO: 14, with a protein consisting of the amino acid sequence of SEQ ID NO: 14 being more preferred. The nucleotide sequence (H) is not particularly limited, as long as it is a nucleotide sequence that encodes such a polypeptide.

[0067] Specifically, examples of the base sequence (H) include the following (h-1) and (h-2): (h-1): a base sequence consisting of the base sequence of SEQ ID NO: 8 (h-2): a base sequence having 85% or more sequence identity with the base sequence of (h-1) and encoding a protein capable of binding to CD46

[0068] The sequence identity may be 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more.

[0069] 2-10. Others The nucleic acid of the present disclosure may further comprise other sequences in addition to the above sequences. Examples of other sequences include a secretory signal peptide coding sequence, a promoter sequence, an enhancer sequence, a repressor sequence, an insulator sequence, a replication origin, a drug resistance gene coding sequence, etc. Furthermore, the nucleic acid of the present disclosure may be a linear nucleic acid or a circular nucleic acid.

[0070] The nucleic acids of the present disclosure can be produced by known methods or methods that can be easily derived from known methods. For example, they can be produced by genetic engineering techniques such as PCR, restriction enzyme cleavage, DNA ligation, and in vitro transcription. Alternatively, the nucleic acids of the present disclosure may be produced by chemical synthesis.

[0071] The oncolytic adenoviral vectors of the present disclosure can exhibit excellent anticancer effects, and therefore the technology of the present disclosure can be suitably used for the prevention, amelioration, treatment, etc. of cancer.

[0072] In the present disclosure, the term "cancer prevention" includes preventing the onset of cancer by applying to a subject at a stage before cancer is diagnosed, and preventing cancer metastasis by applying to tissues in a subject diagnosed with cancer where cancer has not yet been found. In the present disclosure, the term "cancer amelioration" or "cancer treatment" includes suppressing the progression of cancer, halting the progression of cancer, suppressing the proliferation of cancer cells, killing cancer cells, reducing the number of cancer cells, reducing tumor volume, and preventing cancer metastasis by applying to a subject diagnosed with cancer. Furthermore, in the present disclosure, the term "anti-cancer" includes cancer prevention and its effects, cancer amelioration and its effects, and cancer treatment and its effects.

[0073] In the present disclosure, the term "cancer" encompasses cancer, neoplasms, and malignant tumors. Examples of cancer include bladder cancer, breast cancer, prostate cancer, malignant melanoma, kidney cancer, mesothelioma, intraperitoneal disseminated cancer, lung cancer, colorectal cancer, gastric cancer, soft tissue sarcoma, osteosarcoma, brain tumor, neuroblastoma, leukemia, lymphoma, liver cancer, retinoblastoma, ciliary body tumor, basal cell carcinoma, squamous cell carcinoma, malignant soft tissue tumor, chondrosarcoma, ovarian cancer, uterine cancer, and cervical cancer. The cancer to which the technology of the present disclosure is applicable is not particularly limited, but is particularly suitable for use against at least one type selected from the group consisting of bladder cancer, breast cancer, and prostate cancer.

[0074] The technology of the present disclosure can be applied to, for example, humans and non-human mammals (e.g., rats, mice, rabbits, cows, pigs, dogs, cats, sheep, monkeys, etc.), with humans being preferred.

[0075] 4. Application Method The application method of the technology of the present disclosure is not particularly limited as long as the desired effect is achieved. For example, the oncolytic adenoviral vector of the present disclosure or a composition containing the same may be administered directly into a lesion by injection, or may be administered intravenously, intraarterially, intraperitoneally, intramuscularly, subcutaneously, intrapleurally, or by injection or infusion, or may be administered by perfusion via a catheter. Administration by injection is particularly preferred.

[0076] The frequency of application of the technology of the present disclosure is not particularly limited. For example, it may be applied once or multiple times a day, once or multiple times a week, once or multiple times a month, or once or multiple times a year. The application period of the technology of the present disclosure is also not particularly limited, as long as the effects of the present disclosure are achieved. Furthermore, the dose of the oncolytic adenoviral vector of the present disclosure is also not particularly limited, as long as the effects of the present disclosure are achieved. The application frequency, application period, and dose can be adjusted appropriately by those skilled in the art depending on the condition of the subject, the course of treatment, etc.

[0077] The technology of the present disclosure may be optionally combined with other pharmaceutical compositions and / or treatment methods, etc., which are applied to a subject with cancer. When the technology of the present disclosure is combined with other pharmaceutical compositions and / or treatment methods, etc., they may be applied to a subject simultaneously, or may be applied separately at any time.

[0078] In the technology of the present disclosure, the oncolytic adenoviral vector of the present disclosure may be mixed with a pharmaceutically acceptable base, carrier, excipient, diluent, solubilizer, emulsifier, preservative, pH adjuster, adjuvant, chelating agent, etc. These components may be used alone or in combination of two or more.

[0079] Examples of preservatives include parabens such as methylparaben, ethylparaben, propylparaben, and butylparaben, sodium benzoate, phenoxyethanol, alkyldiaminoethylglycine hydrochloride, etc. The preservatives may be used alone or in combination of two or more kinds.

[0080] Examples of pH adjusters include citric acid, phosphoric acid, malic acid, pyrophosphoric acid, lactic acid, tartaric acid, glycerophosphoric acid, acetic acid, nitric acid, and chemically possible salts thereof, as well as sodium hydroxide, potassium hydroxide, etc. pH adjusters can be used alone or in combination of two or more types so that the pH of the composition containing the oncolytic adenoviral vector of the present disclosure is in the range of 4 to 8, preferably 5 to 7.

[0081] The method for preparing a composition containing the oncolytic adenoviral vector of the present disclosure is not particularly limited as long as the effects of the present disclosure are achieved. For example, the composition can be prepared according to a method known in the art. More specifically, the composition can be prepared by mixing the oncolytic adenoviral vector of the present disclosure and other components with sterile distilled water.

[0082] 5. Composition The present disclosure also encompasses an anti-cancer composition comprising an oncolytic adenoviral vector of the present disclosure. Such an anti-cancer composition may be referred to as the "anti-cancer composition of the present disclosure." The items described in "1. Oncolytic adenoviral vector" to "4. Application method" are incorporated by reference into the anti-cancer composition of the present disclosure.

[0083] 6. Presumed Mechanism While not wishing to be bound by theory, it is believed that the anti-cancer activity of the protein encoded by base sequence (I) or base sequence (III) is due to the following mechanism of action. The fusion protein encoded by base sequence (I) or base sequence (III) contains (i) a protein portion having CD44 extracellular function, (ii) a protein portion having Notch core domain function, and (iii) a protein portion having HIF-3α4 function. Therefore, (i) can function as a Decoy receptor and transmit a signal to (iii) via (ii). This is more effective when (i) is fused upstream of (ii). Furthermore, by fusing (iii) downstream of (ii), HIF-1α activated in tumors can be suppressed. This is believed to result in an anti-cancer effect consisting of multiple mechanisms of action.

[0084] It is believed that the base sequence (II) contributes to the efficient anti-cancer activity of the oncolytic adenoviral vector of the present disclosure through the following mechanism of action. As described above, the expression of a gene or protein encoded by a base sequence placed under the control of the COX-2 promoter can be increased in cells in an inflammatory state or in cancer cells. Therefore, it is believed that the expression of the E1A protein and E1B protein under the control of the COX-2 promoter is increased in cancer cells. The expression of the E1A protein and E1B protein causes the oncolytic adenoviral vector of the present disclosure to replicate and proliferate within the cancer cells, resulting in the lysis and death of the cancer cells. Furthermore, the oncolytic adenoviral vector of the present disclosure that has proliferated within the cancer cells can be released from the dead cancer cells and infect other cancer cells in the vicinity. Thereafter, the proliferation of the oncolytic adenoviral vector of the present disclosure, the lysis and death of cancer cells, the release of the oncolytic adenoviral vector, and the infection of nearby cancer cells can occur repeatedly.

[0085] Furthermore, as described above, it is believed that the replication and proliferation of the oncolytic adenoviral vector of the present disclosure in cancer cells increases the abundance of the protein encoded by base sequence (I) or base sequence (III) in a cancer cell-specific manner. That is, in the technology of the present disclosure, by incorporating base sequence (I) or base sequence (III) into an oncolytic adenoviral vector, the anticancer effect of the oncolytic adenoviral vector and the anticancer effect of the fusion protein encoded by base sequence (I) or base sequence (III) are correlated with each other, resulting in the synergistic exertion of an excellent anticancer effect in a cancer cell-specific manner.

[0086] Furthermore, as is clear from the above-described presumed mechanism, the technology of the present disclosure is predicted to exhibit particularly excellent anti-cancer effects against COX-2-positive cancer cells. Therefore, the technology of the present disclosure is particularly suitable for use against COX-2-positive cancer cells. In this disclosure, "COX-2 positive" means that the expression level of COX-2 is elevated compared to the expression level in normal cells that are not in an inflammatory state. Whether a cell is COX-2-positive can be analyzed by a conventionally known method or a method that can be easily derived from a conventionally known method. Examples of such methods include reverse transcription quantitative PCR (RT-qPCR), RNA-seq, Western blotting, immunohistochemical staining, and the like.

[0087] Although the reason is unclear, as described in Test Example 2.5, when the CD44 / Notch / HIF-3α4 fusion gene and the E1A / E1B gene were arranged in opposite directions, neither transcription nor translation of the CD44 / Notch / HIF-3α4 fusion gene was observed. On the other hand, as described in Test Examples 2.6 and 2.7, when the CD44 / Notch / HIF-3α4 fusion gene and the E1A / E1B gene were arranged in the same direction, significant expression of the CD44 / Notch / HIF-3α4 fusion gene was observed. Therefore, in the technology of the present disclosure, it is desirable that the base sequence (I) or the base sequence (III) and the base sequence (II) are arranged in the same direction.

[0088] In this specification, the term "comprising" includes "essentially consisting of" and "consisting of" in addition to "containing." Furthermore, the present disclosure includes any and all combinations of the constituent elements described in this specification.

[0089] Furthermore, the various characteristics (properties, values, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to identify the subject matter encompassed by the present disclosure. In other words, the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein.

