Gene therapy agents for cancer

Abstract

This disclosure provides a cancer gene therapy technique with enhanced cancer suppression effects. The inventors have discovered the possibility of achieving superior cancer suppression using an oncolytic adenovirus vector containing nucleic acids, wherein the nucleic acids comprise an artificial gene obtained by fusing the extracellular portion of CD44, the Notch core region, and HIF-3α4, and E1A and E1B genes placed under the control of a COX-2 promoter, wherein the artificial gene, E1A gene, and E1B gene are aligned in the same direction, thereby completing this disclosure.

Description

Technical Field

[0001] This disclosure relates to, for example, novel gene therapy agents or novel gene therapy methods for cancer, and their active ingredients. All documents described in this specification (including Non-Patent Documents 1-12 listed below as prior art documents) are incorporated herein by reference. Background Technology

[0002] In recent years, gene therapy using viral vectors has gained attention as an emerging treatment for cancer. This method utilizes the properties of various viruses and uses viruses as gene "carriers," i.e., viral vectors. One gene delivery method is in vivo gene therapy, in which recombinant viral vectors integrating therapeutic genes are directly administered into the body to treat the disease. Cancer gene therapy differs from existing treatments such as chemotherapy in its mechanism of action. In cancer gene therapy, genes are introduced into cancer cells to directly inhibit genes involved in cancer development and proliferation, or cancer suppressor genes are introduced into cells to directly induce cell death. Therefore, cancer gene therapy is expected to be effective for cancers that are difficult to treat with existing chemotherapy and radiation therapy.

[0003] Existing technical documents

[0004] Patent documents

[0005] Patent Document 1: WO2021 / 131944

[0006] Non-patent literature

[0007] Non-patent document 1: 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.

[0008] Non-Patent Document 2: Bourguignon L.Y., Zhu H., Shao L., Chen Y.W. (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.

[0009] Non-Patent Document 3: 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.

[0010] Non-Patent Document 4: 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.

[0011] Murakami D., Okamoto I., Nagano O., Kawano Y., Tomita T.,Iwatsubo T., De Strooper B., Yumoto E., Saya H. (2003).Presenilin-dependentgamma-secretase activity mediates the intramembranous cleavage ofCD44.Oncogene.2003 Mar 13; 22(10): 1511-6.

[0012] Sourcebook 6:Ueda M., Saji H.(2014).Radiolabeled Probes TargetingHypoxia-Inducible Factor-1-Active Tumor Microenvironments.Scientific WorldJournal.2014; 2014:165461. doi: 10.1155 / 2014 / 165461.Epub 2014 Aug 18 .

[0013] Arsenite induces HIF-1alpha and VEGF throughPI3K, Akt and reactive oxygen species in DU145 human prostate carcinoma cells.Mol Cell Biochem.2004 Jan; 255(1-2):33-4

[0014] Psychiatry 8:Kopan R., Ilagan MX(2009).The canonical Notch signalingpathway: unfolding the activation mechanism.Cell.2009 Apr 17; 137(2): 216-33.doi:10.1016 / j.cell.2009.03.045.

[0015] Non-patent Document 9: 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.

[0016] Non-patent Document 10: Morsut L., Roybal K.T., Xiong X., Gordley R.M., Coyle S.M., Thomson M., Lim W.A. (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.

[0017] Non-patent Document 11: 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.

[0018] Non-patent Document 12: Maynard M.A., Evans A.J., Hosomi T., Hara S., Jewett M.A., 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) Summary of the Invention

[0019] The problem the invention aims to solve

[0020] The inventors have discovered to date that artificial genes in which CD44 (particularly the extracellular portion), Notch (particularly the core region), and HIF-3α4 are fused can exhibit cancer-suppressive effects (Patent Document 1). The inventors have made further improvements, and the primary objective is to provide a cancer gene therapy technology with enhanced cancer-suppressive effects.

[0021] Solution for solving the problem

[0022] The inventors have discovered that the above-mentioned objective can be achieved using an oncolytic adenovirus vector containing nucleic acids, wherein the nucleic acids comprise an artificial gene fused with the extracellular portion of CD44, the Notch core region, and HIF-3α4, as well as E1A and E1B genes under the control of the COX-2 promoter, wherein the artificial gene, E1A gene, and E1B gene are arranged in the same direction. Further improvements were made by the inventors, leading to the completion of this disclosure.

[0023] This disclosure includes, for example, the subjects described in the following.

[0024] Item 1.

[0025] An oncolytic adenovirus vector containing nucleic acids, said nucleic acids comprising:

[0026] A base sequence (I) having a structure in which the following base sequences are linked in the order (A)-(B)-(C):

[0027] (A) Base sequence encoding a protein with extracellular function of CD44.

[0028] (B) The base sequence encoding a protein that functions in the Notch core region, and

[0029] (C) The base sequence encoding a protein with HIF-3α4 function; and

[0030] The base sequence (II) contains:

[0031] (D) Base sequence containing the COX-2 promoter;

[0032] (E) The base sequence encoding the E1A protein, controlled by the COX-2 promoter; and

[0033] (F) The base sequence encoding the E1B protein, which is placed under the control of the COX-2 promoter.

[0034] The base sequence (I) and the base sequence (II) are aligned in the same direction.

[0035] Item 2.

[0036] An oncolytic adenovirus vector containing nucleic acids, said nucleic acids comprising:

[0037] A base sequence (I) having a structure in which base sequences (A), (B), and (C) are linked in the order (A)-(B)-(C); and

[0038] The base sequence (II) contains:

[0039] (D) Base sequence containing the COX-2 promoter;

[0040] (E) The base sequence encoding the E1A protein, controlled by the COX-2 promoter; and

[0041] (F) The base sequence encoding the E1B protein, which is placed under the control of the COX-2 promoter.

[0042] The base sequence (I) and the base sequence (II) are aligned in the same direction.

[0043] The base sequence (A) is:

[0044] (a-1): A base sequence consisting of the base sequence of SEQ ID NO: 1, or

[0045] (a-2): A base sequence consisting of more than 85% sequence identity with (a-1) and encoding a protein that can bind to hyaluronic acid.

[0046] The base sequence (B) is:

[0047] (b-1): A base sequence consisting of the base sequence of SEQ ID NO: 2, or

[0048] (b-2): A base sequence consisting of more than 85% sequence identity with (b-1) and encoding a protein that can be cleaved by proteases.

[0049] The base sequence (C) is:

[0050] (c-1): A base sequence consisting of the base sequence of SEQ ID NO: 3, or

[0051] (c-2): A base sequence consisting of more than 85% sequence identity with (c-1) and encoding a protein that can bind to HIF-1α.

[0052] The base sequence (D) is:

[0053] (d-1): A base sequence consisting of the base sequence of SEQ ID NO: 4, or

[0054] (d-2): A sequence of bases that has more than 85% sequence identity with (d-1) and encodes the promoter sequence that is induced to express COX-2 under conditions that induce COX-2 expression.

[0055] The base sequence (E) is:

[0056] (e-1): A base sequence consisting of the base sequence of SEQ ID NO: 5, or

[0057] (e-2): A base sequence consisting of more than 85% sequence identity with (e-1) and encoding a protein with E1A function.

[0058] The base sequence (F) is:

[0059] (f-1): A base sequence consisting of the base sequence of SEQ ID NO: 6, or

[0060] (f-2): A base sequence consisting of more than 85% sequence identity with the base sequence of (f-1) and encoding a protein with E1B function.

[0061] Item 3.

[0062] According to the oncolytic adenovirus vector described in item 1

[0063] in,

[0064] The base sequence (A) is:

[0065] (a-1): A base sequence consisting of the base sequence of SEQ ID NO: 1, or

[0066] (a-2): A base sequence consisting of more than 85% sequence identity with the base sequence of (a-1) and encoding a protein that can bind to hyaluronic acid;

[0067] The base sequence (B) is:

[0068] (b-1): A base sequence consisting of the base sequence of SEQ ID NO: 2, or

[0069] (b-2): A nucleic acid that is composed of a base sequence having more than 85% sequence identity with the base sequence of (b-1) and encodes a protein that can be cleaved by a protease in a nucleic acid having a structure in which (A), (B) and (C) are linked in this order.

[0070] The base sequence (C) is:

[0071] (c-1): A nucleic acid consisting of the base sequence of SEQ ID NO: 3, or

[0072] (c-2): A nucleic acid consisting of a base sequence that shares more than 85% sequence identity with (c-1) and encodes a protein that can bind to HIF-1α.

[0073] The base sequence (D) is:

[0074] (d-1): A base sequence consisting of the base sequence of SEQ ID NO: 4, or

[0075] (d-2): A sequence of bases that has more than 85% sequence identity with (d-1) and encodes the promoter sequence that is induced to express COX-2 under conditions that induce COX-2 expression.

[0076] The base sequence (E) is:

[0077] (e-1): A base sequence consisting of the base sequence of SEQ ID NO: 5, or

[0078] (e-2): A base sequence consisting of more than 85% sequence identity with (e-1) and encoding a protein with E1A function.

[0079] The base sequence (F) is:

[0080] (f-1): A base sequence consisting of the base sequence of SEQ ID NO: 6, or

[0081] (f-2): A base sequence consisting of more than 85% sequence identity with the base sequence of (f-1) and encoding a protein with E1B function.

[0082] Item 4.

[0083] An oncolytic adenovirus vector containing nucleic acids, said nucleic acids comprising:

[0084] The base sequence (III) is as follows:

[0085] (g-1) is a base sequence consisting of the base sequence of SEQ ID NO: 7.

[0086] (g-2) is a base sequence consisting of more than 85% sequence identity with the base sequence of SEQ ID NO: 7, and having a structure in which the portions encoding a protein that can bind to hyaluronic acid, a portion encoding a protein that can be cleaved by a protease, and a portion encoding a protein that can bind to HIF-1α are linked in this order, or

[0087] (g-3) is a base sequence consisting of more than 85% sequence identity with the base sequence of SEQ ID NO: 7, and encoding a protein with anticancer activity; and

[0088] The base sequence (II) contains:

[0089] (D) Base sequence containing the COX-2 promoter;

[0090] (E) The base sequence encoding the E1A protein, controlled by the COX-2 promoter; and

[0091] (F) The base sequence encoding the E1B protein, which is placed under the control of the COX-2 promoter.

[0092] The base sequence (III) and the base sequence (II) are aligned in the same direction.

[0093] The base sequence (D) is:

[0094] (d-1): A base sequence consisting of the base sequence of SEQ ID NO: 4, or

[0095] (d-2): A sequence of bases that has more than 85% sequence identity with (d-1) and encodes the promoter sequence that is induced to express COX-2 under conditions that induce COX-2 expression.

[0096] The base sequence (E) is:

[0097] (e-1): A base sequence consisting of the base sequence of SEQ ID NO: 5, or

[0098] (e-2): A base sequence consisting of more than 85% sequence identity with (e-1) and encoding a protein with E1A function.

[0099] The base sequence (F) is:

[0100] (f-1): A base sequence consisting of the base sequence of SEQ ID NO: 6, or

[0101] (f-2): A base sequence consisting of more than 85% sequence identity with the base sequence of (f-1) and encoding a protein with E1B function.

[0102] Item 5.

[0103] The oncolytic adenovirus vector according to any one of items 1 to 4 further comprises (H) a base sequence encoding Ad5 / 35 chimeric fibrin.

[0104] Item 6.

[0105] According to the oncolytic adenovirus vector described in item 5

[0106] The base sequence (H) is described as follows:

[0107] (h-1): A base sequence consisting of the base sequence of SEQ ID NO: 8, or

[0108] (h-2): A base sequence consisting of more than 85% sequence identity with the base sequence of (h-1) and encoding a protein that can bind to CD46.

[0109] Item 7.

[0110] The oncolytic adenovirus vector according to any one of items 2 to 6, wherein the protease is ADAM protease or γ-secretase.

[0111] Item 8.

[0112] An anticancer composition comprising an oncolytic adenovirus vector according to any one of claims 1 to 7.

[0113] Item 9.

[0114] The anticancer composition according to item 8 is an injectable preparation.

[0115] Item 10.

[0116] The anticancer composition according to item 8 or 9 is used to treat COX-2 positive cancers.

[0117] Item 11.

[0118] The anticancer composition according to any one of claims 8 to 10 is used to treat at least one selected from the group consisting of bladder cancer, breast cancer, and prostate cancer.

[0119] Item 12.

[0120] A treatment for cancer comprising administering a therapeutically effective dose of the oncolytic adenovirus vector according to any one of claims 1 to 7 to a subject in need.

[0121] Item 13.

[0122] According to the method described in item 12, the application is an injection.

[0123] Item 14.

[0124] According to the method described in item 12 or 13, the cancer is a COX-2 positive cancer.

[0125] Item 15.

[0126] The method according to any one of items 12 to 14, wherein the cancer is at least one selected from the group consisting of bladder cancer, breast cancer, and prostate cancer.

[0127] Item 16.

[0128] The oncolytic adenovirus vector according to any one of items 1 to 7 is used for the treatment of cancer.

[0129] Item 17.

[0130] According to item 16, the oncolytic adenovirus vector is administered by injection.

[0131] Item 18.

[0132] According to item 16 or 17, the oncolytic adenovirus vector, wherein the cancer is a COX-2 positive cancer.

[0133] Item 19.

[0134] The oncolytic adenovirus vector according to any one of items 16 to 18, wherein the cancer is selected from at least one of the group consisting of bladder cancer, breast cancer, and prostate cancer.

[0135] Item 20.

[0136] Application of the oncolytic adenovirus vector according to any one of items 1 to 7 in the manufacture of cancer therapeutic agents.

[0137] Item 21.

[0138] According to the application described in item 20, the therapeutic agent is an injectable agent.

[0139] Item 22.

[0140] According to the application described in item 20 or 21, the cancer is a COX-2 positive cancer.

[0141] Item 23.

[0142] According to any one of items 20 to 22, the cancer is at least one selected from the group consisting of bladder cancer, breast cancer, and prostate cancer.

[0143] The effects of the invention

[0144] This disclosure provides a novel gene therapy agent for cancer that has a very high cancer-suppressing effect. Attached Figure Description

[0145] Figure 1 This is a schematic diagram of CRAd-synNotch (forward) constructed as an adenovirus vector in an embodiment of this disclosure.

[0146] Figure 2 yes Figure 2 The diagram shown is an excerpt including the CD44 / Notch / HIF-3α4 fusion gene, the Ad5 / 35 chimeric fibrous gene, the COX-2 promoter region, the E1A gene, and the E1B gene.

[0147] Figure 3 This is a schematic diagram of CRAd-GFP constructed as an adenovirus vector in an embodiment of this disclosure.

[0148] Figure 4 The results of the expression analysis of CD46, CAR and CD44 in BT474 cells in Experiment 2.1.1 are shown (n=3, mean ± SE bars, ** p<0.01).

[0149] Figure 5 The results of the expression analysis of CD46, CAR and CD44 in T24 cells in Experiment 2.1.2 are shown (n=3, mean ± SE bars, ** p<0.01).

[0150] Figure 6 The results of the expression analysis of CD46, CAR and CD44 in MDA-MB-231 cells in Experiment 2.1.3 are shown (n=3, mean ± SE bars, ** p<0.01).

[0151] Figure 7 The results of the expression analysis of CD46, CAR and CD44 in DU145 cells in Experiment 2.1.4 are shown (n=3, mean ± SE bars, ** p<0.01).

[0152] Figure 8 The results of quantitative analysis of COX-2 gene expression in T24, MDA-MB-231 and DU145 cells in Experiment 2.2 are shown (n=3, mean ± SE bars, ** p<0.01).