[0090] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0091] 1. <Experimental Method> 1.1. Preparation of CRAd-synNotch (Forward / Reverse) The nucleotide sequence actually designed in this example, in which (A) a nucleotide sequence encoding a protein having CD44 extracellular function, (B) a nucleotide sequence encoding a protein having Notch core domain function, and (C) a nucleotide sequence encoding a protein having HIF-3α4 function are linked in the order (A)-(B)-(C), is sometimes referred to as a "CD44 / Notch / HIF-3α4 fusion gene." The CD44 / Notch / HIF-3α4 fusion gene is a preferred embodiment of the nucleotide sequence (I) or the nucleotide sequence (III) in the technology of the present disclosure.

[0092] In this example, a recombinant oncolytic adenoviral vector in which a COX-2 promoter region is placed upstream of the E1A gene and the E1B gene and a CD44 / Notch / HIF-3α4 fusion gene is inserted is sometimes referred to as CRAd-synNotch (forward / reverse). In CRAd-synNotch (forward), the CD44 / Notch / HIF-3α4 fusion gene is placed in the same orientation (forward) as the COX-2 promoter region, E1A gene, and E1B gene. On the other hand, in CRAd-synNotch (reverse), the CD44 / Notch / HIF-3α4 fusion gene is placed in the reverse orientation to the COX-2 promoter region, E1A gene, and E1B gene. A method for producing CRAd-synNotch (forward / reverse) is described below.

[0093] The vector DNA used in this example was the plasmids pAd1127-04, pAd1128, pAd1129-06, and pAd1130-04 included in the AdenoQuick 2.0 Kit (O.D.260). Cosmid vectors were prepared using Cosmid Construction Kit-2 (O.D.260).

[0094] 1.1.1 Amplification of the COX-2 promoter region and cloning enhancement treatment Using 25 ng of pDrive-hCOX2 (InvivoGen) as a template, PCR was performed with KOD-Plus-Neo (TOYOBO) to amplify the COX-2 promoter region. The composition of the PCR reaction solution and the primers used for PCR are shown in the table below. The nucleotide sequence of the amplified COX-2 promoter region (SEQ ID NO: 4) is shown in Figure 32.

[0095]

[0096]

[0097] 5 μl of the PCR product and 2 μl of Cloning Enhancer (TaKaRa) were reacted at 37° C. for 20 minutes, and then the enzyme was inactivated by heating at 80° C. for 15 minutes.

[0098] 1.1.2 Restriction enzyme treatment of pAd1127-04 plasmid The plasmid pAd1127-04 included in the AdenoQuick 2.0 Kit (O.D.260) was cleaved with the restriction enzymes SalI (TaKaRa) and EcoRI (TaKaRa), and after agarose gel electrophoresis, the fragment was purified using Wizard™ SV Gel and PCR Clean-Up System (Promega). The composition of the restriction enzyme treatment reaction solution is shown in the table below.

[0099]

[0100] 1.1.3 Insertion of the COX-2 promoter region into the pAd1127-04 plasmid and transformation into E. coli To ligate the COX-2 promoter region amplified in 1.1.1 and pAd1127-04, an In-Fusion reaction (50°C, 15 minutes) was carried out using the In-Fusion™ HD Cloning Kit (TaKaRa). Escherichia coli DH5α Competent Cells (TaKaRa) were transformed using this reaction solution. Transformation was carried out according to the method described in the kit manual.

[0101] The transformed E. coli DH5α Competent cells were plated on LB agar medium (Nacalai Tesque) supplemented with 25 μg / ml kanamycin and cultured overnight at 37 ° C. After culture, colonies grown on the LB agar medium were used as templates to perform insert check PCR using KOD-Plus-Neo, and whether the transformation had been successful was confirmed by agarose gel electrophoresis.

[0102] Colonies in which successful transformation was confirmed were subcultured in LB liquid medium supplemented with 25 μg / ml kanamycin, and plasmids were extracted from the culture medium using PureYield™ Plasmid Midiprep System (Promega). The sequences in the extracted plasmids were confirmed using a DNA sequencer.

[0103] 1.1.4 Insertion of CD44 / Notch / HIF-3α4 fusion gene into pAd1129-06 and transformation into E. coli The production of a plasmid carrying the CD44 / Notch / HIF-3α4 fusion gene was outsourced to Gene Universal. The plasmid carrying the fusion gene and the pAd1129-06 plasmid (O.D.260) were each treated with the restriction enzyme EcoRI (TaKaRa), followed by agarose gel electrophoresis and purification. The pAd1129-06 plasmid is an oncolytic adenovirus vector. The composition of each restriction enzyme treatment reaction solution is shown in the table below.

[0104]

[0105]

[0106] The sequence of the CD44 / Notch / HIF-3α4 fusion gene (SEQ ID NO: 7) is shown in Figure 35. The nucleotide sequence encoding a protein having CD44 extracellular function (SEQ ID NO: 1), the nucleotide sequence encoding a protein having Notch core region function (SEQ ID NO: 2), and the nucleotide sequence encoding a protein having HIF-3α4 function (SEQ ID NO: 3), all of which are contained in the CD44 / Notch / HIF-3α4 fusion gene, are shown in Figures 29 to 31, respectively. Furthermore, the amino acid sequence (SEQ ID NO: 13) of the protein encoded by the nucleotide sequence of SEQ ID NO: 7 is shown in Figure 39, and the amino acid sequences (SEQ ID NOs: 10 to 12) of the proteins encoded by the nucleotide sequences of SEQ ID NOs: 1 to 3 are shown in Figure 38.

[0107] Each purified plasmid was mixed with Ligation Mix (TaKaRa) and a ligation reaction was carried out (16°C, 30 minutes). Escherichia coli DH5α Competent Cells (TaKaRa) were transformed using the reaction solution. Transformation was carried out according to the method described in the kit manual.

[0108] The transformed E. coli DH5α Competent cells were plated on LB agar medium (Nacalai Tesque) supplemented with 100 μg / ml ampicillin and cultured overnight at 37 ° C. After culture, colonies grown on the LB agar medium were used as templates to perform insert check PCR using KOD-Plus-Neo, and whether the transformation had been successful was confirmed by agarose gel electrophoresis.

[0109] Colonies in which successful transformation was confirmed were subcultured in LB liquid medium supplemented with 100 μg / ml ampicillin, and plasmids were extracted from the culture medium using PureYield™ Plasmid Midiprep System (Promega). The sequences in the extracted plasmids were confirmed using a DNA sequencer.

[0110] 1.1.5 Preparation of recombinant cosmid vectors and transformation into E. coli pAd1127-04, into which the COX-2 promoter region had been inserted in 1.1.3, and pAd1129-06, into which the CD44 / Notch / HIF-3α4 fusion gene had been inserted in 1.1.4, as well as pAd1128 (O.D.260) and pAd1130-04 (O.D.260), were each cleaved with the restriction enzyme SfiI (New England Biolabs). Each reaction solution was subjected to agarose gel electrophoresis, and the DNA fragments were purified.

[0111] The purified DNA fragments and T4 DNA Ligase (TaKaRa) were mixed and ligated (16°C, overnight). 2.0 μl of this reaction mixture was reacted with 5.0 μl of λ packaging extract included with Cosmid Construction Kit-2 at 30°C for 1 hour. After the reaction, 100 μl of SM medium and 5.0 μl of chloroform (Nacalai Tesque) included with the kit were added, tapped, and centrifuged (15,000 rpm, 1 minute). 20 μl of the centrifuged supernatant, as well as 0.2% maltose and 10 mM MgSO ４ The mixture was mixed with 20 μl of E. coli TOP10 Competent Cells (Invitrogen) culture medium cultured in LB liquid medium supplemented with ampicillin and kanamycin, and incubated at 37 ° C for 30 minutes. After the reaction, the mixture was inoculated onto LB agar medium (Nacalai Tesque) supplemented with 50 μg / ml ampicillin and 25 μg / ml kanamycin, and incubated overnight at 37 ° C. After incubation, colony PCR was performed using colonies grown on the LB agar medium as templates, and colonies harboring a cosmid vector in which the CD44 / Notch / HIF-3α4 fusion gene was linked in the same direction as the COX-2 promoter (forward orientation) and colonies harboring a cosmid vector in which the CD44 / Notch / HIF-3α4 fusion gene was linked in the reverse orientation to the COX-2 promoter were selected. Each selected colony was subcultured in LB liquid medium supplemented with 50 μg / ml ampicillin and 25 μg / ml kanamycin, and the cosmid vector was extracted from the culture medium using PureYield™ Plasmid Midiprep System (Promega).

[0112] In this step, four samples of cosmid vectors were obtained in which the CD44 / Notch / HIF-3α4 fusion gene was linked in the same direction (forward) as the COX-2 promoter (each sample derived from a single colony on the ampicillin- and kanamycin-containing LB agar medium was counted as one sample). In addition, one sample of cosmid vector was obtained in which the CD44 / Notch / HIF-3α4 fusion gene was linked in the opposite direction to the COX-2 promoter.

[0113] 1.1.6 Preparation of Restricted Replication Recombinant Adenovirus Vectors by Transforming HEK293 Cells with Recombinant Cosmids Each recombinant cosmid vector DNA prepared in 1.1.5 was cleaved with the restriction enzyme PacI (New England Biolabs). After restriction enzyme treatment, nucleic acid purification was performed by phenol-chloroform extraction and ethanol precipitation, and the purified nucleic acid was dissolved in 30 μl of sterile purified water. 1 μl of this solution was subjected to agarose gel electrophoresis to confirm digestion by the restriction enzyme PacI.

[0114] 10 μg of the PacI-digested cosmid was mixed with Lipofectamine™ 2000 (Invitrogen) and Opti-MEM™ I Reduced Serum Medium (Gibco), and lipofected into HEK293 cells (National Institutes of Biomedical Innovation, Health and Nutrition) cultured to confluence in a 60 mm tissue culture Petri dish.

[0115] After lipofection, the cultured cells were harvested and seeded onto 16 collagen-coated 96-well microplates with lids, Collagen Type I (AGC TECHNO GLASS) per clone. Four, eight, and 16 days after seeding, 50 μl of Dulbecco's modified Eagle's medium (D-MEM; FUJIFILM Wako Pure Chemical) containing 5% fetal bovine serum (FBS; Sigma-Aldrich) was added to each well, and the cells were cultured with daily observation until all cells were completely degenerated.

[0116] For one clone obtained in 1.1.5 using a cosmid vector in which the CD44 / Notch / HIF-3α4 fusion gene was linked in the reverse orientation to the COX-2 promoter, 22 days after seeding, only one well in which the cells had completely denatured was collected into a 1.5 ml tube. The cells were then frozen in liquid nitrogen and thawed in a 37°C water bath six times.