[0153] Figure 9The results show the quantitative expression of HAS-1, HAS-2 and HAS-3 genes in T24 cells in Experiment 2.3.1 (n=3, mean ± SE bars, ** p<0.01, * p<0.05).

[0154] Figure 10 The results show the quantitative expression of HAS-1, HAS-2 and HAS-3 genes in MDA-MB-231 cells in Experiment Example 2.3.2 (n=3, mean ± SE bars, ** p<0.01).

[0155] Figure 11 The results show the quantitative expression of HAS-1, HAS-2 and HAS-3 genes in DU145 cells in Experiment 2.3.3 (n=3, mean ± SE bars, ** p<0.01).

[0156] Figure 12 The results of CD44 expression analysis performed by Western blotting in Experiment 2.5.1 are shown.

[0157] Figure 13 The results of HIF-3α4 gene expression quantification by real-time RT-PCR in Experiment 2.5.2 are shown (n=3, mean ± SE bars, ** p<0.01).

[0158] Figure 14 The results of CD44 expression analysis by Western blot are shown in CRAd-synNotch (forward) clone 1 in Experimental Example 2.6.1.1.

[0159] Figure 15 The results of CD44 expression analysis by Western blot are shown in CRAd-synNotch (forward) clones 2 and 3 in Experimental Example 2.6.1.2.

[0160] Figure 16 The results of quantification of CD44 and HIF-3α4 gene expression by real-time RT-PCR in Experiment 2.6.2 are shown (n=3, mean ± SE bars, ** p<0.01).

[0161] Figure 17 The results of CD44 expression analysis by Western blot were shown in Experimental Example 2.7.1 in MDA-MB-231 and T24 cells infected with CRAd-synNotch (forward) clone 3 after mass culture.

[0162] Figure 18The results of quantification of CD44 and HIF-3α4 gene expression by real-time RT-PCR in T24 cells infected with CRAd-synNotch (forward) clone 3 after mass culture in Experiment 2.7.2 are shown (n=3, mean ± SE bars, ** p<0.01).

[0163] Figure 19 The results of evaluating the proliferation capacity of the adenovirus vector in Experiment 2.8 are shown (n=3, mean ± SE bars, ** p<0.01). The vertical axis represents the viral copy number (real number). A: wtAd, B: ADX730, C: CRAd-GFP, and D: CRAd-synNotch.

[0164] Figure 20 The results of the evaluation of the inhibitory effect of CRAd-synNotch on the proliferation of BT474 cells in Experiment 2.9.1 are shown (n=3, mean ± SE bars). The vertical axis represents the absorbance measured at a wavelength of 492 nm.

[0165] Figure 21 The results of the evaluation of the inhibitory effect of CRAd-synNotch on T24 cell proliferation in Experiment 2.9.2 are shown (n=3, mean ± SE bars, ** p<0.01). The vertical axis represents the absorbance measured at a wavelength of 492 nm.

[0166] Figure 22 The results of the evaluation of the inhibitory effect of CRAd-synNotch on the proliferation of MDA-MB-231 cells in Experiment 2.9.3 are shown. The vertical axis represents the absorbance measured at a wavelength of 492 nm.

[0167] Figure 23 The results of the evaluation of the inhibitory effect of CRAd-synNotch on the proliferation of DU145 cells in Experiment 2.9.4 are shown (n=3, mean ± SE bars, ** p<0.01, * p<0.05). The vertical axis represents the absorbance measured at a wavelength of 492 nm.

[0168] Figure 24 The results of the evaluation of the VGEF gene suppression effect of CRAd-synNotch in Experiment 2.10.1 are shown (n=3, mean ± SE bars, ** p<0.01).

[0169] Figure 25 The results of the evaluation of the SOX-2 gene suppression effect of CRAd-synNotch in Experiment 2.10.2 are shown (n=3, mean ± SE bars, ** p<0.01).

[0170] Figure 26The results of the evaluation of the CCL2 gene inhibition effect of CRAd-synNotch in Experiment 2.10.3 are shown (n=3, mean ± SE bars, ** p<0.01).

[0171] Figure 27 The results of the in vivo evaluation of the efficacy of CRAd-synNotch in bladder cancer treatment in Case 2.11 are shown. Figure 27 In the figure, the vertical axis represents tumor volume, and the horizontal axis represents the number of days since the start of administration of PBS, ADX730, CRAd-GFP, or CRAd-synNotch (n=5, mean ± SE bars, ** p<0.01).

[0172] Figure 28 The results of the in vivo evaluation of the efficacy of CRAd-synNotch in bladder cancer treatment in Case 2.11 are shown. Figure 28 In the graph, the vertical axis represents survival rate, and the horizontal axis represents the number of days since the application of PBS, ADX730, CRAd-GFP, or CRAd-synNotch (n=5).

[0173] Figure 29 The base sequence of SEQ ID NO: 1 is shown.

[0174] Figure 30 The base sequence of SEQ ID NO: 2 is shown.

[0175] Figure 31 The base sequence of SEQ ID NO: 3 is shown.

[0176] Figure 32 The base sequence of SEQ ID NO: 4 is shown.

[0177] Figure 33 The base sequence of SEQ ID NO: 5 is shown.

[0178] Figure 34 The base sequence of SEQ ID NO: 6 is shown.

[0179] Figure 35 The base sequence of SEQ ID NO: 7 is shown.

[0180] Figure 36 The base sequence of SEQ ID NO: 8 is shown.

[0181] Figure 37 The base sequence of SEQ ID NO: 9 is shown.

[0182] Figure 38The amino acid sequences of SEQ ID NO: 10 to 12 are shown.

[0183] Figure 39 The amino acid sequence of SEQ ID NO: 13 is shown.

[0184] Figure 40 The amino acid sequence of SEQ ID NO: 14 is shown.

[0185] Figure 41 The amino acid sequence of SEQ ID NO: 15 is shown.

[0186] Figure 42 The results of Western blotting using CD44 antibody as primary antibody (left) and HIF3α4 antibody as primary antibody (right) are shown after T24 cells were infected with CRAd-synNotch with MOI 50 and cultured for 24 or 72 hours using the method described in item 1.7.2 of Experiment 2.12.

[0187] Figure 43 The results of Western blotting using CD44 antibody as primary antibody (left) and HIF3α4 antibody as primary antibody (right) are shown after DU145 cells were infected with CRAd-synNotch with an MOI of 25 or 50 and cultured for 24, 48 or 96 hours using the method described in item 1.7.2 of Experiment 2.12.

[0188] Figure 44 The results of the analysis of expression levels of Cortactin, OCT4 and Nanog in T24 cells in Experiment 2.13 are shown (n=3, mean ± SE bars, ** p<0.01).

[0189] Figure 45 The results of the analysis of expression levels of PHD3, GLUT1 and CyclinG2 in T24 cells in Experiment 2.13 are shown (n=3, mean ± SE bars, ** p<0.01, * p<0.05).

[0190] Figure 46 The results of the analysis of expression levels of Cortactin, OCT4, Nanog and SOX-2 in DU145 cells in Experiment 2.13 are shown (n=3, mean ± SE bars, ** p<0.01).

[0191] Figure 47The results of the analysis of expression levels of VEGF, GLUT1, Cyclin G2 and PHD3 in DU145 cells in Experiment 2.13 are shown (n=3, mean ± SE bars, ** p<0.01, * p<0.05).

[0192] Figure 48 The results of immunohistochemical staining of CD44 in the T24 mouse model from Experiment 2.14 are shown. Scale bar = 100 μm. Detailed Implementation

[0193] The embodiments included in this disclosure are described in more detail below. This disclosure preferably includes, for example, oncolytic adenovirus vectors incorporating specific nucleic acids, anticancer compositions comprising the oncolytic adenovirus vector, and methods of treating cancer using the composition; however, this disclosure is not limited thereto. This disclosure includes everything disclosed herein and that may be recognized by those skilled in the art.

[0194] The oncolytic adenovirus vector disclosed herein is preferably an oncolytic adenovirus vector containing nucleic acid, wherein the nucleic acid comprises:

[0195] A base sequence (I) having a structure in which the following base sequences are linked in the order (A)-(B)-(C):

[0196] (A) Base sequence encoding a protein with extracellular function of CD44.

[0197] (B) The base sequence encoding a protein that functions in the Notch core region, and

[0198] (C) The base sequence encoding a protein with HIF-3α4 function; and

[0199] The base sequence (II) contains:

[0200] (D) Base sequence containing the COX-2 promoter;

[0201] (E) The base sequence encoding the E1A protein, controlled by the COX-2 promoter; and

[0202] (F) The base sequence encoding the E1B protein, which is placed under the control of the COX-2 promoter.

[0203] The base sequence (I) and the base sequence (II) are aligned in the same direction.

[0204] The oncolytic adenovirus vector is sometimes referred to as "the oncolytic adenovirus vector of this disclosure". The nucleic acid is sometimes referred to as "the nucleic acid of this disclosure".

[0205] 1. Oncolytic adenovirus vector

[0206] Oncolytic viruses are conditionally proliferating viruses that can specifically multiply within target cancer cells. Oncolytic viruses typically cannot infect normal cells. They attach to specific receptors highly expressed on cancer cells and infect them. Once absorbed into cancer cells, the oncolytic virus replicates intracellularly and produces viral proteins. During this process, the host cancer cells are lysed and killed. The oncolytic virus, which has then multiplied intracellularly, is released from the dead cancer cells into the tumor microenvironment. Oncolytic adenovirus is a representative type of oncolytic virus.

[0207] The oncolytic adenovirus vector used in this disclosure is not particularly limited, as long as the adenovirus derived from the vector can infect and proliferate within cancer cells. However, the adenovirus is preferably an oncolytic adenovirus vector that infects and proliferates within cancer cells expressing the CD46 receptor. Examples of such oncolytic adenovirus vectors include oncolytic adenovirus vectors with human adenovirus type 35 as a backbone. More specific examples include oncolytic adenovirus vectors derived from pAd1129-06 (OD 260 Inc.).

[0208] In a preferred embodiment of the oncolytic adenovirus vector disclosed herein, the nucleic acid of this disclosure is contained in an oncolytic adenovirus vector having human adenovirus type 35 as its backbone. In a more preferred embodiment of the oncolytic adenovirus vector of this disclosure, the nucleic acid of this disclosure is contained in an oncolytic adenovirus vector derived from pAd1129-06.

[0209] The oncolytic adenovirus vector disclosed herein can be manufactured by known methods or methods readily conceived from known methods, such as genetic engineering techniques like nucleic acid synthesis, PCR, restriction enzyme digestion, DNA ligation, and in vitro transcription.

[0210] 2. The nucleic acid disclosed herein

[0211] As described above, the nucleic acid disclosed herein is an oncolytic adenovirus vector containing nucleic acid, said nucleic acid comprising:

[0212] A base sequence (I) having a structure in which the following base sequences are linked in the order (A)-(B)-(C):

[0213] (A) Base sequence encoding a protein with extracellular function of CD44.

[0214] (B) The base sequence encoding a protein that functions in the Notch core region, and

[0215] (C) The base sequence encoding a protein with HIF-3α4 function; and

[0216] The base sequence (II) contains:

[0217] (D) Base sequence containing the Cox-2 promoter;

[0218] (E) The base sequence encoding the E1A protein, placed under the control of the Cox-2 promoter; and

[0219] (F) The base sequence encoding the E1B protein, placed under the control of the Cox-2 promoter.

[0220] The base sequence (I) and the base sequence (II) are aligned in the same direction.

[0221] In this disclosure, the term "nucleic acid" includes DNA, RNA, and PNA, etc. The nucleic acid in this disclosure is preferably DNA or RNA, and particularly preferably DNA. Nucleic acids can also be chemically modified as illustrated below. To prevent degradation by hydrolytic enzymes such as nucleases, for example, the phosphate residues (phosphate esters) of each nucleotide can be replaced with chemically modified phosphate residues such as thiophosphate (PS), methylphosphonate, or dithiophosphate. The hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide can be replaced with -OR (R represents, for example, CH3 (2'-O-Me), CH2CH2OCH3 (2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.). Furthermore, the base moiety (pyrimidine, purine) can be chemically modified. Specifically, such chemical modifications include, for example, introducing a methyl or cationic functional group at the 5-position of the pyrimidine base, and replacing the carbonyl group at the 2-position of the pyrimidine base with a thiocarbonyl group. Non-limiting examples also include nucleic acids in which the phosphate ester moiety and / or hydroxyl moiety are modified, for example, with biotin, amino, lower alkylamino, and acetyl groups. BNA (LNA) and the like may also be preferred, wherein the sugar moiety conformation is fixed in the N-type by bridging the 2' oxygen and 4' carbon of the sugar moiety of the nucleotide.

[0222] 2-1. Base sequence (A)

[0223] The base sequence (A) is the base sequence encoding a protein with extracellular function of CD44. CD44 is a receptor for hyaluronic acid and other substances, and binds to ligands (e.g., hyaluronic acid) to aggregate and transmit signals. This is known to induce intracellular phenomena, including activation of various kinases involved in cell proliferation and migration, such as c-Src, FAK, and MAPK. It is also known that following such signal transduction, the intracellular domain translocates to the nucleus after cleavage by proteases, and the cleaved extracellular domain is released as soluble CD44. Due to these properties, CD44 is highly expressed in many types of cancer cells, such as colon cancer, breast cancer, gastric cancer, pancreatic cancer, and prostate cancer, and has been studied as a marker for cancer stem cells.

[0224] In this disclosure, the extracellular function of CD44 refers to the receptor function of the ligand, and this function preferably includes, for example, hyaluronic acid binding capacity.

[0225] Proteins with extracellular CD44 function can be, for example, proteins comprising the entire extracellular portion of CD44; or, provided the receptor function of the ligand is not impaired, they can comprise the cell membrane portion of CD44, or a portion of the extracellular portion of CD44. Furthermore, one or more amino acids can be deleted, substituted, or added to such proteins as long as the function is maintained. The nucleic acid (A) is not particularly limited, as long as it is a nucleic acid encoding such a polypeptide. Preferred examples of proteins with extracellular CD44 function include proteins comprising the amino acid sequence of SEQ ID NO: 10. More preferred examples include proteins 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 encoding such a polypeptide.

[0226] More specifically, examples of base sequences (A) include (a-1) and (a-2) below.

[0227] (a-1): A base sequence consisting of the base sequence of SEQ ID NO: 1

[0228] (a-2): A base sequence consisting of more than 85% sequence identity with (a-1) and encoding a protein that can bind to hyaluronic acid.

[0229] In this disclosure, the term "identity" of a base sequence refers to the degree of matching between two or more comparable base sequences of two or more comparable nucleotide sequences. Therefore, the higher the degree of matching between two given base sequences, the higher the identity or similarity between these sequences. The level of sequence identity can be determined, for example, using FASTA as a sequence analysis tool with default parameters, or using the BLAST algorithm developed 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); and Karlin S., Altschul SF, “Applications and statistics for multiple high-scoring segments in molecular sequences.” Proc Natl Acad Sci USA, 90: 5873-7 (1993)). The specific techniques used in these analytical methods are known and can be found on the website of the National Center for Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / ). The term "identity" of amino acid sequences is also based on the definition above. The identity of amino acid sequences can be analyzed using a procedure called BLASTX.

[0230] A base sequence X' that is not 100% identical to the base sequence X can be, for example, a base sequence in which one or more bases in the base sequence X are substituted, deleted, added, or inserted (preferably substituted). The term "a plurality of" refers to 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 this range can be 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 or 100.

[0231] In this disclosure, when the base sequence X is a base sequence encoding a polypeptide, a base sequence X' that is not 100% identical to the base sequence X is preferably a base sequence X' that does not cause a frameshift relative 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' and P are identical to each other.

[0232] When the sequence identity of amino acid sequence P' with amino acid sequence P is not 100%, the sequence identity can 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 more than 99%, preferably more than 80%, more preferably more than 85%, even more preferably more than 90%, and particularly preferably more than 95%. Examples of such P' include amino acid sequences in which one or more amino acids in P are substituted, deleted, added, or inserted (preferably substituted, and more preferably conservatively substituted). The term "a plurality of" means, for example, 2 to 20, preferably 2 to 10, more preferably 2 to 5, even more preferably 2 or 3. The upper or lower limit of this range can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0233] The term "conservative substitution" refers to the substitution of an amino acid residue with another amino acid residue having a similar side chain. For example, substitution between amino acid residues with basic side chains, such as lysine, arginine, and histidine, is a conserved substitution. Furthermore, substitutions between the following amino acid residues are also conserved substitutions: amino acid residues with acidic side chains, such as aspartic acid and glutamic acid; amino acid residues with non-polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues with nonpolar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues with β-branched side chains, such as threonine, valine, and isoleucine; and amino acid residues with aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine.

[0234] As described above, the base sequence (a-2) is a base sequence that has at least 85% sequence identity with the base sequence of (a-1) and encodes a protein that can bind to hyaluronic acid. The sequence identity can 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.

[0235] In this disclosure, the ability of a protein to bind to other proteins means that the protein can specifically interact with other proteins. Whether a protein can bind to other proteins can be confirmed by methods known in the art. Examples of such methods include co-immunoprecipitation (Co-IP), pull-down assay, Western blotting, cross-linking, label transfer assays, interaction mapping, surface plasmon resonance (SPR), and fluorescence resonance energy transfer (FRET). Furthermore, the ability of a protein to bind to other proteins can be predicted through computer simulations based on the amino acid sequences of the protein and other proteins.

[0236] 2-2. Base sequence (B)

[0237] The base sequence (B) is the base sequence encoding a protein that functions with the Notch core region. Notch is a receptor expressed on the cell surface, and the Notch signaling system is one of the major signaling systems responsible for intercellular signal transduction. In many cases, the mechanism of intercellular signal transduction involves the signaling cell producing and releasing a soluble ligand, which binds to a receptor on the surface of the signal receiving cell. Furthermore, intracellular signaling pathways, including downstream phosphorylation cascades, are activated on the receiving cell side, and the activity of specific transcription factors changes, thereby regulating gene expression. On the other hand, the Notch signaling system is characterized by signal transduction through direct interactions between adjacent cells. Notch, acting as a receptor, binds to the ligand Delta or Serrate (Jagged in mammals) on the cell surface and undergoes a further conformational change due to applied physical forces, resulting in the dissociation of the Notch intracellular domain by protein-cleaving enzymes (proteases) such as ADAM or γ-secretase. As described above, the intracellular domain of Notch, released from the cell membrane, translocates to the nucleus and regulates transcription of target genes by interacting with transcription factors and coactivators upstream of them.

[0238] In this disclosure, the Notch core region is a region containing sites that are cleaved by the action of a protease, and the Notch core region functions to be cleaved (preferably selectively separated) by a protease capable of cleaving the Notch core region.

[0239] Proteins having Notch core region function can be, for example, proteins containing only a portion of the Notch core region necessary for protease cleavage; or, provided they are cleaved by a protease, proteins containing the portion necessary for protease cleavage and one or more amino acids (e.g., 1 to 30, 1 to 20, 1 to 10, or 1, 2, 3, 4, or 5) before and after the portion necessary for protease cleavage. Furthermore, provided they are cleaved by a protease, one or more amino acids (e.g., 1 to 30, 1 to 20, 1 to 10, or 1, 2, 3, 4, or 5) may be omitted, substituted, or added to such proteins. Preferred examples of proteins having Notch core region function include proteins containing the amino acid sequence of SEQ ID NO: 11. More preferred examples include proteins consisting of the amino acid sequence of SEQ ID NO: 11. The base sequence (B) is not limited, as long as it is a base sequence encoding such a protein.

[0240] As described above, preferred examples of proteases include ADAM proteases and γ-secretases. More preferably, the base sequence encoding a polypeptide cleaved by one or both of these proteases is preferred. ADAM proteases are proteases belonging to the detegrin metalloproteinase family.

[0241] Whether a protein can be cleaved by a protease can be confirmed by treating the protein with the protease and then performing electrophoresis (e.g., SDS-PAGE).

[0242] More specifically, examples of base sequences (B) include (b-1) and (b-2) below.

[0243] (b-1): A base sequence consisting of the base sequence of SEQ ID NO: 2

[0244] (b-2): A sequence of bases that shares more than 85% sequence identity with (b-1) and encodes a protein that can be cleaved by proteases.

[0245] (b-2) is more preferably (b-2'), which is a base sequence consisting of a base sequence having more than 85% sequence identity with the base sequence of (b-1) and encoding a protein encoded by a base sequence having a structure in which (A), (B) and (C) are connected in this order, wherein when the ligand binds to the protein encoded by the base sequence (A), the protein can be cleaved by intracellular proteases.

[0246] The sequence identity can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more.

[0247] 2-3. Base sequence (C)

[0248] The base sequence (C) is the base sequence encoding a protein with HIF-3α4 function. Hypoxia-inducible factor (HIF) is a transcription factor activated in response to intracellular hypoxia and is a heterodimer composed of HIF-1α and HIF-1β. HIF-1α has been shown to be inhibited not only by PHD-mediated degradation under normoxic conditions but also by a transcription factor called inhibitory PAS domain protein (IPAS), found in mice. IPAS has been identified as a splice variant of HIF-3α, one of the HIFs. Although IPAS itself is not transcriptionally active, its interaction with HIF-1α prevents binding to DNA and inhibits HIF-1α function. In humans, HIF-3α4, identified as a splice variant of HIF-3α, has shown similar function to IPAS.

[0249] In this disclosure, the HIF-3α4 function is the function that can inhibit HIF-1α. More specifically, it is the function that interacts with (binds to) HIF-1α.

[0250] Proteins with HIF-3α4 function can be, for example, HIF-3α4 itself, or, provided they can inhibit HIF-1α, proteins with one or more amino acids (e.g., 1 to 30, 1 to 20, 1 to 10, or 1, 2, 3, 4, or 5) further added to HIF-3α4. Furthermore, as long as they can inhibit HIF-1α, one or more amino acids (e.g., 1 to 30, 1 to 20, 1 to 10, or 1, 2, 3, 4, or 5) may be deleted, substituted, or added to such proteins. For example, proteins with HIF-3α4 function are preferably proteins containing the amino acid sequence of SEQ ID NO: 12, and more preferably proteins composed 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 encoding such a protein.

[0251] More specifically, examples of base sequences (C) include (c-1) and (c-2) below.

[0252] (c-1): A base sequence consisting of the base sequence of SEQ ID NO: 3

[0253] (c-2): A base sequence consisting of more than 85% sequence identity with the base sequence of (c-1) and encoding a protein that can bind to HIF-1α.

[0254] The sequence identity can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more.

[0255] 2-4. Base sequence (I)

[0256] The base sequence (I) is a base sequence having a structure in which base sequences (A), (B), and (C) are linked in the order (A)-(B)-(C). In the base sequence (I), the base sequence (A) can be positioned on the 3' side or the 5' side, with the 5' side being preferred. The base sequences (A), (B), and (C) can be directly linked or linked via one or more linkers, with direct linking being preferred. Linkers are not particularly limited, provided they do not impair the effects of this disclosure. For example, linkers consisting of one or two or more bases can be used. When the base sequence (I) contains one or more linkers, the base length of each linker can be from 1 to 1000 bp or from 1 to 500 bp, preferably from 1 to 100 bp or from 1 to 50 bp, and particularly preferably from 1 to 10 bp. The upper or lower limit of this range can be 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, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 bp.

[0257] The base length of the base sequence (I) can be, for example, less than 8000 bp, preferably less than 7500, 7000, 6500 or 6000 bp, more preferably less than 5500, 5000 or 4500 bp, and particularly preferably less than 4000, 3500 or 3100 bp.

[0258] Examples of preferred embodiments of the base sequence (I) include the following (g-1), (g-2), and (g-3). These (g-1), (g-2), and (g-3) are sometimes collectively referred to as "base sequence (III)".

[0259] (g-1) A base sequence consisting of the base sequence of SEQ ID NO: 7.

[0260] (g-2) is a base sequence that has more than 85% sequence identity with the base sequence of SEQ ID NO: 7, and has a structure in which the portions encoding a protein that can bind to hyaluronic acid, a portion encoding a protein that can be cleaved by a protease, and a portion encoding a protein that can bind to HIF-1α are linked in this order.

[0261] (g-3) is a base sequence consisting of more than 85% sequence identity with the base sequence of SEQ ID NO: 7, and encoding a protein with anticancer activity.

[0262] The sequence identity can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more.

[0263] Preferred examples of proteins encoded by the base sequence (I) include proteins comprising the amino acid sequence of SEQ ID NO: 13, and more preferred examples include proteins comprising the amino acid sequence of SEQ ID NO: 13.

[0264] In this disclosure, the term "anticancer effect" includes the effects of inhibiting the proliferation of cancer cells, killing cancer cells, reducing the number of cancer cells, and reducing tumor volume.

[0265] 2-5. Base sequence (D)

[0266] The base sequence (D) is the base sequence containing the cyclooxygenase-2 (COX-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; however, its expression is known to increase in inflammatory states. Furthermore, COX-2 expression is known to increase in many cancer cells. In other words, a gene or protein encoded by a base sequence controlled by the COX-2 promoter can exhibit increased expression in cells under inflammatory conditions or in cancer cells.

[0267] A promoter typically includes a transcription start site, its upstream sequence (5' side), and optionally its downstream sequence (3' side). When the transcription start site of the COX-2 gene in humans is defined as +1, the downstream (3' side) as a positive value, and the upstream (5' side) as a 0 or negative value, examples of COX-2 promoters include any DNA region containing the transcription start site within a DNA region between -10000 and +500, preferably -5000 and +200, more preferably -2000 and +150. When multiple transcription start sites are present, the site with the highest transcription output can be selected. The base sequence (D) has a base length of, for example, 500 to 10000 bp, preferably 8000 to 5000 bp, more preferably 1000 to 3000 bp.

[0268] The COX-2 promoter contained in the base sequence (D) can have a base sequence mutation (e.g., substitution, deletion, insertion, and addition) relative to the endogenous COX-2 promoter of an organism (preferably human), as long as the gene or protein controlled by the COX-2 promoter can exhibit increased expression in cells under inflammatory conditions or in cancer cells. In this case, the COX-2 promoter contained in the base sequence (D) has, for example, at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, still even more preferably at least 97%, and particularly preferably at least 99% identity with the corresponding base sequence of the endogenous promoter. The mutation site is preferably a site other than, for example, known expression regulatory elements (e.g., inflammatory response regions, basic transcription factor binding regions, various activator binding regions, etc.).

[0269] More specifically, examples of base sequences (D) include (d-1) and (d-2) below.

[0270] (d-1): A base sequence consisting of the base sequence of SEQ ID NO: 4.

[0271] (d-2): A sequence of bases that shares more than 85% sequence identity with (d-1) and encodes the promoter that is induced to express COX-2 under conditions that induce COX-2 expression.

[0272] The term "conditions that induce COX-2 expression" refers to conditions, for example, in cells under inflammatory conditions or in cancer cells. The phrase "inducing expression under conditions that induce COX-2 expression" means that the expression level of a gene or protein under the control of a promoter is increased in cells under inflammatory conditions or in cancer cells compared to the expression level in normal cells not under inflammatory conditions.

[0273] In this disclosure, the term "expression level" includes both the amount of transcription product and the amount of translation product. Expression levels can be analyzed using conventionally known methods or methods readily conceived from such known methods. Examples of such methods include quantitative reverse transcription PCR (RT-qPCR), RNA-seq, Western blotting, and immunohistochemical staining.

[0274] The sequence identity can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more.

[0275] 2-6. Base sequence (E)

[0276] The base sequence (E) is the base sequence encoding the E1A protein and is under the control of the COX-2 promoter. Therefore, the base sequence (E) is positioned on the 3' side of the base sequence (D). The base sequence (E) can be directly attached to the 3' end of the base sequence (D), or other base sequences (e.g., adapters, base sequence (F), etc.) can be present between the base sequences (E) and (D). In the latter case, there is no restriction on the base length of the sequence between the base sequences (E) and (D), as long as the base sequence (E) is under the control of the COX-2 promoter.

[0277] The E1A protein, together with the E1B protein described below, is an essential factor for adenovirus replication. More specifically, the E1A protein is a non-structural protein and has the function of initiating viral replication post-transcriptionally and post-translationally during the initial phase of viral replication.

[0278] In this disclosure, similar to the E1A protein, the term "E1A function" refers to the function of initiating viral replication post-transcriptionally and post-translationally during the initial phase of viral replication. A protein having an E1A function can be the E1A protein itself, or a protein similar to the E1A protein that has one or more (e.g., 1 to 30; 1 to 20; 1 to 10; or 1, 2, 3, 4, or 5) amino acids further added to the E1A protein, as long as it can initiate viral replication post-transcriptionally and post-translationally during the initial phase of viral replication. Furthermore, 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 can be deleted, substituted, or added to such proteins, as long as it can initiate viral replication post-transcriptionally and post-translationally during the initial phase of viral replication. The base sequence (E) is not particularly limited, as long as it is a base sequence encoding such a protein.

[0279] More specifically, examples of base sequences (E) include (e-1) and (e-2) below.

[0280] (e-1): A base sequence consisting of the base sequence of SEQ ID NO: 5.

[0281] (e-2): A base sequence consisting of more than 85% sequence identity with (e-1) and encoding a protein with E1A function.

[0282] The sequence identity can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, and particularly preferably 95% or more.

[0283] 2-7. Base sequence (F)

[0284] The base sequence (F) is the base sequence encoding the E1B protein and is under the control of the COX-2 promoter. Therefore, the base sequence (F) is positioned on the 3' side of the base sequence (D). The base sequence (F) can be directly attached to the 3' end of the base sequence (D), or other base sequences (e.g., adapters, base sequence (E), etc.) can be present between the base sequences (F) and (D). In the latter case, there is no restriction on the base length of the sequence between the base sequences (F) and (D), as long as the base sequence (F) is under the control of the COX-2 promoter.

[0285] The E1B protein, along with the E1A protein described above, is an essential factor for adenovirus replication. More specifically, the E1B protein is a protein necessary for the formation of viral replication compartments and controls other functions such as viral genome replication and transcription, as well as late-stage viral mRNA biosynthesis.

[0286] In this disclosure, similar to the E1B protein, the term "E1B function" refers to the function of controlling viral genome replication and transcription, as well as other functions such as late-stage viral mRNA biosynthesis, and participating in the formation of viral replication compartments.

[0287] Proteins with E1B function can be, for example, the E1B protein itself, or proteins similar to the E1B protein, which may have one or more amino acids (e.g., 1 to 30; 1 to 20; 1 to 10; or 1, 2, 3, 4, or 5) added to the E1B protein, as long as they can control viral genome replication and transcription, as well as other functions such as late-stage viral mRNA biosynthesis. Furthermore, proteins similar to the E1B protein may have one or more amino acids (e.g., 1 to 30; 1 to 20; 1 to 10; or 1, 2, 3, 4, or 5) deleted, substituted, or added, as long as they can control viral genome replication and transcription, as well as other functions such as late-stage viral mRNA biosynthesis. The base sequence (F) is not particularly limited, as long as it is a base sequence encoding this type of protein.