[0117] Four cosmid vector samples obtained in 1.1.5, in which the CD44 / Notch / HIF-3α4 fusion gene was linked in the same orientation (forward) as the COX-2 promoter, were transformed into HEK293 cells using the method described above, and cell degeneration was confirmed in three samples. Three wells in which cells had completely degenerated were collected into 1.5 ml tubes 36, 38, and 39 days after seeding, respectively (these samples may be referred to as clones 1 to 3). The cells were then repeatedly frozen in liquid nitrogen and thawed in a 37°C water bath six times.

[0118] After the final freeze-thaw cycle, each clone was centrifuged (5000 rpm, 5 minutes, 4°C) and the supernatant was collected. This supernatant was stored as the primary virus solution. The above method was a modified version of the method described in "Manual for Recombinant Adenovirus Production - Full-Length Genome Transfer Method" (RIKEN BioResource Center, Gene Engineering Division).

[0119] 1.1.7 Purification of high-titer recombinant adenovirus vectors. HEK293 cells (National Institutes of Biomedical Innovation, Health and Nutrition) were cultured in a 24-well collagen-coated microplate with lid, Collagen Type I (AGC TECHNO GLASS) until they reached 80% to 100% confluence. After culturing, the medium was removed, and a mixture of 0.1 ml of Dulbecco's modified eagle medium (D-MEM; FUJIFILM Wako Pure Chemical Co.) containing 5% FBS and 10 μl of the primary virus solution prepared in 1.1.6 was added per well. To infect HEK293 cells with the virus, the cells were incubated in an incubator (37°C, 5% CO ２ ) and gently shake the plate several times every 15 minutes, a total of four times. After 1 hour of infection, 0.4 ml of 5% FBS-E-MEM was added to each well and the cells were cultured for 3 days. After culture, completely denatured cells were collected together with the culture medium. As with the primary virus solution, the collected cells were repeatedly frozen in liquid nitrogen and thawed in a 37°C water bath six times. After the final freeze-thaw, the cells were centrifuged (5000 rpm, 5 minutes, 4°C), and the collected supernatant was stored as the secondary virus solution.

[0120] Next, HEK293 cells were cultured in a 60 mm tissue culture Petri dish until they reached 80-100% confluence, and 0.4 ml of 5% FBS-D-MEM and 100 μl of the secondary virus solution were added to infect the cells with the virus. During the infection, the cells were incubated in an incubator (37°C, 5% CO ２ ) and gently shake the plate several times every 15 minutes, a total of four times. After 1 hour of infection, 2.5 ml of 5% FBS-D-MEM was added and the cells were cultured for 3 days. After the culture, it was confirmed that the cells were completely denatured, and the cells were collected together with the culture medium. As with the primary virus solution, the collected cells were repeatedly frozen in liquid nitrogen and thawed in a 37°C warm bath six times. After the final freeze-thaw, the cells were centrifuged (3000 rpm, 10 minutes, 4°C), and the collected supernatant was stored as the tertiary virus solution.

[0121] 1.1.8 Mass culture and purification of recombinant adenovirus vector The recombinant adenovirus vector prepared in 1.1.7 was mass cultured and purified. The specific method is shown below.

[0122] HEK293 cells were cultured at 80-100% confluence in a Corning™ 225cm ２ The HEK293 cells were seeded onto 10 flasks (Corning). 5 ml of 5% FBS-D-MEM and 500 μl of the tertiary virus solution prepared in 1.1.7 were added to the HEK293 cells, and the cells were infected with the virus. During the infection, the cells were incubated in an incubator (37°C, 5% CO ２ ) and gently shake the flask several times every 15 minutes, a total of four times. After 1 hour of infection, 30 ml of 5% FBS-D-MEM was added and the cells were cultured for two days. After culture, it was confirmed that all cells had denatured, and the cells were collected together with the culture medium. After collection, the cells were centrifuged (3000 rpm, 10 minutes, 4°C), the supernatant was removed, fresh 10% FBS-D-MEM was added, and the cell pellet was resuspended. Then, freezing in liquid nitrogen and thawing in a 37°C warm bath were repeated six times.

[0123] After the final freeze-thaw, the cells were again collected by centrifugation (3000 rpm, 10 minutes, 4°C). Only the supernatant was collected and purified by the ultracentrifugation method described below.

[0124] Using a 5 ml syringe with a needle, 3 ml of sterile 2.2 M CsCl solution was poured into the bottom of a Beckman tube (Beckman Coulter), and then 3 ml of sterile 4.0 M CsCl solution was slowly added to the bottom of the tube. Using a 5 ml syringe with a needle, 2.5 ml of the above-mentioned centrifugal supernatant was gently layered on top of the above-mentioned 2.2 M CsCl solution. The composition of each solution is shown in the table below.

[0125]

[0126]

[0127]

[0128] The tubes were sealed using a Beckman Coulter cordless tube topper (Beckman Coulter) and subjected to ultracentrifugation (30,000 rpm, 4°C, 18 hours). After ultracentrifugation, the needle of a 5 ml syringe with a 23 G needle was inserted just below the intermediate layer (band) observed in the tube, and 1.5 ml of liquid was collected, taking care to collect as much of the band as possible. The collected liquid was stored in a 2 ml tube.

[0129] 1.1.9 Measurement of the titer of the recombinant adenovirus vector The titer of the recombinant adenovirus vector prepared in 1.1.8 was measured. The specific method is shown below.

[0130] HEK293 cells were seeded onto 12-well cell culture plates with a flat bottom (CORNING). The virus solution, which had been mass-cultured and purified in accordance with 1.1.8, was added dropwise to each well at 10-fold dilutions ranging from 100-fold to 10,000,000-fold dilutions, and cultured for two days. After culture, the medium was removed, the cells were allowed to dry slightly, and then 1 ml of -20°C methanol (FUJIFILM Wako Pure Chemical Co.) was added dropwise and the cells were incubated at -20°C for 10 minutes. After incubation, all of the methanol was removed, and the plate was washed three times with Dulbecco's PBS(-) "Nissui" (PBS; Nissui Pharmaceutical) containing 1% bovine serum albumin (BSA; Sigma-Aldrich).

[0131] After washing, titration was performed using the Adeno-X™ Rapid Titer Kit (TaKaRa). Specifically, 0.5 ml of mouse anti-hexon antibody diluted 1000-fold with PBS containing 1% BSA was added dropwise, and the plate was incubated (37°C, 1 hour) with shaking on a shaker. After the reaction, the plate was washed three times with PBS containing 1% BSA, and 0.5 ml of HRP-conjugate rat anti-mouse antibody diluted 500-fold with PBS containing 1% BSA was added dropwise, and the plate was incubated (37°C, 1 hour) with shaking on a shaker. After the reaction, the cells were washed three times with PBS containing 1% BSA, and 0.5 ml of 10x DAB Substrate diluted 10-fold with 1x Stable Peroxidase Buffer was added dropwise and incubated (room temperature, 10 minutes). After the reaction, the entire reaction solution was removed, 1 ml of PBS was added, and the titer was determined by observing the stained cells under a microscope. The above steps were performed according to the Adeno-X™ Rapid Titer Kit (TaKaRa) manual.

[0132] 1.2 Preparation of CRAd-GFP As a negative control for the CRAd-synNotch (forward) prepared in 1.1, a conditionally replicative adenoviral vector was prepared by inserting the AcGFP1 (sometimes referred to simply as "GFP" in this disclosure) gene in place of the CD44 / Notch / HIF-3α4 fusion gene of CRAd-synNotch (forward). This adenoviral vector may also be referred to as "CRAd-GFP." The nucleotide sequence (SEQ ID NO: 9) of the GFP gene used in this example is shown in Figure 37, and the amino acid sequence (SEQ ID NO: 15) of the protein encoded by the nucleotide sequence of SEQ ID NO: 9 is shown in Figure 41. A specific method for preparing CRAd-GFP is described below.

[0133] The pAd1129-06 plasmid (O.D.260) and pAcGFP1 Vector (TaKaRa) were digested with the restriction enzymes SalI (TaKaRa) and EcoRI (TaKaRa), respectively, and purified after agarose gel electrophoresis. The composition of the restriction enzyme digestion reaction solution is shown in the table below.

[0134]

[0135]

[0136] Each purified nucleic acid fragment was mixed with Ligation Mix (TaKaRa) and a ligation reaction was carried out (16°C, 30 minutes). Escherichia coli DH5α Competent Cells (TaKaRa) were transformed using the reaction solution. Transformation was carried out according to the method described in the kit manual.

[0137] The transformed E. coli DH5α Competent cells were plated on LB agar medium (Nacalai Tesque) supplemented with 100 μg / ml ampicillin and cultured overnight at 37 ° C. After culture, colonies grown on the LB agar medium were used as templates to perform insert check PCR using KOD-Plus-Neo, and whether the transformation had been successful was confirmed by agarose gel electrophoresis.

[0138] Colonies that were confirmed to be successfully transformed were subcultured in LB liquid medium supplemented with 100 μg / ml ampicillin, and plasmids were extracted from the culture medium using the PureYield™ Plasmid Midiprep System (Promega). The sequences in the extracted plasmids were confirmed using a DNA sequencer. The primers used for sequencing are shown in the table below.

[0139]

[0140] Thereafter, CRAd-GFP was produced in the same manner as in 1.1 except for 1.1.4.

[0141] 1.3 Various Adenovirus Vectors The adenovirus vectors used in this example are as follows: CRAd-synNotch (forward) and CRAd-synNotch (reverse) prepared in 1.1; CRAd-GFP prepared in 1.2; ADX730, a non-replicating type 5 recombinant adenovirus vector in which a CD44 / Notch / HIF-3α4 fusion gene has been incorporated into the E1 region, as described in Patent Document 1; wild-type type 5 adenovirus (wtAd), a virus that replicates independently of COX-2; and a recombinant adenovirus vector in which the LacZ gene has been incorporated into the E1 region (Ad-LacZ).