[0288] More specifically, examples of base sequences (F) include the following (f-1) and (f-2).

[0289] (f-1): A base sequence consisting of the base sequence of SEQ ID NO: 6

[0290] (f-2): A sequence of bases that shares more than 85% sequence identity with (f-1) and encodes a protein with E1B function.

[0291] The sequence identity can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more.

[0292] 2-8. Base sequence (II)

[0293] The base sequence (II) is a base sequence comprising base sequences (D), (E), and (F). As described above, base sequences (E) and (F) are under the control of the COX-2 promoter contained in base sequence (D). Therefore, base sequence (D) is configured upstream (5' side) of base sequences (E) and (F). Either base sequence (E) or base sequence (F) is configured on the 5' side. In other words, the base sequence (II) can have a structure in which the base sequences are linked from the 5' side in the order (D)-(E)-(F) or in which the base sequences are linked from the 5' side in the order (D)-(F)-(E). Base sequences (D), (E), and (F) can be directly linked or linked via one or more linkers. There are no particular limitations on linkers, provided that they do not impair the effects of this disclosure. For example, linkers consisting of one or more bases can be used. When the base sequence (II) contains more than one linker, the base length of each linker can 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 this range can be 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, 6 9, 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, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 bp.

[0294] In the nucleic acids disclosed herein, base sequence (I) or base sequence (III) is aligned with base sequence (II) in the same direction. In this disclosure, "aligned with each other" means that transcription of each base sequence proceeds in the same direction. In the case of double-stranded nucleic acids, this can also mean that the same nucleic acid strand is used as a template for transcription (i.e., the sense strand is identical). Either base sequence (I) or base sequence (III) can be positioned upstream of base sequence (II).

[0295] 2-9. Base sequence (H)

[0296] Although not specifically limited, in addition to the base sequences (A) to (F), the nucleic acid of this disclosure preferably also includes the base sequence (H), which is the base sequence encoding Ad5 / 35 chimeric fibrin. In this disclosure, the term "Ad5 / 35 chimeric fibrin" refers to a chimeric fibrin comprising a portion or all of fibrin derived from adenovirus serotype 35 (Ad35) and a portion or all of fibrin derived from adenovirus serotype 5 (Ad5). In this disclosure, Ad5 / 35 chimeric fibrin or the base sequence encoding Ad5 / 35 chimeric fibrin is sometimes simply described as "Ad5 / 35 fibrin".

[0297] In order to introduce nucleic acids into target cells using the Ad5 vector, which is widely used in gene therapy, an adenovirus receptor (CAR) must be expressed in the target cells. Therefore, in gene therapy using the Ad5 vector, sufficient therapeutic effect cannot be expected in target cells where CAR is not expressed or where CAR expression levels are low. Meanwhile, Ad35 fibrin can bind to CD46, a membrane protein distinct from CAR. When the nucleic acid of this disclosure contains a base sequence encoding an Ad5 / 35 chimeric fibrin, the nucleic acid can be introduced into cells where CAR is not expressed or where CAR expression levels are low via binding to CD46. In other words, from the viewpoint of expecting high anticancer activity, the nucleic acid of this disclosure preferably contains the base sequence (H), i.e., the base sequence encoding the Ad5 / 35 chimeric fibrin, regardless of the presence or absence of CAR expression in the target cells and the level of CAR expression.

[0298] As described above, in this disclosure, the term "Ad5 / 35 chimeric fibrin" refers to a chimeric fibrin comprising part or all of a fibrin derived from Ad35 and part or all of a fibrin derived from Ad5, and its specific structure is not particularly limited, as long as it can bind to CD46. For example, it can be a chimeric fibrin comprising a knob portion and a shaft portion of a fibrin derived from Ad35 and a tail portion of a fibrin derived from Ad5. Furthermore, one or more amino acids may be missing, substituted, or added to such proteins, as long as they can bind to CD46. Preferred examples of Ad5 / 35 chimeric fibrin include proteins comprising the amino acid sequence of SEQ ID NO: 14. More preferred examples include proteins consisting of the amino acid sequence of SEQ ID NO: 14. The base sequence (H) is not limited, as long as it is a base sequence encoding such a polypeptide.

[0299] Specifically, examples of base sequences (H) include (h-1) and (h-2) below.

[0300] (h-1): A base sequence consisting of the base sequence of SEQ ID NO: 8

[0301] (h-2): A sequence of bases that shares more than 85% sequence identity with (h-1) and encodes a protein that binds to CD46.

[0302] The sequence identity can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more.

[0303] 2-10. Others

[0304] In addition to the sequences described above, the nucleic acids disclosed herein may further include other sequences. Examples of other sequences include signal peptide coding sequences, promoter sequences, enhancer sequences, repressor sequences, insulator sequences, origin of replication sequences, and drug resistance gene coding sequences. The nucleic acids disclosed herein may be linear or circular nucleic acids.

[0305] The nucleic acids disclosed herein can be manufactured by known methods or methods readily conceived from known methods. For example, they can be manufactured using genetic engineering techniques such as PCR, restriction enzyme digestion, DNA ligation, and in vitro transcription. The nucleic acids disclosed herein can also be manufactured through chemical synthesis.

[0306] 3. Uses

[0307] The oncolytic adenovirus vector disclosed herein exhibits excellent anticancer activity. Therefore, the technology disclosed herein is applicable to, for example, the prevention, improvement, and treatment of cancer.

[0308] In this disclosure, the term "cancer prevention" includes preventing cancer by applying treatment to a subject before the subject is diagnosed with cancer, and preventing cancer metastasis by applying treatment to undetected tissue in a subject diagnosed with cancer. In this disclosure, the terms "cancer improvement" or "cancer treatment" include, when applied to a subject diagnosed with cancer, inhibiting cancer progression, preventing cancer progression, inhibiting cancer cell proliferation, killing cancer cells, reducing the number of cancer cells, reducing tumor volume, and preventing cancer metastasis. Furthermore, in this disclosure, the term "anti-cancer" includes cancer prevention and its effects, cancer improvement and its effects, and cancer treatment and its effects.

[0309] In this disclosure, the term "cancer" includes cancer, neoplasm, and malignant tumor. Examples of cancer include bladder cancer, breast cancer, prostate cancer, malignant melanoma, kidney cancer, mesothelioma, intraperitoneal disseminated carcinoma, 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, endometrial cancer, and cervical cancer. The techniques of this disclosure are not particularly limited to any particular type of cancer, but are particularly suitable for application to at least one selected from the group consisting of bladder cancer, breast cancer, and prostate cancer.

[0310] Examples of subjects to whom the techniques of this disclosure may be applied include humans and non-human mammals (e.g., rats, mice, rabbits, cattle, pigs, dogs, cats, sheep, and monkeys), with humans being preferred.

[0311] 4. Application Method

[0312] The application of the technology disclosed herein is not particularly limited, as long as the desired effect is achieved. For example, the oncolytic adenovirus vector or composition containing it disclosed herein can be administered directly to the lesion by injection, or by injection or infusion via intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, or intrapleural route, and can also be administered via perfusion through a catheter. Injection is particularly preferred.

[0313] There are no particular limitations on the frequency of application of the technology disclosed herein. For example, the technology can be applied once or more daily, once or more weekly, once or more monthly, or once or more annually. There are also no particular limitations on the application period of the technology disclosed herein, as long as the desired effects are achieved. Furthermore, there are no particular limitations on the dosage of the oncolytic adenovirus vector disclosed herein, as long as the desired effects are achieved. Those skilled in the art can appropriately adjust the application frequency, application period, and dosage according to the subject's condition and treatment process.

[0314] The techniques disclosed herein can optionally be combined with other pharmaceutical compositions and / or treatments applied to subjects suffering from cancer. When the techniques disclosed herein are combined with other pharmaceutical compositions and / or treatments, they can be applied to the subject simultaneously, or each can be applied separately at any given time.

[0315] In the technology disclosed herein, the oncolytic adenovirus vector can be mixed with pharmaceutically acceptable base agents, carriers, excipients, diluents, solubilizers, emulsifiers, preservatives, pH adjusters, adjuvants, chelating agents, etc. These components can be used alone or in combination of two or more.

[0316] Examples of preservatives include parabens such as methylparaben, ethylparaben, propylparaben, and butylparaben; sodium benzoate; phenoxyethanol; and alkyl diaminoethyl glycine hydrochloride. These preservatives can be used alone or in combination of two or more.

[0317] Examples of pH adjusters include citric acid, phosphoric acid, malic acid, pyrophosphate, lactic acid, tartaric acid, glycerophosphate, acetic acid, nitric acid, chemically acceptable salts of these acids, sodium hydroxide, and potassium hydroxide. These pH adjusters can be used alone or in combination of two or more, such that the pH of the composition containing the oncolytic adenovirus vector of this disclosure is in the range of 4 to 8, preferably 5 to 7.

[0318] There are no particular limitations on the preparation method of the composition containing the oncolytic adenovirus vector of this disclosure, as long as the effects of this disclosure are achieved. For example, the composition can be prepared according to methods known in the art. More specifically, for example, the composition can be prepared by mixing the oncolytic adenovirus vector of this disclosure and other components with sterile distilled water.

[0319] 5. Composition

[0320] This disclosure also includes anticancer compositions comprising the oncolytic adenovirus vector of this disclosure. Such anticancer compositions are sometimes referred to as "anticancer compositions of this disclosure". The matters described in sections "1. Oncolytic adenovirus vector" through "4. Application method" are incorporated into the anticancer compositions of this disclosure.

[0321] 6. Presumption Mechanism

[0322] Without being bound by theory, it is assumed that proteins encoded by base sequence (I) or base sequence (III) exhibit anticancer effects through the following mechanism of action. Fusion proteins encoded by base sequence (I) or base sequence (III) comprise (i) a protein moiety with extracellular CD44 function, (ii) a protein moiety with Notch core region function, and (iii) a protein moiety with HIF-3α4 function. Therefore, (i) can act as a decoy receptor, and the signal can be transmitted to (iii) via (ii). This becomes more effective when (i) is fused upstream of (ii). HIF-1α activated in tumors can be inhibited by fusing downstream of (ii) with (iii). It is hypothesized that this anticancer effect is achieved based on multiple mechanisms of action.

[0323] It is hypothesized that the base sequence (II) contributes to the effective anticancer effect of the oncolytic adenovirus vector of this disclosure through the following mechanism of action. As described above, genes or proteins encoded by base sequences controlled by the COX-2 promoter can exhibit increased expression in cells under inflammatory conditions or in cancer cells. Therefore, it is believed that E1A and E1B proteins controlled by the COX-2 promoter exhibit increased expression in cancer cells. The expression of E1A and E1B proteins leads to the replication and proliferation of the oncolytic adenovirus vector of this disclosure in cancer cells, resulting in cancer cell lysis and death. Furthermore, the oncolytic adenovirus vector of this disclosure, which has already proliferated within cancer cells, is released from the dead cancer cells and can infect other nearby cancer cells. Subsequently, the proliferation of the oncolytic adenovirus vector of this disclosure, the lysis and death of cancer cells, the release of the oncolytic adenovirus vector, and the infection of nearby cancer cells can be repeated.

[0324] Furthermore, as described above, it is presumed that the replication and proliferation of the oncolytic adenovirus vector of this disclosure within cancer cells increases the abundance of the protein encoded by the base sequence (I) or the base sequence (III) in a cancer cell-specific manner. In other words, in the technology of this disclosure, it is presumed that the incorporation of the base sequence (I) or the base sequence (III) into the oncolytic adenovirus vector leads to an interrelation between the anticancer effect of the oncolytic adenovirus vector and the anticancer effect of the fusion protein encoded by the base sequence (I) or the base sequence (III), thereby exerting a synergistically enhanced anticancer effect in a cancer cell-specific manner.

[0325] As is clearly evident from the above-described presumed mechanism, the technology of this disclosure is expected to exert excellent anti-cancer effects against COX-2 positive cancer cells. Therefore, the technology of this disclosure is particularly suitable for COX-2 positive cancer cells. In this disclosure, the term "COX-2 positive" means that the COX-2 expression level is increased in cells under inflammation or in cancer cells compared to the COX-2 expression level in normal cells not under inflammation. Whether a given cell is COX-2 positive can be analyzed by conventionally known methods or methods readily conceivable from such known methods. Examples of such methods include reverse transcription quantitative PCR (RT-qPCR), RNA-seq, Western blotting, and immunohistochemical staining.

[0326] Although the reason is unclear, as described in Example 2.5, when the CD44 / Notch / HIF-3α4 fusion gene and the E1A / E1B gene are reversed, no expression of the CD44 / Notch / HIF-3α4 fusion gene is detected in either transcription or translation. In contrast, as described in Examples 2.6 and 2.7, when the CD44 / Notch / HIF-3α4 fusion gene and the E1A / E1B gene are aligned, significant levels of expression of the CD44 / Notch / HIF-3α4 fusion gene are detected. Therefore, in the art of this disclosure, it is desirable for the base sequence (I) or base sequence (III) to be aligned with the base sequence (II).

[0327] In this specification, the term "comprising" includes both "consisting essentially of" and "consisting of". Furthermore, this disclosure includes all arbitrary combinations of the features described herein.

[0328] Furthermore, when specifying the main subject matter included in this disclosure, the various characteristics (properties, values, functions, etc.) described in the various embodiments of this disclosure described above can be combined in any way. In other words, this disclosure includes all main subject matter comprising all combinations of the composable characteristics described in this specification.

[0329] Example

[0330] The embodiments of this disclosure are described in more detail below while providing examples; however, the embodiments of this disclosure are not limited to the following embodiments.

[0331] 1. Experimental Methods

[0332] 1.1. Construction of CRAd-synNotch (forward / reverse)

[0333] In this embodiment, the designed base sequence having (A) encoding a protein with extracellular CD44 function, (B) encoding a protein with Notch core region function, and (C) encoding a protein with HIF-3α4 function linked in the order of (A)-(B)-(C) can be called a "CD44 / Notch / HIF-3α4 fusion gene". The CD44 / Notch / HIF-3α4 fusion gene is a preferred embodiment of base sequence (I) or base sequence (III) in the present disclosure.

[0334] In this embodiment, the recombinant oncolytic adenovirus vector in which the COX-2 promoter region is located upstream of the E1A and E1B genes and the CD44 / Notch / HIF-3α4 fusion gene is inserted can be referred to as "CRAd-synNotch (forward / reverse)". In CRAd-synNotch (forward), the CD44 / Notch / HIF-3α4 fusion gene is aligned in the same direction (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 aligned in reverse relative to the COX-2 promoter region, E1A gene, and E1B gene. The construction method of CRAd-synNotch (forward / reverse) is described below.

[0335] The plasmids pAd1127-04, pAd1128, pAd1129-06, and pAd1130-04 included in the AdenoQuick 2.0 Kit (OD260 Inc.) were used as the vector DNA in this example. The Cosmid Construction Kit-2 (OD260 Inc.) was used to construct the cosmid vector.

[0336] 1.1.1 Amplification and Clonal Enhancement of the COX-2 Promoter Region

[0337] Using 25 ng of pDrive-hCOX2 (InvivoGen) as a template, PCR was performed with KOD-Plus-Neo (ToyoboCo., Ltd.) 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. Furthermore, the base sequence of the amplified COX-2 promoter region (SEQ ID NO: 4) is shown in [Table data missing]. Figure 32 middle.