[0142] As described above, CRAd-synNotch (forward / reverse) is a recombinant oncolytic adenoviral vector in which a COX-2 promoter region is placed upstream of the E1A gene and the E1B gene, and a CD44 / Notch / HIF-3α4 fusion gene is inserted. In CRAd-synNotch (forward), the CD44 / Notch / HIF-3α4 fusion gene is placed in the same orientation (forward) as the COX-2 promoter region, E1A gene, and E1B gene. On the other hand, in CRAd-synNotch (reverse), the CD44 / Notch / HIF-3α4 fusion gene is placed in the reverse orientation to the COX-2 promoter region, E1A gene, and E1B gene. The base sequences of the E1A gene (SEQ ID NO: 5) and the E1B gene (SEQ ID NO: 6) used in this example are shown in Figures 33 and 34, respectively.

[0143] CRAd-GFP is a recombinant oncolytic adenoviral vector in which the GFP gene has been inserted in place of the CD44 / Notch / HIF-3α4 fusion gene of CRAd-synNotch (forward orientation). ADX730 is a recombinant adenoviral vector in which the CD44 / Notch / HIF-3α4 fusion gene has been inserted, as described in Patent Document 1, but which does not have the E1A gene and the E1B gene controlled by the COX-2 promoter. wtAd is an adenoviral vector that does not have either the CD44 / Notch / HIF-3α4 fusion gene or the E1A gene and the E1B gene controlled by the COX-2 promoter. Ad-LacZ is a recombinant adenoviral vector in which the LacZ gene has been inserted in place of the CD44 / Notch / HIF-3α4 fusion gene of ADX730.

[0144] Note that CRAd-synNotch (forward / reverse) and CRAd-GFP are oncolytic, whereas ADX730, wtAd, and Ad-LacZ are not. Furthermore, CRAd-synNotch (forward / reverse) and CRAd-GFP have an Ad5 / 35 chimeric fiber gene containing the knob and shaft portions of the fiber protein derived from Ad35 and the tail portion of the fiber protein derived from Ad5, whereas other adenoviral vectors do not have an Ad5 / 35 chimeric fiber gene. Schematic diagrams of CRAd-synNotch (forward) are shown in Figures 1 and 2, and a schematic diagram of CRAd-GFP is shown in Figure 3. The nucleotide sequence (SEQ ID NO: 8) of the Ad5 / 35 chimeric fiber gene used in this example is shown in FIG. 36, and the amino acid sequence (SEQ ID NO: 14) of the protein encoded by the nucleotide sequence of SEQ ID NO: 8 is shown in FIG. 40.

[0145] CRAd-synNotch (forward / reverse), CRAd-GFP, ADX730, wtAd, and Ad-LacZ were cultured and purified in large quantities, and then transformed into a 500-kDa solution containing Tris, sucrose, NaCl, and MgCl using an Amicon Ultra-15 (Merck). ２The buffer was replaced with an adenovirus stock solution containing Tween (trademark) 80, EDTA, and ethanol at pH 8.0, and appropriate amounts of each solution were dispensed and stored at -80°C until use in subsequent experiments. The composition of the adenovirus stock solution is shown in the table below.

[0146]

[0147] 1.4 Cells and Medium HEK293 cells, human triple-negative breast cancer cell line MDA-MB-231 cells (The European Collection of Cell Cultures), human normal urothelial cell line SV-HUC-1 cells (American Type Culture Collection), and COX-2 protein-negative human breast cancer cell line BT474 cells (American Type Culture Collection) were cultured in D-MEM medium containing 10% FBS and 1% 100 U / ml penicillin and 100 mg / ml streptomycin (P / S; Nacalai Tesque).

[0148] Human bladder cancer cell line T24 cells (American Type Culture Collection) were cultured in Eagle's minimal essential medium (E-MEM; FUJIFILM Wako Pure Chemical Co.) containing 10% FBS and 1% P / S.

[0149] Human prostate cancer cell line DU145 cells (American Type Culture Collection) were cultured in Roswell Park Memorial Institute 1640 medium (RPMI 1640; FUJIFILM Wako Pure Chemical Co.) containing 10% FBS and 1% P / S.

[0150] Unless otherwise stated, cell culture was performed at 37°C and 5% CO ２、 21% O ２ The experiment was carried out under the following conditions.

[0151] 1.5 Analysis of CD46, CAR, and CD44 by Flow Cytometry The expression levels of CD46, CAR, and CD44 in BT474, T24, MDA-MB-231, and DU145 cells were analyzed by flow cytometry. The specific method is shown below.

[0152] 1 x 10 ６ Each cell line was centrifuged and washed with PBS. A flow cytometry reaction solution was prepared using FITC Anti-Human CD46 (BioLegend), PE-Anti-Car (Merck), and APC Anti-Mouse / Human CD44 (BioLegend). This was added to each cell line and incubated on ice for 30 minutes in the dark. After incubation, the cells were centrifuged and washed twice with PBS, and analyzed using Guava™ easyCyte™ (Merck). As a control, 10 μl of PBS was added to each cell line instead of the flow cytometry reaction solution, incubated on ice for 30 minutes in the dark, washed twice with PBS, and analyzed in the same manner. The data obtained was analyzed using the accompanying software, InCyte.

[0153]

[0154] 1.6 Real-time RT-PCR 1.6.1 Expression Analysis of COX-2, HAS-1, HAS-2, and HAS-3 Genes Expression of the COX-2, HAS-1, HAS-2, and HAS-3 genes in T24 cells, MDA-MB-231 cells, and DU145 cells was analyzed by real-time RT-PCR. The COX-2 gene was analyzed for relative expression level, with the expression level in BT474 cells set to 1. The HAS-1, HAS-2, and HAS-3 genes were analyzed for relative expression level, with the expression level in SV-HUC-1 cells set to 1.

[0155] Specifically, T24 cells, MDA-MB-231 cells, DU145 cells, BT474 cells, and SV-HUC-1 cells were cultured in a 6-well Cell Culture Plate Flat Bottom (CORNING) at 5 × 10 ５Cells were seeded at 1000 kJ / well and cultured. The day after seeding, cells were harvested, and total RNA was extracted using NucleoSpin™ RNA (TaKaRa). cDNA was synthesized from the extracted total RNA using PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa). Using this as a template, PCR reactions were performed in three wells using TB Green™ Premix Ex Taq™ II (TaKaRa) and analysis was performed by the ΔΔCt method. mRNA levels were normalized by the expression level of the control gene TATA-binding protein (TBP). The primers used for PCR are shown in the table below.

[0156]

[0157] 1.6.2 Expression Analysis of HIF-3α4, VEGF, CCL2, and SOX-2 Genes The expression of HIF-3α4, VEGF, CCL2, and SOX-2 genes in T24 cells and MDA-MB-231 cells was analyzed by real-time RT-PCR. Specifically, each cell was plated at 1.0 × 10 in a 6-well Cell Culture Plate Flat Bottom (CORNING). ５ The cells were seeded at 1000 cells / well and cultured overnight. The next day, they were infected with CRAd-synNotch (forward) or ADX730 at an MOI of 50. ２ ) or hypoxic conditions (2% O ２ ) for 48 hours, and the cells were harvested. The expression levels of HIF-3α4, VEGF, CCL2, and SOX-2 genes were analyzed in the same manner as in 1.6.1 except for the PCR primers used. The PCR primers are shown in the table in 1.6.1.

[0158] 1.6.3 Expression Analysis of SOX-2, Cortactin, OCT4, Nanog, VEGF, PHD3, GLUT1, and Cyclin G2 Genes The expression of SOX-2, Cortactin, OCT4, Nanog, VEGF, PHD3, GLUT1, and Cyclin G2 genes in T24 cells and DU145 cells was analyzed by real-time RT-PCR. Specifically, each cell was plated at 5.0 × 10 in a 6-well Cell Culture Plate Flat Bottom (CORNING). ５ The cells were seeded at 1000 x g / well and infected with CRAd-synNotch (forward orientation) or CRAd-GFP at an MOI of 50. Then, the cells were cultured under normal oxygen conditions (21% O ２ ) or hypoxic conditions (2% O ２ ) for 72 hours for T24 cells and 96 hours for DU145 cells, respectively, and the cells were harvested. The expression levels of SOX-2, Cortactin, OCT4, Nanog, VEGF, PHD3, GLUT1, and Cyclin G2 genes were analyzed in the same manner as in 1.6.1 except for the PCR primers. The PCR primers are shown in the table in 1.6.1.

[0159] 1.7 Western Blotting 1.7.1 CRAd-synNotch (forward) or ADX730 was infected in vitro into T24 cells or MDA-MB-231 cells, and the increase or decrease in the expression level of the CD44 region in the fusion gene carried was evaluated by Western blotting.

[0160] Specifically, each cell was placed in a 6-well plate (Corning) at 1.0 × 10 ５ Cells were seeded per well and cultured overnight. After culture, the cells were infected with a predetermined amount of adenovirus and cultured for an additional 48 hours. After culture, the cells were harvested and mixed with sample buffer (for SDS-PAGE, 6x concentrated, containing a reducing agent) (Nacalai Tesque, Cat. No. 09499-14), heated at 95°C for 5 minutes, and then cooled on ice to prepare a sample.

[0161] Each sample was subjected to SDS-polyacrylamide gel electrophoresis, and the gel was then blotted onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was washed with a washing solution (PBS containing 0.1% Tween™ 20, hereafter referred to as "PBS-T") and then blocked with PBS-T containing 5% skim milk (Nacalai Tesque) at room temperature for 1 hour.

[0162] After blocking, the membrane was washed and incubated overnight with a primary antibody, using CD44 (E7K2Y) XP™ Rabbit mAb (Cell Signaling Technology) diluted 1000-fold with Can Get Signal Immunoreaction Enhancer Solution 1 (TOYOBO) or β-Actin (C4) (SANTA CRUZ BIOTECHNOLOGY) diluted 1000-fold with PBS-T containing 5% Blocking One (Nacalai Tesque), at 4°C with shaking.

[0163] After the primary antibody reaction, the membrane was washed, and a secondary antibody reaction was carried out for 1 hour with shaking using anti-IgG (H+L chain) (Rabbit) p-Ab-HRP (Medical & Biological Laboratories) diluted 1000-fold with Can Get Signal Immunoreaction Enhancer Solution 2 (TOYOBO) or anti-IgG (H+L chain) (Mouse) pAb-HRP (Medical & Biological Laboratories) diluted 1000-fold with PBS-T containing 5% Blocking One (Nacalai Tesque). Then, chemiluminescence was detected using Chemi-Lumi One L (Nacalai Tesque).

[0164] 1.7.2 T24 cells and DU145 cells were infected with CRAd-synNotch (forward orientation), and the increase or decrease in the expression level of the CD44 region in the fusion gene carried by the infected cells was evaluated by Western blotting. Specifically, the amount of adenovirus used for infection was 25 MOI or 50 MOI, and the incubation time after infection was 24, 48, 72, or 96 hours. For the primary antibody reaction, CD44 (E7K2Y) XP™ Rabbit mAb (Cell Signaling Technology) diluted 1000-fold with Can Get Signal Immunoreaction Enhancer Solution 1 (TOYOBO), HIF3α4 polyclonal antibody (Hokudo) diluted 1000-fold with Can Get Signal Immunoreaction Enhancer Solution 1, or β-Actin (C4) (SANTA) diluted 1000-fold with PBS-T containing 5% Blocking One (Nacalai Tesque) were used. The other experimental conditions were the same as those in 1.7.1.

[0165] 1.8 Evaluation of Adenoviral Vector Propagation Ability COX-2-positive T24 cells and DU145 cells, and COX-2-negative BT474 cells and MDA-MB-231 cells were infected in vitro with wtAd, ADX730, CRAd-GFP, or CRAd-synNotch (forward orientation), and the viral proliferation ability driven by the COX-2 promoter and E1 promoter was evaluated by PCR.

[0166] Specifically, each cell line was placed in a 6-well Cell Culture Plate Flat Bottom (CORNING) at 5 × 10 ５ Cells / well were seeded, 37°C, 5% CO ２ After the incubation, the medium was replaced with 300 μl of medium containing 50 MOI of each viral vector, and the cells were incubated at 37°C and 5% CO ２ After infection, 1.7 ml of medium was added, and the cells were incubated at 37°C, 5% CO ２The cells were cultured for 24 hours under the conditions. After culturing, the cells were collected and DNA was extracted using NucleoSpin™ Tissue (MACHEREY-NAGEL). Quantification and analysis were carried out by PCR using the extracted DNA as a template and TB Green™ Premix Ex Taq™ II (TaKaRa). The primers used for PCR are shown in the table below.

[0167]

[0168] 1.9 Cancer cell proliferation inhibitory effect of CRAd-synNotch (forward orientation) BT474 cells, T24 cells, MDA-MB-231 cells, and DU145 cells were cultured in a 96-well Cell Culture Plate Flat Bottom (CORNING) at 5 × 10 ３ Cells / well were seeded and cultured overnight. The medium was then replaced with 100 μl of medium supplemented with 50 MOI of ADX730, CRAd-GFP, or CRAd-synNotch (forward), or fresh medium (mock) without recombinant adenovirus. The cells were maintained under normoxic conditions (Normoxia: 21% O ２ ) or hypoxic conditions (Hypoxia: 2% O ２ ) for 72 hours. 20 μl of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) (Promega) was added to the culture supernatant as a colorimetric reagent, and after 4 hours of incubation, the absorbance was measured at a wavelength of 492 nm.

[0169] 1.10 Bladder cancer treatment experiment using mice Six-week-old female BALB / c-nu / nu mice were purchased from CLEA Japan. ６Mice were inoculated into the right flank with 140 μl of a 1:1 mixture of T24 cells (T24 cells) and Matrigel (Becton Dickinson). 13 days after inoculation, tumor formation was confirmed, and 20 mice were randomly divided into four groups (n=5): a CRAd-synNotch (forward) intratumoral administration group, a CRAd-GFP intratumoral administration group, an ADX730 intratumoral administration group, and a PBS intratumoral administration group. ９ Infectious units (ifu) / 50 μl PBS of CRAd-synNotch (forward), CRAd-GFP, or ADX730, or 50 μl PBS, were administered into the tumor at 3-day intervals for a total of 6 times (Day 0, 4, 8, 12, 16, and 20). The start of treatment was Day 0, and the short diameter (W) and long diameter (L) of the tumor were measured. ２ The tumor volume was calculated using the formula: (L × L) / 2. Tumor volume 300 mm ３ was used as the endpoint.

[0170] 1.11 Immunohistochemical staining 1×10 ６ T24 cells were subcutaneously inoculated into nude mice to form tumors, and then PBS, 1 × 10 ９ IFU of CRAd-GFP, ADX730, or CRAd-synNotch (forward) was administered intratumorally. Tumor tissues were excised and fixed with paraformaldehyde. Paraffin-embedded T24 tumor tissue sections were deparaffinized and rehydrated. Antigen retrieval was performed in Bond epitope retrieval buffer (pH 6.0; Leica Microsystems) at 98°C for 20 minutes. Immunohistochemical staining was performed using an automated tissue processor (Leica Microsystems Bond) according to the manufacturer's standard protocol. Tissue sections were incubated with anti-CD44 antibody (1:600, Catalog #: 37259S, Cell Signaling Technology) at room temperature for 15 minutes. After washing, the sections were incubated with peroxidase-labeled secondary antibodies. After further washing, the sections were incubated with 3,3'-diaminobenzidine (Muto Pure Chemicals Co., Ltd.) and counterstained with hematoxylin. The obtained tissue slides were observed under a fluorescence microscope BZ-X710 (Keyence).

[0171] 2. <Results> 2.1 Analysis of CD46, CAR, and CD44 expression in BT474 cells, T24 cells, MDA-MB-231 cells, and DU145 cells by flow cytometry 2.1.1 Analysis of CD46, CAR, and CD44 expression in BT474 cells Flow cytometry was performed on BT474 cells using the method described in 1.5. The results are shown in Figure 4.

[0172] When the flow cytometry reaction solution was used, the mean fluorescence intensity (MFI) of BT474 cells at the CD46 fluorescence wavelength was 157.56 ± 2.41. On the other hand, the MFI of the control group using PBS was 17.68 ± 0.09. The significantly increased fluorescence intensity compared to the control group confirmed high expression of CD46 in BT474 cells.

[0173] When the flow cytometry reaction solution was used, the MFI of BT474 cells at the CAR fluorescence wavelength was 23.83 ± 0.31. Meanwhile, the MFI of the control group was 15.48 ± 0.05. The significantly increased fluorescence intensity compared to the control group confirmed high expression of CAR in BT474 cells.

[0174] When using the flow cytometry reaction solution, the MFI of BT474 cells at the CD44 fluorescence wavelength was 22.67 ± 0.09. In contrast, the MFI of the control group was 30.54 ± 0.05. Since there was no significant difference in fluorescence intensity compared to the control group, it was confirmed that CD44 was not highly expressed in BT474 cells.

[0175] 2.1.2 Analysis of CD46, CAR, and CD44 Expression in T24 Cells Flow cytometry was performed on T24 cells using the method described in 1.5. The results are shown in Figure 5.

[0176] When the flow cytometry reaction solution was used, the MFI of T24 cells at the CD46 fluorescence wavelength was 826.67 ± 5.50. On the other hand, the MFI of the control group using PBS was 11.59 ± 0.07. The significantly increased fluorescence intensity compared to the control group confirmed the high expression of CD46 in T24 cells.

[0177] When the flow cytometry reaction solution was used, the MFI of T24 cells at the CAR fluorescence wavelength was 57.90±2.63. Meanwhile, the MFI of the control group was 19.67±0.09. The significantly increased fluorescence intensity compared to the control group confirmed that CAR was highly expressed in T24 cells.

[0178] When the flow cytometry reaction solution was used, the MFI of T24 cells at the CD44 fluorescence wavelength was 3058.09 ± 107.04. On the other hand, the MFI of the control group was 4.97 ± 0.01. The significantly increased fluorescence intensity compared to the control group confirmed high expression of CD44 in T24 cells.

[0179] 2.1.3 Analysis of CD46, CAR, and CD44 Expression in MDA-MB-231 Cells Flow cytometry was performed on MDA-MB-231 cells using the method described in 1.5. The results are shown in Figure 6.

[0180] When the flow cytometry reaction solution was used, the MFI of MDA-MB-231 cells at the CD46 fluorescence wavelength was 838.49 ± 10.80. On the other hand, the MFI of the control group using PBS was 38.60 ± 0.25. The significantly increased fluorescence intensity compared to the control group confirmed high expression of CD46 in MDA-MB-231 cells.

[0181] When the flow cytometry reaction solution was used, the MFI of MDA-MB-231 cells at the CAR fluorescence wavelength was 33.36 ± 0.49. On the other hand, the MFI of the control group was 67.55 ± 0.39. Since there was no significant difference in fluorescence intensity compared to the control group, it was confirmed that CAR was not highly expressed in the MDA-MB-231 cells.

[0182] When the flow cytometry reaction solution was used, the MFI of MDA-MB-231 cells at the CD44 fluorescence wavelength was 5869.71 ± 32.15. On the other hand, the MFI of the control group was 5.62 ± 0.02. The significantly increased fluorescence intensity compared to the control group confirmed high expression of CD44 in MDA-MB-231 cells.

[0183] 2.1.4 Analysis of CD46, CAR, and CD44 Expression in DU145 Cells Flow cytometry was performed on DU145 cells using the method described in 1.5. The results are shown in Figure 7.

[0184] When the flow cytometry reaction solution was used, the MFI of DU145 cells at the CD46 fluorescence wavelength was 881.86 ± 6.87. On the other hand, the MFI of the control group using PBS was 24.16 ± 0.62. The significantly increased fluorescence intensity compared to the control group confirmed the high expression of CD46 in DU145 cells.

[0185] When the flow cytometry reaction solution was used, the MFI of DU145 cells at the CAR fluorescence wavelength was 188.65±6.18. Meanwhile, the MFI of the control group was 23.97±0.45. The significantly increased fluorescence intensity compared to the control group confirmed that CAR was highly expressed in DU145 cells.

[0186] When the flow cytometry reaction solution was used, the MFI of DU145 cells at the CD44 fluorescence wavelength was 3434.24 ± 38.66. On the other hand, the MFI of the control group was 4.79 ± 0.04. The significantly increased fluorescence intensity compared to the control group confirmed high expression of CD44 in DU145 cells.

[0187] 2.2 Quantification of COX-2 gene expression in T24 cells, MDA-MB-231 cells, and DU145 cells Real-time RT-PCR was performed using the method described in 1.6.1, and the expression levels of the COX-2 gene in T24 cells, MDA-MB-231 cells, and DU145 cells were evaluated by comparing them with the expression level of the COX-2 gene in the COX-2 protein-negative human breast cancer cell line BT474 cells. The results are shown in Figure 8.