[0338] [Table 1]

[0339]

[0340] [Table 2]

[0341]

[0342] 5 μl of PCR product and 2 μl of Cloning Enhancer (TaKaRa) were reacted at 37 °C for 20 min, and then the enzyme was inactivated by heating at 80 °C for 15 min.

[0343] 1.1.2 Restriction enzyme treatment of pAd1127-04 plasmid

[0344] The pAd1127-04 plasmid contained in the AdenoQuick 2.0 Kit (OD260 Inc.) was digested with SalI (TaKaRa) and EcoRI (TaKaRa) restriction enzymes, and purified using a Wizard (trademark) SV Gel and PCR Clean-Up System (Promega Corporation) after agarose gel electrophoresis. The composition of the restriction enzyme-treated reaction solution is shown in the table below.

[0345] [Table 3]

[0346]

[0347] 1.1.3 Insertion of the COX-2 promoter region into the pAd1127-04 plasmid and into *E. coli* ( E. coli ) In Transformation

[0348] To ligate the COX-2 promoter region amplified in section 1.1.1 to pAd1127-04, an In-Fusion reaction was performed using the In-Fusion (trademark) HD ​​Cloning Kit (TaKaRa) (50°C, 15 min). The reaction solution was used to transform *E. coli* DH5α competent cells (TaKaRa). Transformation was performed according to the instructions in the kit manual.

[0349] Transformed E. coli DH5α competent cells were seeded on LB agar (Nacalai Tesque) supplemented with 25 μg / ml kanamycin and incubated overnight at 37°C. After incubation, colonies grown on LB agar were used as templates for PCR testing using the KOD-Plus-Neo insert fragment, and the success of the transformation was confirmed by agarose gel electrophoresis.

[0350] The colonies confirmed to have undergone successful transformation were subcultured in LB liquid medium supplemented with 25 μg / ml kanamycin, and plasmids were extracted from the culture using the PureYield Plasmid Midiprep System (Promega Corporation). The sequences in the extracted plasmids were confirmed using a DNA sequencer.

[0351] 1.1.4 Insertion of the CD44 / Notch / HIF-3α4 fusion gene into pAd1129-06 and into E. coli Transformation

[0352] The construction of the 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 (OD260 Inc.) were treated with EcoRI (TaKaRa) restriction enzymes, followed by agarose gel electrophoresis and purification. The pAd1129-06 plasmid is an oncolytic adenovirus vector. The composition of the restriction enzyme treatment reaction solution is shown in the table below.

[0353] [Table 4]

[0354]

[0355] [Table 5]

[0356]

[0357] Figure 35 The sequence of the CD44 / Notch / HIF-3α4 fusion gene is shown (SEQ ID NO: 7). Figures 29 to 31 The base sequences encoding proteins with extracellular CD44 function (SEQ ID NO: 1), proteins with Notch core region function (SEQ ID NO: 2), and proteins with HIF-3α4 function (SEQ ID NO: 3) contained in the CD44 / Notch / HIF-3α4 fusion gene are shown, respectively. Furthermore, Figure 39 The amino acid sequence of the protein encoded by the base sequence of SEQ ID NO: 7 is shown (SEQ ID NO: 13), and Figure 38 The amino acid sequences (SEQ ID NO: 10 to 12) of the proteins encoded by the base sequences of SEQ ID NO: 1 to 3 are shown.

[0358] The purified plasmid and Ligation Mix (TaKaRa) were mixed and ligation was performed (16°C, 30 min). The reaction mixture was used to transform *E. coli* DH5α competent cells (TaKaRa). Transformation was performed according to the kit manual.

[0359] Transformed E. coli DH5α competent cells were seeded on LB agar (Nacalai Tesque) supplemented with 100 μg / ml ampicillin and incubated overnight at 37°C. After incubation, colonies grown on LB agar were used as templates for PCR using KOD-Plus-Neo insert fragment detection, and the transformation was confirmed by agarose gel electrophoresis.

[0360] The colonies confirmed to have undergone successful transformation were subcultured in LB liquid medium supplemented with 100 μg / ml ampicillin, and plasmids were extracted from the culture using the PureYield Plasmid Midiprep System (Promega Corporation). The sequences in the extracted plasmids were confirmed using a DNA sequencer.

[0361] 1.1.5 Construction of recombinant clay vectors and transformation into Escherichia coli

[0362] The pAd1127-04 plasmid containing the COX-2 promoter region (from item 1.1.3), the pAd1129-06 plasmid containing the CD44 / Notch / HIF-3α4 fusion gene (from item 1.1.4), as well as pAd1128 (OD260 Inc.) and pAd1130-04 (OD260 Inc.), were digested with SfiI (New England Biolabs) restriction enzymes. After agarose gel electrophoresis of each reaction solution, the DNA fragments were purified.

[0363] The purified DNA fragment was mixed with T4 DNA ligase (TaKaRa) and ligated (16°C, overnight). 2.0 μl of the reaction solution and 5.0 μl of the λ-packaged extract contained in the Cosmid Construction Kit-2 were incubated at 30°C for 1 hour. After the reaction, 100 μl of SM medium and 5.0 μl of chloroform (NacalaiTesque) contained in the kit were added, the mixture was gently tapped, and then centrifuged (15000 rpm, 1 min). 20 μl of the centrifuged supernatant was mixed with 20 μl of *E. coli* TOP10 competent cells (Invitrogen) cultured in LB liquid medium supplemented with 0.2% maltose and 10 mM MgSO4 and incubated at 37°C for 30 minutes. After the reaction, the cells were inoculated onto LB agar medium (NacalaiTesque) supplemented with 50 μg / ml ampicillin and 25 μg / ml kanamycin and incubated overnight at 37°C. After culturing, colonies grown on LB agar were used as templates for colony PCR. Colonies carrying a granular vector with the CD44 / Notch / HIF-3α4 fusion gene linked in the same direction (forward) to the COX-2 promoter and colonies carrying a granular vector with the CD44 / Notch / HIF-3α4 fusion gene linked in the opposite direction 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 granular vector was extracted from the culture using the PureYield Plasmid Midiprep System (Promega Corporation).

[0364] In this step, four samples of a granular vector in which the CD44 / Notch / HIF-3α4 fusion gene is linked in the same direction (forward) to the COX-2 promoter were obtained (samples with single colonies derived from the above-mentioned LB agar medium containing ampicillin and kanamycin were counted as one sample). Additionally, one sample of a granular vector in which the CD44 / Notch / HIF-3α4 fusion gene is linked in the opposite direction to the COX-2 promoter was obtained.

[0365] 1.1.6 Construction of a conditionally proliferating recombinant adenovirus vector by transforming HEK293 cells with recombinant clomiphene

[0366] The recombinant coliform vector DNAs prepared in section 1.1.5 were digested using PacI (New England Biolabs) restriction enzyme. Following restriction enzyme treatment, nucleic acid purification was performed by phenol-chloroform extraction and ethanol precipitation, and the purified nucleic acids were dissolved in 30 μl of sterile pure water. 1 μl of the resulting solution was subjected to agarose gel electrophoresis to confirm digestion with PacI restriction enzyme.

[0367] 10 μg of PacI-digested granules were mixed with Lipofectamine 2000 (Invitrogen) and Opti-MEM I Reduced Serum Medium (Gibco) and then transfected with liposomes onto HEK293 cells (National Institutes of Biomedical Innovation, Health and Nutrition) cultured to confluence in 60 mm tissue culture dishes.

[0368] Following liposome transfection, cultured cells were collected and each cell clone was seeded onto 16 capped 96-well microplates (AGC TECHNO GLASS) containing type I collagen. At 4, 8, and 16 days post-seeding, 50 μl of DuPont modified Eagle medium (D-MEM; FUJIFILMWako Pure Chemical Corporation) containing 5% fetal bovine serum (FBS; Sigma Aldrich) was added to each well, and cells were cultured and observed daily until all cells exhibited complete cytopathic effects.

[0369] For a clone obtained using the granular vector obtained in section 1.1.5, in which the CD44 / Notch / HIF-3α4 fusion gene is reverse-linked relative to the COX-2 promoter, only one well from which cells exhibited complete cytopathic effect was collected into a 1.5 ml tube 22 days post-inoculation. This process was subsequently repeated 6 times with liquid nitrogen freezing and thawing at 37°C.

[0370] Using the method described above, each of the four granular vector samples obtained in section 1.1.5, in which the CD44 / Notch / HIF-3α4 fusion gene and the COX-2 promoter were linked in the same direction (forward direction), was transformed into HEK293 cells. Three of these samples confirmed cytopathic effects. At days 36, 38, and 39 post-inoculation, the three wells showing complete cytopathic effects were collected into 1.5 ml tubes (which may be referred to as "clones 1 to 3"). Subsequently, the freezing and thawing processes were repeated six times using liquid nitrogen and a 37°C incubator.

[0371] After the final freeze-thaw cycle, each clone was centrifuged (5000 rpm, 5 minutes, 4°C) to collect the supernatant. The supernatant was stored as the primary virus solution. The above method is a modified version of the method described in Kumikae AdenouirusuSakusei Manyuaru: Kanzencho Genomu Donyuho [Recombinant Adenovirus Production Manual: Full-Length Genome Introduction Method] (Genetic Engineering Department, Bioresource Center, RIKEN).

[0372] 1.1.7 Purification of high-titer recombinant adenovirus vector

[0373] HEK293 cells (National Institutes of Biomedical Innovation, Health and Nutrition) were cultured in 24-well microplates (with caps) containing type I collagen until 80% to 100% confluence. After culture, the medium was removed, and 0.1 ml of a mixture of DuPont modified Eagle medium (D-MEM; FUJIFILM Wako Pure Chemical Corporation) containing 5% FBS and 10 μl of the viral solution prepared in section 1.1.6 was added to each well. To infect HEK293 cells with the virus, the plates were gently agitated several times in an incubator (37°C, 5% CO2), and this operation was repeated four times every 15 minutes. One hour after infection, 0.4 ml of 5% FBS-E-MEM was added to each well, and the cells were cultured for 3 days. After culture, cells exhibiting complete cytopathic effects were collected along with the culture medium. For the collected cells, the same process as with the primary virus solution was repeated 6 times: freezing in liquid nitrogen and thawing at 37°C. After the final freeze-thaw cycle, the cells were centrifuged (5000 rpm, 5 minutes, 4°C), and the collected supernatant was stored as the secondary virus solution.

[0374] Next, HEK293 cells were cultured in 60 mm tissue culture dishes until 80-100% confluence, and 0.4 ml of 5% FBS-D-MEM and 100 μl of secondary virus solution were added to infect the cells with the virus. For infection, the plates were gently shaken several times in an incubator (37°C, 5% CO2), and this operation was repeated four times every 15 minutes. One hour after infection, 2.5 ml of 5% FBS-D-MEM was added, and the cells were cultured for 3 days. After culture, cells exhibiting complete cytopathic effect were confirmed, and the cells were collected along with the culture medium. For the collected cells, the same freezing and thawing process as with the primary virus solution was repeated 6 times using liquid nitrogen. After the final freeze-thaw cycle, the cells were centrifuged (3000 rpm, 10 min, 4°C), and the collected supernatant was stored as the tertiary virus solution.

[0375] 1.1.8 Large-scale culture and purification of recombinant adenovirus vector

[0376] Large-scale culture and purification of the recombinant adenovirus vector constructed in section 1.1.7 were carried out. The specific methods are described below.

[0377] HEK293 cells were placed in 10 Corning (trademark) 225cm 2 Cells were seeded in Corning flasks to 80-100% confluence. 5 ml of 5% FBS-D-MEM and 500 μl of the three-stage virus solution prepared in section 1.1.7 were added to HEK293 cells to infect them with the virus. For infection, each flask was gently shaken several times in an incubator (37°C, 5% CO2), and this operation was repeated four times every 15 minutes. One hour after infection, 30 ml of 5% FBS-D-MEM was added, and the cells were cultured for 2 days. After culture, once all cells showed complete cytopathic effects, the cells were collected along with the culture medium. After collection, the cells were centrifuged (3000 rpm, 10 min, 4°C), the supernatant was removed, and fresh 10% FBS-D-MEM was added to resuspend the cell pellet. The cells were then frozen in liquid nitrogen and thawed at 37°C, repeated six times.

[0378] After the final freeze-thaw cycle, the cells were centrifuged (3000 rpm, 10 min, 4 °C) and collected again. Only the supernatant was collected and purified by ultracentrifugation as described below.

[0379] Using a 5ml syringe with a needle, inject 3ml of sterile 2.2M CsCl solution into a Beckman Coulter tube from the bottom, then slowly add 3ml of sterile 4.0M CsCl solution from the bottom of the tube. Using the same 5ml syringe, gently layer 2.5ml of the supernatant from the centrifugation solution on top of the 2.2M CsCl solution. The composition of the solutions is shown in the table below.

[0380] [Table 6]

[0381]

[0382] [Table 7]

[0383]

[0384] [Table 8]

[0385]

[0386] Seal the tube using a Beckman Coulter cordless tube topper and ultracentrifuge (30,000 rpm, 4°C, 18 hours). After ultracentrifugation, insert a 23G needle attached to a 5ml syringe slightly below the observed middle layer (band) in the tube and collect 1.5ml of liquid to collect as much of the band as possible. Store the collected liquid in a 2ml tube.

[0387] 1.1.9 Titer Measurement of Recombinant Adenovirus Vector

[0388] The titer of the recombinant adenovirus vector constructed in section 1.1.8 is measured. The specific method is described below.

[0389] HEK293 cells were seeded in 12-well flat-bottom cell culture plates (Corning), and the viral solution, diluted 100-fold to 10 million-fold in increments from 100-fold to 10 million-fold, was added dropwise to each well, and the cells were cultured for 2 days. After culture, the entire culture medium was removed, the cells were lightly dried, and then 1 ml of methanol (FUJIFILM Wako Pure Chemical Corporation) was added dropwise at -20°C and incubated (-20°C, 10 min). After incubation, all methanol was removed, and the cells were washed three times with Dulbecco's PBS(-) "Nissui" (PBS; Nissui Pharmaceutical Co., Ltd.) containing 1% bovine serum albumin (BSA; Sigma Aldrich).

[0390] After washing, titers were measured 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 mixture was incubated with shaking (37°C, 1 h). After the reaction, the mixture was washed three times with PBS containing 1% BSA, and 0.5 ml of HRP-conjugated rat anti-mouse antibody diluted 500-fold with PBS containing 1% BSA was added dropwise, and the mixture was incubated with shaking (37°C, 1 h). After the reaction, the mixture was washed three times with PBS containing 1% BSA, and 0.5 ml of 10× DAB substrate diluted 10-fold with 1× stable peroxidase buffer was added dropwise, and the mixture was incubated (room temperature, 10 min). After the reaction, all 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 procedure was performed according to the method in the Adeno-X (trademark) Rapid Titration Kit (TaKaRa) manual.

[0391] 1.2 Construction of CRAd-GFP

[0392] As a negative control for the CRAd-synNotch (positive) constructed in section 1.1, a conditionally proliferating adenovirus vector was constructed by inserting the AcGFP1 gene (which may be simply referred to as "GFP" in this disclosure) instead of the CD44 / Notch / HIF-3α4 fusion gene of the CRAd-synNotch (positive). This adenovirus vector may be referred to as "CRAd-GFP". Figure 37 The base sequence of the GFP gene used in this embodiment is shown (SEQ ID NO: 9). Figure 41 The amino acid sequence of the protein encoded by the base sequence of SEQ ID NO: 9 is shown (SEQ ID NO: 15). The specific construction method of CRAd-GFP is described below.