[0188] When the expression level of the COX-2 gene in BT474 cells was set to 1, the relative expression levels of the COX-2 gene in T24 cells, MDA-MB-231 cells, and DU145 cells were all significantly higher. These results confirmed that the COX-2 gene was highly expressed in T24 cells, MDA-MB-231 cells, and DU145 cells.

[0189] 2.3 Quantification of HAS-1, HAS-2, and HAS-3 Gene Expression in T24 Cells, MDA-MB-231 Cells, and DU145 Cells Real-time RT-PCR was performed using the method described in 1.6.1 to evaluate the expression levels of HAS-1, HAS-2, and HAS-3 genes (i.e., hyaluronic acid-producing ability) in T24 cells, MDA-MB-231 cells, and DU145 cells, compared with those of SV-HUC-1 cells, a normal human urothelial cell line. The results are shown in Figures 9 to 11.

[0190] 2.3.1 T24 cells When the expression level in SV-HUC-1 cells was set to 1, the relative expression levels of the hyaluronic acid production genes HAS-1 and HAS-3 in T24 cells were significantly higher, while the relative expression level of HAS-2 was not significantly different (Figure 9).

[0191] 2.3.2 MDA-MB-231 cells The relative expression level of the hyaluronic acid production gene HAS-2 in MDA-MB-231 cells was significantly higher when the expression level in SV-HUC-1 cells was set to 1. On the other hand, there was no significant difference in the relative expression levels of HAS-1 and HAS-3 (Figure 10).

[0192] 2.3.3 DU145 cells The relative expression levels of the hyaluronic acid production genes HAS-2 and HAS-3 in DU145 cells were significantly higher when the expression level in SV-HUC-1 cells was set to 1. On the other hand, there was no significant difference in the relative expression level of HAS-1 (Figure 11).

[0193] 2.4 Titer measurement of recombinant adenoviral vectors The titer of the prepared adenoviral vectors was measured by the method described in 1.1.9. The titer of CRAd-synNotch (reverse) was 1.7 x 10 １０ The titer of CRAd-synNotch (forward) was 1.2 x 10 １２ The titer of CRAd-GFP was 1.05 x 10 １１ ifu / ml.

[0194] 2.5 Analysis of CD44 / Notch / HIF-3α4 Fusion Gene Expression in MDA-MB-231 Cells Infected with CRAd-synNotch (Reverse) Expression of the CD44 / Notch / HIF-3α4 fusion gene was assessed by Western blotting and real-time RT-PCR for MDA-MB-231 cells infected with CRAd-synNotch (reverse) prepared in 1.1. Note that CRAd-synNotch (reverse) and other adenoviral vectors were mass-cultured and purified from the tertiary virus solution using the method described in 1.1.8. Specific results are shown below.

[0195] 2.5.1 Analysis of CD44 Expression by Western Blotting Western blotting was performed on MDA-MB-231 cells (cell only) not infected with adenovirus, MDA-MB-231 cells infected with Ad-LacZ at 100 MOI (multiplicity of infection), MDA-MB-231 cells infected with ADX730 at 100 MOI, MDA-MB-231 cells infected with CRAd-GFP at 50 MOI, and MDA-MB-231 cells infected with CRAd-synNotch (reverse) at 50 MOI, using the method described in 1.7.1. The results are shown in Figure 12.

[0196] A band (approximately 80 kDa) attributed to endogenous CD44 was observed in all samples, whereas a band (approximately 105 kDa) thought to be a CD44 / Notch / HIF-3α4 fusion protein was observed only in ADX730-infected cells, but not in CRAd-synNotch (reverse)-infected cells.

[0197] 2.5.2 Quantification of HIF-3α4 Gene Expression by Real-time RT-PCR Real-time RT-PCR was performed on MDA-MB-231 cells not infected with adenovirus (cell only), MDA-MB-231 cells infected with 100 MOI of ADX730, and MDA-MB-231 cells infected with 50 MOI of CRAd-synNotch (reverse orientation) using the method described in 1.6.2 to quantify the expression level of the HIF-3α4 gene. The results are shown in Figure 13.

[0198] The expression level of the HIF-3α4 gene in MDA-MB-231 cells infected with ADX730 was significantly increased compared to MDA-MB-231 cells not infected with adenovirus, whereas the expression level of the HIF-3α4 gene in MDA-MB-231 cells infected with CRAd-synNotch (reverse orientation) was not significantly different compared to MDA-MB-231 cells not infected with adenovirus.

[0199] 2.5.3 Summary Generally, when constructing an adenoviral vector, if it is intended to express two sets of genes (in this test example, the CD44 / Notch / HIF-3α4 fusion gene and the E1A / E1B genes) under the control of separate promoters (in this test example, the β-actin promoter and the COX-2 promoter), the two sets of genes are often arranged in opposite orientations. For this reason, the present inventors first investigated CRAd-synNotch (reverse orientation), in which the CD44 / Notch / HIF-3α4 fusion gene and the E1A / E1B genes are arranged in opposite orientations. However, as described above, no expression of the CD44 / Notch / HIF-3α4 fusion gene was observed from CRAd-synNotch (reverse orientation).

[0200] Therefore, the present inventors investigated CRAd-synNotch (forward orientation), in which the CD44 / Notch / HIF-3α4 fusion gene and the E1A / E1B genes were arranged in the same direction (forward orientation). The results are shown in 2.6 and subsequent sections.

[0201] 2.6 Analysis of CD44 / Notch / HIF-3α4 fusion gene expression in MDA-MB-231 cells infected with CRAd-synNotch (forward orientation) Expression of the CD44 / Notch / HIF-3α4 fusion gene was evaluated in MDA-MB-231 cells infected with CRAd-synNotch (forward orientation) by Western blotting and RT-PCR in the same manner as in 2.5. Note that a secondary virus solution was used for CRAd-synNotch (forward orientation). Specific results are shown below.

[0202] 2.6.1 Analysis of CD44 Expression by Western Blotting 2.6.1.1 CRAd-synNotch (Forward) Clone 1 Western blotting was performed using the method described in 1.7.1 on MDA-MB-231 cells infected with 10 μl, 20 μl, or 50 μl of the secondary virus solution of CRAd-synNotch (Forward) Clone 1, MDA-MB-231 cells not infected with adenovirus (cell only), and MDA-MB-231 cells infected with ADX730 at 100 MOI. The results are shown in Figure 14.

[0203] A band (approximately 80 kDa) attributed to endogenous CD44 was observed in all samples, whereas a band (approximately 105 kDa) thought to be a CD44 / Notch / HIF-3α4 fusion protein was observed only in ADX730-infected cells, but not in CRAd-synNotch (forward) clone 1-infected cells.

[0204] 2.6.1.2 CRAd-synNotch (forward) clone 2 and clone 3 Western blotting was performed using the method described in 1.7.1 on MDA-MB-231 cells infected with 20 μl or 40 μl of the secondary virus solution from CRAd-synNotch (forward) clone 2, MDA-MB-231 cells infected with 20 μl or 40 μl of the secondary virus solution from CRAd-synNotch (forward) clone 3, MDA-MB-231 cells not infected with adenovirus (cell only), and MDA-MB-231 cells infected with 100 MOI of ADX730. The results are shown in Figure 15.

[0205] A band (approximately 80 kDa) assigned to endogenous CD44 was observed in all samples. In addition, a band (approximately 105 kDa) thought to be a CD44 / Notch / HIF-3α4 fusion protein was observed in cells infected with CRAd-synNotch (forward) clone 2, CRAd-synNotch (forward) clone 3, and ADX730.

[0206] 2.6.2 Quantification of CD44 Gene and HIF-3α4 Gene Expression by Real-time RT-PCR Real-time RT-PCR was performed using the method described in 1.6.2 to quantify the expression levels of the HIF-3α4 gene and CD44 gene for MDA-MB-231 cells (cell only) not infected with adenovirus, MDA-MB-231 cells infected with 100 MOI of ADX730, MDA-MB-231 cells infected with 20 μl of CRAd-synNotch (forward) clone 2, and MDA-MB-231 cells infected with 20 μl of CRAd-synNotch (forward) clone 3. The results are shown in Figure 16.

[0207] In MDA-MB-231 cells infected with ADX730, MDA-MB-231 cells infected with CRAd-synNotch (forward) clone 2, and MDA-MB-231 cells infected with CRAd-synNotch (forward) clone 3, the expression levels of HIF-3α4 gene and CD44 gene were significantly increased compared to MDA-MB-231 cells not infected with adenovirus.

[0208] Because the expression levels of the HIF-3α4 gene and CD44 gene were particularly high in MDA-MB-231 cells infected with CRAd-synNotch (forward) clone 3, the present inventors decided to use CRAd-synNotch (forward) clone 3 in subsequent tests, and CRAd-synNotch (forward) clone 3 was mass-cultured and purified by the method described in 1.1.8.

[0209] 2.7 Analysis of CD44 / Notch / HIF-3α4 Fusion Gene Expression in MDA-MB-231 and T24 Cells Infected with CRAd-synNotch (Forward) Clone 3 After Mass Cultivation MDA-MB-231 and T24 cells infected with CRAd-synNotch (Forward) Clone 3, which had been mass-cultured and purified by the method described in 1.1.8, were evaluated for CD44 / Notch / HIF-3α4 fusion gene expression by Western blotting and real-time RT-PCR. Specific results are shown below. In the following test examples, CRAd-synNotch (Forward) Clone 3 will be referred to simply as "CRAd-synNotch."

[0210] 2.7.1 Analysis of CD44 Expression by Western Blotting Western blotting was performed on MDA-MB-231 cells (cell only) not infected with adenovirus, MDA-MB-231 cells infected with CRAd-synNotch at 50, 100, 200, or 400 MOI, and MDA-MB-231 cells infected with ADX730 at 100 MOI, using the method described in 1.7.1. Western blotting was also performed on T24 cells (cell only) not infected with adenovirus, T24 cells infected with CRAd-synNotch at 50, 100, 200, or 400 MOI, and T24 cells infected with ADX730 at 100 MOI, using the method described in 1.7.1. The results are shown in Figure 17.

[0211] A band (approximately 80 kDa) assigned to endogenous CD44 was observed in all samples. In addition, a band (approximately 105 kDa) thought to be a CD44 / Notch / HIF-3α4 fusion protein was observed in MDA-MB-231 and T24 cells infected with CRAd-synNotch and in MDA-MB-231 and T24 cells infected with ADX730.

[0212] 2.7.2 Quantification of CD44 Gene and HIF-3α4 Gene Expression by Real-time RT-PCR Real-time RT-PCR was performed on T24 cells not infected with adenovirus (Cell only), T24 cells infected with ADX730 at 100 MOI, and T24 cells infected with CRAd-synNotch at 50 MOI, using the method described in 1.6.2, to quantify the expression levels of the HIF-3α4 gene and CD44 gene. The results are shown in Figure 18.

[0213] The expression level of the HIF-3α4 gene was significantly increased in both T24 cells infected with ADX730 and T24 cells infected with CRAd-synNotch compared to T24 cells not infected with adenovirus.

[0214] The expression level of the CD44 gene was not significantly different in T24 cells infected with ADX730 compared to T24 cells not infected with adenovirus, whereas it was significantly increased in T24 cells infected with CRAd-synNotch compared to T24 cells not infected with adenovirus.

[0215] 2.8 Evaluation of the proliferation ability of adenovirus vectors The proliferation ability of wtAd, ADX730, CRAd-GFP, and CRAd-synNotch in BT474 cells, T24 cells, MDA-MB-231 cells, and DU145 cells was evaluated by the method described in 1.8. The results are shown in Figure 19.

[0216] 2.8.1 wtAd The copy number of wtAd infected in BT474, T24, and DU145 cells was significantly increased at 24 h postinfection compared to 1 h postinfection, whereas the copy number of wtAd infected in MDA-MB-231 cells was not significantly different at 24 h postinfection compared to 1 h postinfection (Figure 19A).

[0217] 2.8.2 ADX730 The copy number of ADX730 infected into each of the cancer cells, BT474 cells, T24 cells, MDA-MB-231 cells, and DU145 cells, showed no significant difference 24 hours after infection compared to 1 hour after infection (Figure 19B).

[0218] 2.8.3 CRAd-GFP and CRAd-synNotch The copy numbers of CRAd-GFP and CRAd-synNotch infected in COX-2-positive T24 and DU145 cells were significantly increased at 24 hours post-infection compared to 1 hour post-infection. In contrast, the copy numbers of CRAd-GFP and CRAd-synNotch infected in COX-2-negative BT474 and MDA-MB-231 cells were not significantly different at 24 hours post-infection compared to 1 hour post-infection (Figures 19C and 19D).

[0219] 2.9 Evaluation of the cancer cell proliferation inhibitory effect of CRAd-synNotch The cell proliferation inhibitory effect of CRAd-synNotch on BT474 cells, T24 cells, MDA-MB-231 cells, and DU145 cells was evaluated in vitro using the method described in 1.9. The results are shown in Figures 20 to 23. Note that "Cell only" refers to a mock treatment in which adenovirus-free medium was added instead of recombinant adenovirus-added medium.

[0220] 2.9.1 BT474 cells under normoxic conditions (21% O ２ ) and hypoxic conditions (Hypoxia: 2% O ２ ) In all of the above groups, ADX730, CRAd-GFP, and CRAd-synNotch did not show any significant cell growth inhibitory effect on BT474 cells compared to the Cell only group (FIG. 20).

[0221] 2.9.2 T24 cells Under both normoxic and hypoxic conditions, ADX730, CRAd-GFP, and CRAd-synNotch all exhibited significant cell growth inhibitory effects compared to the Cell only group. In particular, CRAd-synNotch exhibited a significantly higher cell growth inhibitory effect than ADX730 and CRAd-GFP (Figure 21).

[0222] 2.9.3 MDA-MB-231 cells Under both normoxic and hypoxic conditions, ADX730, CRAd-GFP, and CRAd-synNotch did not show any significant inhibitory effect on cell proliferation in MDA-MB-231 cells compared to the Cell only group (Figure 22).

[0223] 2.9.4 DU145 cells Under normoxic conditions, only CRAd-synNotch exhibited a significant inhibitory effect on DU145 cells compared to the Cell only group, whereas ADX730 and CRAd-GFP did not. Under hypoxic conditions, ADX730 and CRAd-synNotch exhibited a significant inhibitory effect on DU145 cells compared to the Cell only group, whereas CRAd-GFP did not exhibit a significant inhibitory effect on DU145 cells compared to the Cell only group (Figure 23).

[0224] 2.10 Evaluation of the inhibitory effect of CRAd-synNotch on HIF-1α downstream genes and CD44 downstream genes Using the method described in 1.6.2, the expression of VEGF, a downstream gene of HIF-1α, and SOX-2 and CCL2, downstream genes of CD44, was analyzed in T24 cells not infected with adenovirus (cell only), T24 cells infected with ADX730 at 50 MOI, and T24 cells infected with CRAd-synNotch at 50 MOI. The results are shown in Figures 24 to 26.

[0225] 2.10.1 VEGF Gene Under normoxic conditions, expression levels of the HIF-1α downstream gene VEGF were low in T24 cells not infected with adenovirus (cell only), T24 cells infected with ADX730, and T24 cells infected with CRAd-synNotch. On the other hand, under hypoxic conditions, high VEGF expression was observed in T24 cells not infected with adenovirus (cell only) and T24 cells infected with ADX730. However, VEGF expression levels were significantly lower in T24 cells infected with CRAd-synNotch compared to T24 cells not infected with adenovirus (cell only) and T24 cells infected with ADX730 (Figure 24).

[0226] 2.10.2 SOX-2 Gene Under normoxic conditions, the expression level of the CD44 downstream gene SOX-2 was significantly lower in T24 cells infected with CRAd-synNotch than in T24 cells not infected with adenovirus (Cell only) and T24 cells infected with ADX730. Under hypoxic conditions, the expression level of the CD44 downstream gene SOX-2 was significantly lower in T24 cells infected with ADX730 and T24 cells infected with CRAd-synNotch than in T24 cells not infected with adenovirus (Cell only) (Figure 25).

[0227] 2.10.3 CCL2 gene Under normoxic conditions, no significant difference was observed in the expression level of the CD44 downstream gene CCL2 in T24 cells infected with CRAd-synNotch compared to T24 cells not infected with adenovirus (Cell only). On the other hand, under hypoxic conditions, the expression level of the CD44 downstream gene CCL2 was significantly lower in T24 cells infected with CRAd-synNotch compared to T24 cells not infected with adenovirus (Cell only) (Figure 26).

[0228] 2.10.4 Summary These results suggest that the decoy function of the extracellular portion of CD44 contained in CRAd-synNotch suppresses the expression of downstream genes of CD44, and that the HIF-3α4 portion contained in CRAd-synNotch suppresses the expression of downstream genes of HIF-1α.

[0229] 2.11 Evaluation of the in vivo therapeutic effect of CRAd-synNotch on bladder cancer Nude mice transplanted with T24 cells were intratumorally administered PBS, ADX730, CRAd-GFP, or CRAd-synNotch using the method described in 1.10, and the mice were monitored over time. The results are shown in Figures 27 and 28.

[0230] At 24 days (Day 24) after the start of administration of PBS, ADX730, CRAd-GFP, or CRAd-synNotch, tumor growth was significantly suppressed in the CRAd-synNotch group compared to the PBS and CRAd-GFP groups. Furthermore, the CRAd-synNotch group tended to have a greater tumor growth suppression effect than the ADX730 group (Figure 27).

[0231] After six doses were administered, observations were continued and the survival time was significantly longer in the CRAd-synNotch group compared to the PBS and CRAd-GFP groups. Furthermore, the CRAd-synNotch group tended to have a longer survival time compared to the ADX730 group (Figure 28).

[0232] 2.12 Analysis of Fusion Protein Expression by Western Blotting Using the method described in 1.7.2, T24 cells were infected with CRAd-synNotch at a concentration of 50 MOI and cultured for 24 or 72 hours. After culture, Western blotting was performed using a CD44 antibody as the primary antibody, and the results are shown in the left side of Figure 42. Western blotting was performed using a HIF-3α4 antibody as the primary antibody, and the results are shown in the right side of Figure 42. As shown in the left side of Figure 42, an endogenous CD44 band (approximately 80 kDa) was observed in all samples. Furthermore, in CRAd-synNotch-infected cells, a band (approximately 105 kDa) believed to be the CD44-ECD / Notch / HIF-3α4 fusion protein was observed in both the 24-hour and 72-hour cultures after infection. A lower molecular weight band was observed in the 72-hour culture. These results suggest that HIF-3α4 was cleaved from the fusion protein. As shown on the right side of Figure 42, when an HIF-3α4 antibody was used as the primary antibody, a band (approximately 105 kDa) believed to represent the CD44-ECD / Notch / HIF-3α4 fusion protein was observed in CRAd-synNotch-infected cells cultured for 24 hours after infection, but this band was not observed in CRAd-synNotch-infected cells cultured for 72 hours after infection. This result also suggests that HIF-3α4 was cleaved from the fusion protein.

[0233] DU145 cells were infected with CRAd-synNotch at 25 MOI or 50 MOI using the method described in 1.7.2 and cultured for 24, 48, or 96 hours. After culture, Western blotting was performed using a CD44 antibody as the primary antibody, and the results are shown in the left side of Figure 43. Western blotting was performed using a HIF-3α4 antibody as the primary antibody, and the results are shown in the right side of Figure 43. As shown in the left side of Figure 43, an endogenous CD44 band (approximately 80 kDa) was observed in all samples. Furthermore, in CRAd-synNotch-infected cells cultured for 24, 48, and 96 hours after infection, a band believed to be CD44-ECD / Notch / HIF-3α4 fusion protein (approximately 105 kDa) was observed in all samples. A band at a lower molecular weight was observed in cells cultured for 96 hours after infection. These results suggest that HIF-3α4 was cleaved from the fusion protein. As shown on the right side of Figure 43, when an HIF-3α4 antibody was used as the primary antibody, a band (approximately 105 kDa) that appeared to be the CD44-ECD / Notch / HIF-3α4 fusion protein was observed in CRAd-synNotch-infected cells cultured for 24 hours after infection, but this band was not observed in CRAd-synNotch-infected cells cultured for 96 hours after infection. These results also suggest that HIF-3α4 was cleaved from the fusion protein.