[0393] The pAd1129-06 plasmid (OD260 Inc.) and pAcGFP1 vector (TaKaRa) were digested with SalI (TaKaRa) and EcoRI (TaKaRa) restriction enzymes, respectively, followed by agarose gel electrophoresis and purification. The composition of the restriction enzyme treatment reaction solution is shown in the table below.

[0394] [Table 9]

[0395]

[0396] [Table 10]

[0397]

[0398] The purified nucleic acid fragments were mixed with the Ligation Mix kit (TaKaRa) and a ligation reaction was performed (16°C, 30 min). The reaction mixture was then used to transform *E. coli* DH5α competent cells (TaKaRa). Transformation was performed according to the kit manual.

[0399] Transformed E. coli DH5α competent cells were seeded on LB agar (Nacalai Tesque) supplemented with 100 μg / ml ampicillin and incubated overnight at 37°C. After incubation, colonies grown on LB agar were used as templates for PCR using KOD-Plus-Neo insert fragment detection, and the transformation was confirmed by agarose gel electrophoresis.

[0400] The colonies confirmed to have undergone successful transformation were subcultured in LB liquid medium supplemented with 100 μg / ml ampicillin, and plasmids were extracted from the culture using the PureYield Plasmid Midiprep System (Promega Corporation). The sequences in the extracted plasmids were confirmed using a DNA sequencer. Primers used for sequencing are shown in the table below.

[0401] [Table 11]

[0402]

[0403] Except for item 1.1.4, proceed with the subsequent procedures in the same manner as in item 1.1 to prepare CRAd-GFP.

[0404] 1.3 Various adenovirus vectors

[0405] The adenovirus vectors used in this embodiment are as follows: CRAd-synNotch (forward) and CRAd-synNotch (reverse) constructed in item 1.1; CRAd-GFP constructed in item 1.2; the ADX730 non-replicating type 5 recombinant adenovirus vector described in Patent Document 1, wherein the CD44 / Notch / HIF-3α4 fusion gene is incorporated into the E1 region; wild-type type 5 adenovirus (wtAd), which exhibits COX-2-independent replication; and the recombinant adenovirus vector, wherein the LacZ gene is incorporated into the E1 region (Ad-LacZ).

[0406] As described above, CRAd-synNotch (forward / reverse) is a recombinant oncolytic adenovirus vector in which the COX-2 promoter region is positioned upstream of the E1A and E1B genes, and the CD44 / Notch / HIF-3α4 fusion gene is inserted. In CRAd-synNotch (forward), the CD44 / Notch / HIF-3α4 fusion gene is aligned in the same direction (forward) as the COX-2 promoter region, E1A, and E1B genes. On the other hand, in CRAd-synNotch (reverse), the CD44 / Notch / HIF-3α4 fusion gene is reversed relative to the COX-2 promoter region, E1A, and E1B genes. Figure 33 and 34 The base sequences of the E1A gene (SEQ ID NO: 5) and the E1B gene (SEQ ID NO: 6) used in this embodiment are shown respectively.

[0407] CRAd-GFP is a recombinant oncolytic adenovirus vector in which the GFP gene is inserted to replace the CD44 / Notch / HIF-3α4 fusion gene in CRAd-synNotch (forward). As described in Patent Document 1, ADX730 is a recombinant adenovirus vector in which the CD44 / Notch / HIF-3α4 fusion gene is inserted, but it does not contain the E1A or E1B gene controlled by the COX-2 promoter. wtAd is an adenovirus vector that does not possess the CD44 / Notch / HIF-3α4 fusion gene or whose E1A or E1B genes are all controlled by the COX-2 promoter. Ad-LacZ is a recombinant adenovirus vector in which the LacZ gene is inserted to replace the CD44 / Notch / HIF-3α4 fusion gene of ADX730.

[0408] CRAd-synNotch (forward / reverse) and CRAd-GFP are oncolytic, while ADX730, wtAd, and Ad-LacZ are not. CRAd-synNotch (forward / reverse) and CRAd-GFP possess the Ad5 / 35 chimeric fibrillary gene, which includes the knob and shaft portions of fibrin derived from Ad35 and the tail portion of fibrin derived from Ad5, while other adenoviral vectors do not possess the Ad5 / 35 chimeric fibrillary gene. Figure 1 and Figure 2 This is a schematic diagram of CRAd-synNotch (forward), and Figure 3 This is a schematic diagram of CRAd-GFP. Figure 36 The base sequence of the Ad5 / 35 chimeric fiber gene used in this embodiment is shown (SEQ ID NO: 8). Figure 40The amino acid sequence of the protein encoded by the base sequence of SEQ ID NO: 8 is shown (SEQ ID NO: 14).

[0409] CRAd-synNotch (forward / reverse), CRAd-GFP, ADX730, wtAd, and Ad-LacZ were cultured and purified in large quantities, then buffer-replaced using an Amicon Ultra-15 (Merck) to prepare a pH 8.0 adenovirus preservation solution containing Tris, sucrose, NaCl, MgCl2, Tween 80 (trademark), EDTA, and ethanol. Each solution was aliquoted and stored at -80°C until use in subsequent experiments. The composition of the adenovirus preservation solution is shown in the table below.

[0410] [Table 12]

[0411]

[0412] 1.4 Cells and Culture Media

[0413] HEK293 cells, human triple-negative breast cancer cell line MDA-MB-231 cells (European Center for Cell Culture Collection), human normal urothelial cell line SV-HUC-1 cells (American Center for Type Culture Collection), and COX-2 protein-negative human breast cancer cell line BT474 cells (American Center for Type Culture Collection) were cultured in D-MEM medium containing 10% FBS, 1% 100 U / ml penicillin, and 100 mg / ml streptomycin (P / S).

[0414] Human bladder cancer cell line T24 cells were cultured using Eagle's Minimal Essential Medium (E-MEM; FUJIFILM Wako PureChemical Corporation) containing 10% FBS and 1% P / S (American Center for Type Culture Collection).

[0415] Human prostate cancer cell line DU145 cells were cultured using Roswell Park Memorial Institute 1640 (RPMI1640; FUJIFILM Wako Pure Chemical Corporation) containing 10% FBS and 1% P / S (American Center for Type Culture Collection).

[0416] Unless otherwise specified, cell culture is performed at 37°C, 5% CO2 and 21% O2.

[0417] 1.5 Analysis of CD46, CAR and CD44 by flow cytometry

[0418] The expression levels of CD46, CAR, and CD44 in BT474, T24, MDA-MB-231, and DU145 cells were analyzed using flow cytometry. The specific methods are described below.

[0419] Each cell line was washed with PBS by centrifugation (1×10⁻⁶). 6 (Number of cells). Flow cytometry reaction solutions were prepared using FITC anti-human CD46 (BioLegend), PE anti-CAR (Merck), and APC anti-mouse / human CD44 (BioLegend), added to each cell line, and incubated on ice in the dark for 30 minutes. After the reaction, the cells were washed twice with PBS by centrifugation and analyzed using Guava (trademark) easyCyte (trademark) (Merck). As a control, 10 μl of PBS was added to each cell line instead of the flow cytometry reaction solution, and the cells were incubated on ice in the dark for 30 minutes. After washing twice with PBS, the cells were analyzed in the same manner. The obtained data were analyzed using the accompanying InCyte software.

[0420] [Table 13]

[0421]

[0422] 1.6 Real-time RT-PCR

[0423] 1.6.1 Expression analysis of COX-2, HAS-1, HAS-2 and HAS-3 genes

[0424] The expression of COX-2, HAS-1, HAS-2, and HAS-3 genes was analyzed by real-time RT-PCR in T24, MDA-MB-231, and DU145 cells. For the COX-2 gene, relative expression levels were analyzed, with the expression level in BT474 cells set as 1. For the HAS-1, HAS-2, and HAS-3 genes, relative expression levels were analyzed, with the expression level in SV-HUC-1 cells set as 1.

[0425] Specifically, T24 cells, MDA-MB-231 cells, DU145 cells, BT474 cells, and SV-HUC-1 cells were cultured at a concentration of 5 × 10⁻⁶ cells / mL. 5Cells were seeded per well in 6-well flat-bottom cell culture plates (Corning) and cultured. Cells were harvested the day after seeding, and total RNA was extracted using NucleoSpin RNA (TaKaRa). cDNA was synthesized from the extracted total RNA using the PrimeScript RT Reagent Kit (TaKaRa) with gDNA Eraser, and cDNA was used as a template for PCR reactions in triplicate wells using TB Green Premix Ex Taq II (TaKaRa) and analyzed by the ΔΔCt method. mRNA levels were normalized to the expression level of the control gene TATA-binding protein (TBP). Primers used for PCR are shown in the table below.

[0426] [Table 14a]

[0427]

[0428] [Table 14b]

[0429]

[0430] 1.6.2 Expression analysis of HIF-3α4, VEGF, CCL2 and SOX-2 genes

[0431] The expression of HIF-3α4, VEGF, CCL2, and SOX-2 genes in T24 and MDA-MB-231 cells was analyzed by real-time RT-PCR. Specifically, each cell line was subjected to 1.0 × 10⁻⁶ cells / cells. 5 Cells were seeded per well in 6-well flat-bottom cell culture plates (Corning) and cultured overnight. The following day, cells were infected with CRAd-synNotch (forward) or ADX730 at an MOI of 50. Cells were cultured for 48 hours under normoxic (21% O2) or hypoxic (2% O2) conditions and then harvested. Subsequently, except for the PCR primers, the expression levels of HIF-3α4, VEGF, CCL2, and SOX-2 genes were analyzed in the same manner as described in Section 1.6.1. PCR primers are shown in the table in Section 1.6.1.

[0432] 1.6.3 Expression of SOX-2, Cortactin, OCT4, Nanog, VEGF, PHD3, GLUT1 and Cyclin G2 genes analyze

[0433] The expression of SOX-2, Cortactin, OCT4, Nanog, VEGF, PHD3, GLUT1, and Cyclin G2 genes in T24 and DU145 cells was analyzed by real-time RT-PCR. Specifically, each cell line was cultured at 5.0 × 10⁻⁶ cells / cells. 5Cells / well were seeded in 6-well flat-bottom cell culture plates (Corning) and infected with CRAd-synNotch (forward) or CRAd-GFP at an MOI of 50. T24 cells were then cultured for 72 hours under normoxic (21% O2) or hypoxic (2% O2) conditions, and DU145 cells for 96 hours. After cell collection, the expression levels of SOX-2, Cortactin, OCT4, Nanog, VEGF, PHD3, GLUT1, and CyclinG2 genes were analyzed in the same manner as described in Section 1.6.1, except for the PCR primers. PCR primers are shown in the table in Section 1.6.1.

[0434] 1.7 Western blot 1.7.1

[0436] T24 cells or MDA-MB-231 cells were infected in vitro with CRAd-synNotch (forward) or ADX730, and the increase or decrease in the expression level of the CD44 region in the fusion gene carried by the infection was evaluated by Western blotting.

[0437] Specifically, each cell line was fed at a concentration of 1.0 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates (Corning) and cultured overnight. After culture, cells were infected with a predetermined amount of adenovirus and cultured for another 48 hours. After culture, cells were collected, mixed with sample buffer (for SDS-PAGE, 6-fold concentrated, containing reducing agent) (Nacalai Tesque, catalog number 09499-14), heated at 95°C for 5 minutes, cooled on ice, and used as samples.

[0438] Each sample was subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, the gel was imprinted onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was washed with washing buffer (PBS containing 0.1% Tween (trademark) 20, hereinafter referred to as "PBS-T"), and then blocked with PBS-T containing 5% skim milk (Nacalai Tesque) at room temperature for 1 hour.

[0439] After blocking, the membrane was washed and subjected to primary antibody reaction using CD44 (E7K2Y) XP (trademark) Rabbit mAb (Cell Signaling Technology) diluted 1000-fold with Can Get Signal Immunoreaction Enhancer Solution 1 (Toyobo Co., Ltd.) or β-actin (C4) (Santa Cruz Biotechnology) diluted 1000-fold with PBS-T containing 5% Blocking One (Nacalai Tesque), with shaking overnight at 4°C.

[0440] After the primary antibody reaction, the membrane was washed and a secondary antibody reaction was performed using either anti-IgG (H+L chain) (rabbit) p-Ab-HRP (Medical & Biological Laboratories) diluted 1000-fold with Can Get Signal Immunoreaction Enhancer Solution 2 (Toyobo Co., Ltd.) or anti-IgG (H+L chain) (mouse) pAb-HRP (Medical & Biological Laboratories) diluted 1000-fold with PBS-T containing 5% Blocking One (Nacalai Tesque), with shaking for 1 hour. Chemiluminescence was then detected using Chemi-Lumi One L (Nacalai Tesque). 1.7.2

[0442] In addition, T24 and DU145 cells were infected with CRAd-synNotch (forward), and the increase or decrease in the expression level of the CD44 region in the fusion gene carried by the infection was evaluated by Western blotting. Specifically, the amount of adenovirus used for infection was set at 25 MOI or 50 MOI, and the post-infection culture time was set at 24 h, 48 h, 72 h, or 96 h. For the primary antibody reaction, CD44 (E7K2Y) XP (trademark) Rabbit mAb (Cell Signaling Technology) diluted 1000-fold with Can Get Signal Immunoreaction Enhancer Solution 1 (Toyobo Co., Ltd.), HIF3α4 polyclonal antibody (Hokudo) diluted 1000-fold with Can Get Signal Immunoreaction Enhancer Solution 1, or β-actin (C4) (Santa Cruz Biotechnology) diluted 1000-fold with PBS-T containing 5% Blocking One (Nacalai Tesque) was used. Other experimental conditions were the same as in section 1.7.1.

[0443] 1.8 Evaluation of adenovirus vector proliferation capacity

[0444] COX-2-positive T24 and DU145 cells, COX-2-negative BT474 cells, and MDA-MB-231 cells were infected in vitro with wtAd, ADX730, CRAd-GFP, or CRAd-synNotch (forward), and the viral replication capacity driven by the COX-2 promoter and E1 promoter was evaluated by PCR.

[0445] Specifically, each cell line was divided into 5 × 10 5 Cells / well were seeded in 6-well flat-bottom cell culture plates (Corning) and cultured overnight at 37°C and 5% CO2. After culture, the medium was replaced with 300 μl of medium containing the corresponding viral vector with an MOI of 50, and infection was performed at 37°C and 5% CO2 for 1 hour. After infection, 1.7 ml of medium was added, and culture was performed at 37°C and 5% CO2 for 24 hours. After culture, cells were collected, and DNA was extracted using NucleoSpin (trademark) tissue (MACHEREY-NAGEL). The extracted DNA was used as a template for PCR quantification and analysis using TB Green (trademark) premix Ex Taq (trademark) II (TaKaRa). Primers used for PCR are shown in the table below.

[0446] [Table 15]

[0447]

[0448] 1.9 Inhibitory effect of CRAd-synNotch (positive) on cancer cell proliferation

[0449] Each of the following cell lines: BT474, T24, MDA-MB-231, and DU145, at 5 × 10⁻⁶ 3 Cells / well were seeded in 96-well flat-bottom cell culture plates (Corning) and cultured overnight. Subsequently, the medium was replaced with 100 μl of ADX730, CRAd-GFP, or CRAd-synNotch (forward) supplemented with an MOI of 50, or fresh medium without recombinant adenovirus (Mock), and cultured under normoxic (21% O2) or hypoxic (2% O2) conditions for 72 hours. As a colorimetric reagent, 20 μl of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole inner salt (MTS) (Promega Corporation) was added to the culture supernatant, and absorbance was measured at 492 nm after 4 hours of culture.

[0450] 1.10 Bladder cancer treatment experiment using mice

[0451] Six-week-old female BALB / c-nu / nu mice were purchased and used from CLEA Japan. 140 μl of 1×10⁻⁶ g of [a specific antibiotic] ​​was administered. 6 A 1:1 mixture of T24 cells and matrix gel (Becton Dickinson) was inoculated into the right flank of each mouse. Tumor formation was confirmed 13 days post-inoculation, and 20 mice were randomly divided into four groups: CRAd-synNotch (positive) intratumoral administration group, CRAd-GFP intratumoral administration group, ADX730 intratumoral administration group, and PBS intratumoral administration group (n=5). 1×10⁶ T24 cells were inoculated into the right flank of each mouse. 9 Infection units (ifu) / 50 μl PBS of CRAd-synNotch (positive), CRAd-GFP, or ADX730, or 50 μl PBS, were administered intratumorally for a total of six times at 3-day intervals (days 0, 4, 8, 12, 16, and 20). The short diameter (W) and long diameter (L) of the tumor were measured, and the treatment start date was designated as day 0. The formula (W... 2 Calculate the tumor volume using (×L) / 2. Take 300mm... 3 The tumor volume was set as the endpoint.

[0452] 1.11 Immunohistochemical staining

[0453] 1×10 6One T24 cell was subcutaneously inoculated into nude mice, and after tumor formation, PBS or 1×10⁻⁶ cells were administered intratumorally. 9 Ifu's CRAd-GFP, ADX730, or CRAd-synNotch (positive). Tumor tissue was excised and fixed with paraformaldehyde. Paraffin-embedded T24 tumor tissue sections were dewaxed and rehydrated. Antigen retrieval was performed for 20 minutes at 98°C using Bond epitope retrieval buffer (pH 6.0; Leica Microsystems). 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 number: 37259S, Cell Signaling Technology) at room temperature for 15 minutes. After washing, sections were incubated with peroxidase-labeled secondary antibody. Additionally, after washing, sections were incubated with 3,3'-diaminobenzidine (Muto PureChemicals Co., Ltd.) and counterstained with hematoxylin. The obtained tissue sections were observed using a BZ-X710 fluorescence microscope (Keyence).

[0454] 2. Results

[0455] 2.1 BT474 cells, T24 cells, MDA-MB-231 cells, and DU145 cells analyzed by flow cytometry Expression analysis of CD46, CAR and CD44

[0456] 2.1.1 Expression analysis of CD46, CAR and CD44 in BT474 cells

[0457] BT474 cells were subjected to flow cytometry using the method described in item 1.5. Figure 4 The results are displayed.

[0458] When using flow cytometry reaction solution, the mean fluorescence intensity (MFI) of BT474 cells at the fluorescence wavelength of CD46 was 157.56 ± 2.41. The MFI of the control group using PBS was 17.68 ± 0.09. The significant increase in fluorescence intensity compared to the control group confirmed the high expression of CD46 in BT474 cells.

[0459] When using flow cytometry reaction solution, the MFI of CAR at the fluorescence wavelength in BT474 cells was 23.83 ± 0.31. The MFI of the control group was 15.48 ± 0.05. The significant increase in fluorescence intensity compared to the control group confirmed the high expression of CAR in BT474 cells.

[0460] When using flow cytometry reaction solution, the mean fluorescence intensity (MFI) of CD44 in BT474 cells was 22.67 ± 0.09 at the specified fluorescence wavelength. The MFI in 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.

[0461] 2.1.2 Expression analysis of CD46, CAR and D44 in T24 cells

[0462] T24 cells were subjected to flow cytometry using the method described in item 1.5. Figure 5 The results are displayed.

[0463] When using flow cytometry reaction solution, the molecular weight fluorescence index (MFI) of CD46 in T24 cells was 826.67 ± 5.50 at the specified fluorescence wavelength. The MFI in the control group using PBS was 11.59 ± 0.07. This significant increase in fluorescence intensity compared to the control group confirmed high expression of CD46 in T24 cells.

[0464] When using flow cytometry reaction solution, the MFI of CAR at the fluorescence wavelength in T24 cells was 57.90 ± 2.63. The MFI of the control group was 19.67 ± 0.09. The significant increase in fluorescence intensity compared to the control group confirmed the high expression of CAR in T24 cells.

[0465] When using flow cytometry reaction solution, the mean fluorescence intensity (MFI) of CD44 in T24 cells was 3058.09 ± 107.04 at the specified fluorescence wavelength. The MFI in the control group was 4.97 ± 0.01. This significant increase in fluorescence intensity compared to the control group confirmed the high expression of CD44 in T24 cells.

[0466] 2.1.3 Expression analysis of CD46, CAR and CD44 in MDA-MB-231 cells

[0467] MDA-MB-231 cells were subjected to flow cytometry using the method described in item 1.5. Figure 6 The results are displayed.

[0468] When using flow cytometry reaction solution, the MFI of CD46 in MDA-MB-231 cells at the specified fluorescence wavelength was 838.49 ± 10.80. The MFI in the control group using PBS was 38.60 ± 0.25. This significant increase in fluorescence intensity compared to the control group confirmed the high expression of CD46 in MDA-MB-231 cells.

[0469] When using flow cytometry reaction solution, the MFI of MDA-MB-231 cells at the fluorescence wavelength of CAR was 33.36 ± 0.49. The MFI of the control group was 67.55 ± 0.39. Since there was no significant difference in fluorescence intensity compared with the control group, it was confirmed that CAR was not highly expressed in MDA-MB-231 cells.

[0470] When using flow cytometry reaction solution, the MFI of CD44 in MDA-MB-231 cells was 5869.71 ± 32.15 at the specified fluorescence wavelength. The MFI in the control group was 5.62 ± 0.02. The significant increase in fluorescence intensity compared to the control group confirmed the high expression of CD44 in MDA-MB-231 cells.

[0471] 2.1.4 Expression analysis of CD46, CAR and CD44 in DU145 cells

[0472] DU145 cells were subjected to flow cytometry using the method described in item 1.5. Figure 7 The results are displayed.

[0473] When using flow cytometry reaction solution, the mean fluorescence intensity (MFI) of CD46 in DU145 cells was 881.86 ± 6.87 at the specified fluorescence wavelength. The MFI in the control group using PBS was 24.16 ± 0.62. This significant increase in fluorescence intensity compared to the control group confirmed high expression of CD46 in DU145 cells.

[0474] When using flow cytometry reaction solution, the MFI of DU145 cells at the fluorescence wavelength of CAR was 188.65 ± 6.18. The MFI of the control group was 23.97 ± 0.45. The significant increase in fluorescence intensity compared to the control group confirmed the high expression of CAR in DU145 cells.

[0475] When using flow cytometry reaction solution, the mean fluorescence intensity (MFI) of CD44 in DU145 cells was 3434.24 ± 38.66 at the specified fluorescence wavelength. The MFI in the control group was 4.79 ± 0.04. This significant increase in fluorescence intensity compared to the control group confirmed the high expression of CD44 in DU145 cells.

[0476] 2.2 Quantitative analysis of COX-2 gene expression in T24, MDA-MB-231, and DU145 cells

[0477] Real-time PT-PCR was performed using the method described in section 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 levels of the COX-2 gene in BT474 cells (a COX-2 protein-negative human breast cancer cell line). Figure 8 The results are displayed.

[0478] 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 were significantly higher in T24 cells, MDA-MB-231 cells, and DU145 cells. These results confirm that the COX-2 gene is highly expressed in T24 cells, MDA-MB-231 cells, and DU145 cells.

[0479] 2.3 Expression of HAS-1, HAS-2, and HAS-3 genes in T24, MDA-MB-231, and DU145 cells quantity

[0480] Real-time PT-PCR was performed using the method described in section 1.6.1, and the expression levels of HAS-1, HAS-2, and HAS-3 genes (in other words, hyaluronic acid production capacity) in T24, MDA-MB-231, and DU145 cells were evaluated by comparing them with the expression levels of HAS-1, HAS-2, and HAS-3 genes in SV-HUC-1 cells (a normal human urothelial cell line). Figure 9-11 The results are displayed.

[0481] 2.3.1 T24 cells

[0482] When the expression level in SV-HUC-1 cells was set to 1, the relative expression levels of hyaluronic acid production genes HAS-1 and HAS-3 were significantly higher in T24 cells. On the other hand, no significant difference was observed in the relative expression level of HAS-2. Figure 9 ).

[0483] 2.3.2 MDA-MB-231 cells

[0484] When the expression level in SV-HUC-1 cells was set to 1, the relative expression level of the hyaluronic acid production gene HAS-2 was significantly higher in MDA-MB-231 cells. On the other hand, no significant difference was observed in the relative expression levels of HAS-1 and HAS-3. Figure 10 ).

[0485] 2.3.3 DU145 cells

[0486] When the expression level in SV-HUC-1 cells was set to 1, the relative expression levels of hyaluronic acid production genes HAS-2 and HAS-3 were significantly higher in DU145 cells. On the other hand, no significant difference was observed in the relative expression level of HAS-1. Figure 11 ).

[0487] 2.4 Titer Measurement of Recombinant Adenovirus Vector

[0488] The titer of each constructed adenovirus vector was measured using the method described in section 1.1.9. The titer of CRAd-synNotch (reverse) was 1.7 × 10⁻⁶. 10 ifu / ml. The titer of CRAd-synNotch (positive) is 1.2 × 10⁻⁶. 12 ifu / ml. The titer of CRAd-GFP was 1.05 × 10⁻⁶. 11 ifu / ml.

[0489] 2.5 Infection with CRAd-synNotch ( Reverse ) CD44 / Notch / HIF-3α4 fusion gene in MDA-MB-231 cells Analysis of the expression of cause

[0490] The expression of the CD44 / Notch / HIF-3α4 fusion gene in MDA-MB-231 cells infected with CRAd-synNotch (reverse) constructed in Section 1.1 was evaluated by Western blotting and real-time RT-PCR. CRAd-synNotch (reverse) and other adenovirus vectors were obtained from three large-scale cultures and purifications of viral fluid as described in Section 1.1.8. Specific results are shown below.

[0491] 2.5.1 CD44 Expression Analysis by Western Blotting

[0492] Western blotting was performed on uninfected adenovirus MDA-MB-231 cells (cells only), MDA-MB-231 cells infected with Ad-LacZ with an MOI of 100, MDA-MB-231 cells infected with ADX730 with an MOI of 100, MDA-MB-231 cells infected with CRAd-GFP with an MOI of 50, and MDA-MB-231 cells infected with CRAd-synNotch (reverse) with an MOI of 50 using the method described in section 1.7.1. Figure 12 The results are displayed.

[0493] In all samples, a band belonging to endogenous CD44 (approximately 80 kDa) was observed. On the other hand, a band believed to be the CD44 / Notch / HIF-3α4 fusion protein (approximately 105 kDa) was observed only in cells infected with ADX730, and not in cells infected with CRAd-synNotch (reverse).

[0494] 2.5.2 Quantitative analysis of HIF-3α4 gene expression by real-time RT-PCR

[0495] Real-time RT-PCR was performed on uninfected adenovirus-infected MDA-MB-231 cells (cells only), MDA-MB-231 cells infected with ADX730 at MOI 100, and MDA-MB-231 cells infected with CRAd-synNotch (reverse) at MOI 50 using the method described in section 1.6.2, and the expression level of the HIF-3α4 gene was quantified. Figure 13 The results are displayed.

[0496] The expression level of the HIF-3α4 gene in MDA-MB-231 cells infected with ADX730 was significantly higher than that in MDA-MB-231 cells uninfected with adenovirus. On the other hand, the expression level of the HIF-3α4 gene in MDA-MB-231 cells infected with CRAd-synNotch (reverse) did not show a significant difference compared with that in MDA-MB-231 cells uninfected with adenovirus.

[0497] 2.5.3. Summary

[0498] Typically, in the construction of adenoviral vectors, when the aim is to express two sets of genes (the CD44 / Notch / HIF-3α4 fusion gene and the E1A / E1B gene in this example) under the control of different promoters (the β-actin promoter and the COX-2 promoter in this experimental example), the usual practice is to reverse the arrangement of the two sets of genes. Therefore, the inventors first investigated CRAd-synNotch (reverse), in which the CD44 / Notch / HIF-3α4 fusion gene and the E1A / E1B gene are reversed. However, as mentioned above, expression of the CD44 / Notch / HIF-3α4 fusion gene was not observed from CRAd-synNotch (reverse).

[0499] Therefore, the inventors investigated CRAd-synNotch (forward), in which the CD44 / Notch / HIF-3α4 fusion gene and the E1A / E1B gene are aligned in the same direction (forward). The results are shown in section 2.6 and subsequent sections.

[0500] 2.6 Infection with CRAd-synNotch ( positive ) CD44 / Notch / HIF-3α4 fusion gene in MDA-MB-231 cells Analysis of the expression of cause

[0501] As in section 2.5, the expression of the CD44 / Notch / HIF-3α4 fusion gene in MDA-MB-231 cells infected with CRAd-synNotch (positive) was evaluated by Western blotting and RT-PCR. For CRAd-synNotch (positive), a secondary viral load was used. Specific results are shown below.

[0502] 2.6.1 CD44 Expression Analysis by Western Blotting

[0503] 2.6.1.1 CRAd-synNotch (forward) cloning 1

[0504] Western blotting was performed on MDA-MB-231 cells infected with 10 μl, 20 μl, or 50 μl of secondary viral fluid of CRAd-synNotch (forward) clone 1, uninfected MDA-MB-231 cells (cells only), and MDA-MB-231 cells infected with ADX730 at MOI of 100, using the method described in section 1.7.1. Figure 14 The results are displayed.

[0505] In all samples, a band belonging to endogenous CD44 (approximately 80 kDa) was observed. On the other hand, a band believed to be the CD44 / Notch / HIF-3α4 fusion protein (approximately 105 kDa) was observed only in cells infected with ADX730, and not in cells infected with CRAd-synNotch (forward) clone 1.

[0506] 2.6.1.2 CRAd-synNotch (forward) clones 2 and 3

[0507] Western blotting was performed on MDA-MB-231 cells infected with 20 μl or 40 μl of secondary viral fluid of CRAd-synNotch (forward) clone 2, MDA-MB-231 cells infected with 20 μl or 40 μl of secondary viral fluid of CRAd-synNotch (forward) clone 3, uninfected MDA-MB-231 cells (cells only), and MDA-MB-231 cells infected with ADX730 at MOI of 100 using the method described in section 1.7.1. Figure 15 The results are displayed.

[0508] In all samples, a band belonging to endogenous CD44 (approximately 80 kDa) was observed. Additionally, a band believed to be the CD44 / Notch / HIF-3α4 fusion protein (approximately 105 kDa) was observed in cells infected with CRAd-synNotch (forward) clone 2, cells infected with CRAd-synNotch (forward) clone 3, and cells infected with ADX730.

[0509] 2.6.2 Quantitative analysis of CD44 and HIF-3α4 gene expression by real-time RT-PCR

[0510] Real-time RT-PCR was performed on uninfected adenovirus-infected MDA-MB-231 cells (cells only), MDA-MB-231 cells infected with ADX730 at MOI 100, 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 using the method described in section 1.6.2, and the expression levels of HIF-3α4 and CD44 genes were quantified. Figure 16 The results are displayed.