[0234] 2.13 Suppression of CD44 downstream genes and HIF-1α downstream genes by CRAd-synNotch As described in 1.6.3, T24 cells were infected with 50 MOI of CRAd-GFP or 50 MOI of CRAd-synNotch, and DU145 cells were infected with 25 MOI of CRAd-GFP or 25 MOI of CRAd-synNotch, and then cultured under normal oxygen conditions (Normoxia, 21% O ２ ) or hypoxic conditions (Hypoxia, 2% O ２) conditions for 72 or 96 hours. After culturing, the expression levels of CD44 downstream genes (Cortactin, OCT4, Nanog, and SOX-2) and HIF-1α downstream genes (VEGF, GLUT1, Cyclin G2, and PHD3) were analyzed by real-time RT-PCR. The results are shown in Figures 44 to 47.

[0235] Figure 44 shows the results of analyzing the expression levels of Cortactin (left), OCT4 (center), and Nanog (right), which are downstream genes of CD44, in T24 cells. As shown in Figure 44, under hypoxic conditions, the expression levels of Cortactin, OCT4, and Nanog in CRAd-synNotch-infected cells were significantly reduced compared to uninfected cells and CRAd-GFP.

[0236] Figure 45 shows the results of analyzing the expression levels of PHD3 (left), GLUT1 (center), and CyclinG2 (right), which are downstream genes of HIF-1α, in T24 cells. As shown in Figure 45, under hypoxic conditions, the expression levels of PHD3, GLUT1, and CyclinG2 in CRAd-synNotch-infected cells were significantly reduced compared to uninfected cells and CRAd-GFP.

[0237] Figure 46 shows the results of analyzing the expression levels of Cortactin (upper left in Figure 46), OCT4 (upper right in Figure 46), Nanog (lower left in Figure 46), and SOX-2 (lower right in Figure 46), which are downstream genes of CD46 in DU145 cells. As shown in Figure 46, under hypoxic conditions, the expression levels of Cortactin, OCT4, Nanog, and SOX-2 in CRAd-synNotch-infected cells were significantly reduced compared to uninfected cells and CRAd-GFP.

[0238] Figure 47 shows the results of analyzing the expression levels of VEGF (Figure 47, upper left), GLUT1 (Figure 47, upper right), CyclinG2 (Figure 47, lower left), and PHD3 (Figure 47, lower right), which are downstream genes of HIF-1α, in DU145 cells. As shown in Figure 47, under hypoxic conditions, the expression levels of VEGF, GLUT1, CyclinG2, and PHD3 in CRAd-synNotch-infected cells were significantly reduced compared to uninfected cells and CRAd-GFP.

[0239] 2.14 Immunohistochemical staining of CD44 in T24 mouse model. As described in 1.11, 1 x 10 ６ T24 cells were subcutaneously inoculated into nude mice to form tumors. ９ IFU of ADX730, CRAd-GFP, or CRAd-synNotch was administered intratumorally. 24 hours after administration, tumors were excised and CD44 expression was confirmed by immunohistochemical staining. The results are shown in Figure 48. As shown at the bottom of Figure 48, CD44 expression was significantly increased on the cell membrane of tumors administered with CRAd-synNotch or ADX730 compared to tumors administered with CRAd-GFP or PBS.

[0240] 3. Discussion These results demonstrate that CRAd-synNotch has an antitumor effect. Furthermore, it was suggested that the antitumor effect may be due to the suppression of expression of downstream genes of CD44 by the decoy function of the extracellular portion of CD44 contained in CRAd-synNotch, and the suppression of expression of downstream genes of HIF-1α by the HIF-3α4 portion contained in CRAd-synNotch. Furthermore, it was suggested that the HIF-3α4 portion is cleaved from the fusion protein after expression.

Claims

1. An oncolytic adenovirus vector comprising a nucleic acid comprising: a base sequence (I): a base sequence having a structure in which (A) a base sequence encoding a protein having a CD44 extracellular function, (B) a base sequence encoding a protein having a Notch core region function, and (C) a base sequence encoding a protein having a HIF-3α4 function are linked in the order of (A)-(B)-(C); and a base sequence (II): a base sequence comprising (D) a COX-2 promoter, (E) a base sequence encoding an E1A protein placed under the control of the COX-2 promoter, and (F) a base sequence encoding an E1B protein placed under the control of the COX-2 promoter, wherein the base sequence (I) and the base sequence (II) are arranged in the same orientation.

2. An oncolytic adenovirus vector comprising: a base sequence (I): a base sequence having a structure in which the base sequence (A), the base sequence (B), and the base sequence (C) are linked in the order of (A)-(B)-(C); and a base sequence (II): a base sequence comprising: (D) a base sequence including a COX-2 promoter, (E) a base sequence encoding an E1A protein placed under the control of the COX-2 promoter, and (F) a base sequence encoding an E1B protein placed under the control of the COX-2 promoter, wherein the base sequence (I) and the base sequence (II) are arranged in the same orientation, and the base sequence (A) is: (a-1): a base sequence consisting of the base sequence of SEQ ID NO: 1, or (a-2): a base sequence having a sequence identity of 85% or more with the base sequence of (a-1) and encoding a protein capable of binding to hyaluronic acid, and the base sequence (B) is: (b-1): a base sequence consisting of the base sequence of SEQ ID NO: 2, or (b-2): a base sequence having a sequence identity of 85% or more with the base sequence of (b-1) and encoding a protein that can be cleaved by a protease, said base sequence (C) being: (c-1): a base sequence having the base sequence of SEQ ID NO: 3, or (c-2): a base sequence having a sequence identity of 85% or more with the base sequence of (c-1) and encoding a protein capable of binding to HIF-1α, said base sequence (D) being: (d-1): a base sequence having the base sequence of SEQ ID NO: 4, or (d-2): a base sequence having a sequence identity of 85% or more with the base sequence of (d-1) and encoding a promoter whose expression is induced under COX-2 expression-inducing conditions, said base sequence (E) being: (e-1): a base sequence having the base sequence of SEQ ID NO: 5, or (e-2): a base sequence having 85% or more sequence identity with the base sequence of (e-1) and encoding a protein having E1A function, and the base sequence (F) is a nucleic acid comprising: (f-1): a base sequence having the base sequence of SEQ ID NO: 6; or (f-2): a base sequence having 85% or more sequence identity with the base sequence of (f-1) and encoding a protein having E1B function.

3. The base sequence (A) is (a-1): a base sequence consisting of the base sequence of SEQ ID NO: 1, or (a-2): a base sequence consisting of a base sequence having a sequence identity of 85% or more with the base sequence of (a-1), and encoding a protein capable of binding to hyaluronic acid, the base sequence (B) is (b-1): a base sequence consisting of the base sequence of SEQ ID NO: 2, or (b-2): a base sequence having a sequence identity of 85% or more with the base sequence of (b-1), and encoding a protein that can be cleaved by a protease, in a protein encoded by a nucleic acid having a structure in which the base sequences (A)-(B)-(C) are linked in this order, the base sequence (C) is (c-1): a nucleic acid consisting of the base sequence of SEQ ID NO: 3, or (c-2): a nucleic acid having a sequence identity of 85% or more with the base sequence of (c-1), and encoding a protein capable of binding to HIF-1α, and the base sequence (D) is 2. The oncolytic adenovirus vector according to claim 1, wherein the base sequence (E) is: (d-1): a base sequence consisting of the base sequence of SEQ ID NO: 4; or (d-2): a base sequence having a sequence identity of 85% or more with the base sequence of (d-1), and encoding a promoter whose expression is induced under COX-2 expression-inducing conditions; wherein the base sequence (E) is: (e-1): a base sequence consisting of the base sequence of SEQ ID NO: 5; or (e-2): a base sequence having a sequence identity of 85% or more with the base sequence of (e-1), and encoding a protein having E1A function; and wherein the base sequence (F) is: (f-1): a base sequence consisting of the base sequence of SEQ ID NO: 6; or (f-2): a base sequence having a sequence identity of 85% or more with the base sequence of (f-1), and encoding a protein having E1B function.

4. An oncolytic adenovirus vector comprising a base sequence (III): (g-1) consisting of the base sequence of SEQ ID NO: 7; or (g-2) consisting of a base sequence having a sequence identity of 85% or more with the base sequence of SEQ ID NO: 7, and having a structure in which a portion encoding a protein capable of binding to hyaluronic acid, a portion encoding a protein that can be cleaved by a protease, and a portion encoding a protein capable of binding to HIF-1α are linked in this order; or (g-3) consisting of a base sequence having a sequence identity of 85% or more with the base sequence of SEQ ID NO: 7, and encoding a protein having anti-cancer activity; and a base sequence (II): comprising: (D) a base sequence including a COX-2 promoter, (E) a base sequence encoding an E1A protein placed under the control of the COX-2 promoter, and (F) a base sequence encoding an E1B protein placed under the control of the COX-2 promoter, wherein the base sequence (III) and the base sequence (II) are arranged in the same direction, and the base sequence (D) is An oncolytic adenovirus vector comprising a nucleic acid, the nucleic acid comprising: (d-1): a base sequence consisting of the base sequence of SEQ ID NO: 4; or (d-2): a base sequence having a sequence identity of 85% or more with the base sequence of (d-1), and encoding a promoter whose expression is induced under COX-2 expression-inducing conditions; the nucleic acid sequence (E) is: (e-1): a base sequence consisting of the base sequence of SEQ ID NO: 5; or (e-2): a base sequence having a sequence identity of 85% or more with the base sequence of (e-1), and encoding a protein having E1A function; and the nucleic acid sequence (F) is: (f-1): a base sequence consisting of the base sequence of SEQ ID NO: 6; or (f-2): a base sequence having a sequence identity of 85% or more with the base sequence of (f-1), and encoding a protein having E1B function.

5. An oncolytic adenovirus vector according to any one of claims 1 to 4, further comprising a base sequence encoding an (H)Ad5 / 35 chimeric fiber protein.

6. The oncolytic adenovirus vector according to claim 5, wherein the base sequence (H) is: (h-1): a base sequence of SEQ ID NO: 8; or (h-2): a base sequence having 85% or more sequence identity with the base sequence of (h-1), and encoding a protein capable of binding to CD46.

7. An oncolytic adenoviral vector according to any one of claims 2 to 4 and 6, wherein the protease is an ADAM protease or γ-secretase.

8. An anti-cancer composition comprising an oncolytic adenoviral vector according to any one of claims 1 to 4 and 6.

9. The anti-cancer composition according to claim 8, which is an injectable preparation.

10. The anti-cancer composition according to claim 8, which is for treating COX-2 positive cancer.

11. The anti-cancer composition according to claim 8, which is for treating at least one cancer selected from the group consisting of bladder cancer, breast cancer, and prostate cancer.

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