[0511] In all 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 and CD44 genes were significantly higher than those in MDA-MB-231 cells not infected with adenovirus.

[0512] Since the expression levels of HIF-3α4 and CD44 genes are particularly high in MDA-MB-231 cells infected with CRAd-synNotch (forward) clone 3, the inventors used CRAd-synNotch (forward) clone 3 in subsequent experiments and carried out large-scale culture and purification of CRAd-synNotch (forward) clone 3 by means of the method described in item 1.1.8.

[0513] 2.7 Large-scale culture of MDA-MB-231 and T24 cells infected with CRAd-synNotch (forward) clone 3 Expression analysis of CD44 / Notch / HIF-3α4 fusion gene

[0514] The expression of the CD44 / Notch / HIF-3α4 fusion gene was evaluated by Western blotting and real-time RT-PCR in MDA-MB-231 and T24 cells infected with CRAd-synNotch (forward) clone 3, which had been cultured and purified in large quantities according to the methods described in section 1.1.8. Specifically, the results are shown below. In subsequent experimental examples, CRAd-synNotch (forward) clone 3 is simply referred to as "CRAd-synNotch".

[0515] 2.7.1 CD44 Expression Analysis by Western Blotting

[0516] Western blotting was performed on uninfected adenovirus-infected MDA-MB-231 cells (cells only), MDA-MB-231 cells infected with CRAd-synNotch at MOIs of 50, 100, 200, or 400, and MDA-MB-231 cells infected with ADX730 at MOI of 100, using the method described in section 1.7.1. Additionally, Western blotting was performed on uninfected adenovirus-infected T24 cells (cells only), T24 cells infected with CRAd-synNotch at MOIs of 50, 100, 200, or 400, and T24 cells infected with ADX730 at MOI of 100, using the method described in section 1.7.1. Figure 17 The results are displayed.

[0517] In all samples, a band belonging to endogenous CD44 (approximately 80 kDa) was observed. Furthermore, a band believed to be the CD44 / Notch / HIF-3α4 fusion protein (approximately 105 kDa) was observed in MDA-MB-231 and T24 cells infected with CRAd-synNotch, and in MDA-MB-231 and T24 cells infected with ADX730.

[0518] 2.7.2 Quantitative analysis of CD44 and HIF-3α4 gene expression by real-time RT-PCR

[0519] Real-time RT-PCR was performed on uninfected adenovirus T24 cells (cells only), T24 cells infected with ADX730 at MOI 100, and T24 cells infected with CRAd-synNotch at MOI 50 using the method described in section 1.6.2, and the expression levels of HIF-3α4 and CD44 genes were quantified. Figure 18 The results are displayed.

[0520] The expression level of HIF-3α4 gene was significantly higher in T24 cells infected with ADX730 and T24 cells infected with CRAd-synNotch than in T24 cells not infected with adenovirus.

[0521] The expression level of the CD44 gene in T24 cells infected with ADX730 was not significantly different from that in T24 cells not infected with adenovirus. On the other hand, the expression level of the CD44 gene in T24 cells infected with CRAd-synNotch was significantly higher than that in T24 cells not infected with adenovirus.

[0522] 2.8 Evaluation of the proliferation capacity of adenovirus vectors

[0523] The proliferation capacity of wtAd, ADX730, CRAd-GFP, and CRAd-synNotch in BT474, T24, MDA-MB-231, and DU145 cells was evaluated using the method described in section 1.8. Figure 19 The results are displayed.

[0524] 2.8.1 wtAd

[0525] In each of the infected BT474, T24, and DU145 cell lines, the copy number of wtAd increased significantly at 24 hours post-infection compared to 1 hour post-infection. On the other hand, in infected MDA-MB-231 cells, the copy number of wtAd did not show a significant difference at 24 hours post-infection compared to 1 hour post-infection. Figure 19 A).

[0526] 2.8.2 ADX730

[0527] In each of the infected BT474, T24, MDA-MB-231, and DU145 cancer cell lines, the copy number of ADX730 did not show a significant difference between 24 hours and 1 hour post-infection. Figure 19 B).

[0528] 2.8.3 CRAd-GFP and CRAd-synNotch

[0529] The copy numbers of CRAd-GFP and CRAd-synNotch in infected COX-2-positive T24 and DU145 cells were significantly increased at 24 hours post-infection compared to 1 hour post-infection. On the other hand, the copy numbers of CRAd-GFP and CRAd-synNotch in infected COX-2-negative BT474 and MDA-MB-231 cells showed no significant difference at 24 hours post-infection compared to 1 hour post-infection. Figure 19 C and Figure 19 D).

[0530] 2.9 Evaluation of the inhibitory effect of CRAd-synNotch on cancer cell proliferation

[0531] The inhibitory effect of CRAd-synNotch on the cell proliferation of BT474, T24, MDA-MB-231 and DU145 cells was evaluated in vitro using the method described in item 1.9. Figures 20 to 23 The results are shown. Note that "cells only" refers to cases where, as a mock treatment, a medium without adenovirus was added instead of a medium containing recombinant adenovirus.

[0532] 2.9.1 BT474 cells

[0533] Under normoxic (normative: 21% O2) or hypoxic (hypoxic: 2% O2) conditions, no significant cell proliferation inhibition effect was observed in BT474 cells for any of ADX730, CRAd-GFP, and CRAd-synNotch compared to the "cells only" group. Figure 20 ).

[0534] 2.9.2 T24 cells

[0535] Under both normoxic and hypoxic conditions, significant cell proliferation inhibition was observed in T24 cells for each of ADX730, CRAd-GFP, and CRAd-synNotch compared to the "cells only" group. In particular, CRAd-synNotch showed a significantly higher cell proliferation inhibition effect than ADX730 and CRAd-GFP. Figure 21 ).

[0536] 2.9.3 MDA-MB-231 cells

[0537] Under normoxic or hypoxic conditions, no significant inhibitory effect on cell proliferation was observed in MDA-MB-231 cells for any of ADX730, CRAd-GFP, and CRAd-synNotch compared to the "cells only" group. Figure 22 ).

[0538] 2.9.4 DU145 cells

[0539] Under normoxic conditions, for DU145 cells, only CRAd-synNotch showed a significant inhibitory effect on cell proliferation compared to the "cells only" group, while neither ADX730 nor CRAd-GFP showed a significant inhibitory effect on cell proliferation compared to the "cells only" group. Under hypoxic conditions, for DU145 cells, ADX730 and CRAd-synNotch showed significant inhibitory effects on cell proliferation compared to the "cells only" group, while CRAd-GFP did not show a significant inhibitory effect on cell proliferation compared to the "cells only" group. Figure 23 ).

[0540] 2.10 Evaluation of the inhibitory effects of CRAd-synNotch on downstream genes of HIF-1α and CD44.

[0541] Using the method described in section 1.6.2, the expression of VEGF as a downstream gene of HIF-1α and SOX-2 and CCL2 as downstream genes of CD44 were analyzed in uninfected adenovirus T24 cells (cells only), T24 cells infected with ADX730 at MOI 50, and T24 cells infected with CRAd-synNotch at MOI 50. Figures 24 to 26 The results are displayed.

[0542] 2.10.1 VEGF gene

[0543] Under normoxic conditions, the expression level of VEGF, a downstream gene of HIF-1α, was low in all of the following cell types: uninfected T24 cells (cells only), T24 cells infected with ADX730, and T24 cells infected with CRAd-synNotch. Conversely, under hypoxic conditions, high VEGF expression was observed in uninfected T24 cells (cells only) and T24 cells infected with ADX730; however, the VEGF expression level in T24 cells infected with CRAd-synNotch was significantly lower than that in uninfected T24 cells (cells only) and T24 cells infected with ADX730. Figure 24 ).

[0544] 2.10.2 SOX-2 gene

[0545] Under normoxic conditions, the expression level of SOX-2, a downstream gene of CD44, in CRAd-synNotch-infected T24 cells was significantly lower than that in uninfected T24 cells (cells only) and T24 cells infected with ADX730. Under hypoxic conditions, the expression level of SOX-2, a downstream gene of CD44, in T24 cells infected with ADX730 and CRAd-synNotch was significantly lower than that in uninfected T24 cells (cells only). Figure 25 ).

[0546] 2.10.3 CCL2 gene

[0547] Under normoxic conditions, the expression level of CCL2, a downstream gene of CD44, in CRAd-synNotch-infected T24 cells showed no significant difference compared to uninfected T24 cells (cells only). On the other hand, under hypoxic conditions, the expression level of CCL2, a downstream gene of CD44, in CRAd-synNotch-infected T24 cells was significantly lower than that in uninfected T24 cells (cells only). Figure 26 ).

[0548] 2.10.4 Summary

[0549] These results indicate that the decoy function of the extracellular portion of CD44 contained in CRAd-synNotch inhibits the expression of downstream genes of CD44, and the HIF-3α4 portion contained in CRAd-synNotch inhibits the expression of downstream genes of HIF-1α.

[0550] 2.11 Evaluation of the efficacy of CRAd-synNotch in vivo for the treatment of bladder cancer

[0551] Using the method described in item 1.10, PBS, ADX730, CRAd-GFP, or CRAd-synNotch were administered intratumorally to nude mice transplanted with T24 cells, and the mice were monitored. Figure 27 and 28 The results are displayed.

[0552] Twenty-four days after the start of administration of PBS, ADX730, CRAd-GFP, or CRAd-synNotch (day 24), tumor growth was significantly inhibited in the CRAd-synNotch group compared to the PBS and CRAd-GFP groups. Furthermore, the CRAd-synNotch group tended to have a higher tumor growth inhibition effect than the ADX730 group. Figure 27 ).

[0553] Continued observation after a total of six administrations revealed significantly prolonged survival in the CRAd-synNotch administration group compared to the PBS and CRAd-GFP administration groups. Furthermore, the CRAd-synNotch administration group tended to show longer survival than the ADX730 administration group. Figure 28 ).

[0554] 2.12 Expression analysis of fusion proteins by Western blotting

[0555] Using the method described in section 1.7.2, T24 cells were infected with CRAd-synNotch at an MOI of 50 and cultured for 24 or 72 hours. After culture, Western blotting was performed. Results of Western blotting using CD44 antibody as the primary antibody were shown... Figure 42 On the left side. Results of Western blotting using HIF-3α4 antibody as the primary antibody are shown... Figure 42 The right side. For example... Figure 42 As shown on the left, an endogenous CD44 band (approximately 80 kDa) was observed in all samples. Furthermore, in CRAd-synNotch-infected cells, a band believed to be the CD44-ECD / Notch / HIF-3α4 fusion protein (approximately 105 kDa) was observed in both cells cultured for 24 hours and 72 hours post-infection. In cells cultured for 72 hours post-infection, a band was observed on the lower molecular weight side. This result indicates that HIF-3α4 is cleaved from the fusion protein. Figure 42As shown on the right, when the HIF-3α4 antibody was used as the primary antibody, a band (approximately 105 kDa) believed to be the CD44-ECD / Notch / HIF-3α4 fusion protein was observed in CRAd-synNotch-infected cells cultured for 24 hours post-infection, but this band was not observed in CRAd-synNotch-infected cells cultured for 72 hours post-infection. This result also indicates that HIF-3α4 is cleaved from the fusion protein.

[0556] Using the method described in section 1.7.2, DU145 cells were infected with CRAd-synNotch at an MOI of 25 or 50 and cultured for 24, 48, or 96 hours. After culture, Western blotting was performed. Results of Western blotting using CD44 antibody as the primary antibody were shown... Figure 43 On the left side. Results of Western blotting using HIF-3α4 antibody as the primary antibody are shown... Figure 43 The right side. For example... Figure 43 As shown on the left, an endogenous CD44 band (approximately 80 kDa) was observed in all samples. Furthermore, in CRAd-synNotch-infected cells, a band believed to be the CD44-ECD / Notch / HIF-3α4 fusion protein (approximately 105 kDa) was observed in all cells cultured for 24 hours, 48 ​​hours, and 96 hours post-infection. In cells cultured for 96 hours post-infection, a band was observed on the lower molecular weight side. This result indicates that HIF-3α4 is cleaved from the fusion protein. Figure 43 As shown on the right, when the HIF-3α4 antibody was used as the primary antibody, a band (approximately 105 kDa) believed to be the CD44-ECD / Notch / HIF-3α4 fusion protein was observed in CRAd-synNotch-infected cells cultured for 24 hours post-infection, but this band was not observed in CRAd-synNotch-infected cells cultured for 96 hours post-infection. This result also indicates that HIF-3α4 is cleaved from the fusion protein.

[0557] 2.13 Inhibition of CD44 downstream genes and HIF-1α downstream genes by CRAd-synNotch

[0558] As described in section 1.6.3, T24 cells were infected with CRAd-GFP or CRAd-synNotch with an MOI of 50, and DU145 cells were infected with CRAd-GFP or CRAd-synNotch with an MOI of 25. Cells were then cultured under normoxic (21% O2) or hypoxic (2% O2) conditions for 72 or 96 hours. After culture, 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. Figures 44 to 47 The results are displayed.

[0559] Figure 44 This shows the analysis of Cortactin, a downstream gene of CD44, in T24 cells. Figure 44 (left)), OCT4( Figure 44 (Chinese) and Nanog Figure 44 (Right)) The result of the expression level. For example Figure 44 As shown, under hypoxic conditions, the expression levels of Cortactin, OCT4, and Nanog in CRAd-synNotch-infected cells were significantly lower than those in uninfected cells and CRAd-GFP-infected cells.

[0560] Figure 45 This shows the analysis of PHD3 (a downstream gene of HIF-1α) in T24 cells. Figure 45 (left)), GLUT1( Figure 45 (in Chinese) and CyclinG2 Figure 45 (Right)) The result of the expression level. For example Figure 45 As shown, under hypoxic conditions, the expression levels of PHD3, GLUT1, and CyclinG2 in CRAd-synNotch-infected cells were significantly lower than those in uninfected cells and CRAd-GFP-infected cells.

[0561] Figure 46 This shows the analysis of Cortactin (a downstream gene of CD46) in DU145 cells. Figure 46 (top left)), OCT4 Figure 46 (top right)), Nanog Figure 46 (bottom left) and SOX-2 Figure 46 (Lower right) shows the result of the expression level. For example... Figure 46As shown, under hypoxic conditions, the expression levels of Cortactin, OCT4, Nanog, and SOX-2 in CRAd-synNotch-infected cells were significantly lower than those in uninfected cells and CRAd-GFP-infected cells.

[0562] Figure 47 This shows the analysis of VEGF (a downstream gene of HIF-1α) in DU145 cells. Figure 47 (top left)), GLUT1 Figure 47 (top right)), CyclinG2 Figure 47 (bottom left) and PHD3 Figure 47 (Lower right) shows the result of the expression level. For example... Figure 47 As shown, under hypoxic conditions, the expression levels of VEGF, GLUT1, Cyclin G2, and PHD3 in CRAd-synNotch-infected cells were significantly lower than those in uninfected cells and CRAd-GFP-infected cells.

[0563] 2.14 Immunohistochemical staining of CD44 in the T24 mouse model

[0564] As described in item 1.11, 1×10 6 T24 cells were subcutaneously inoculated into nude mice to induce tumor formation. Subsequently, PBS or 1 × 10⁶ T24 cells were administered intratumorally. 9 Ifu's ADX730, CRAd-GFP, or CRAd-synNotch were administered. Tumors were resected 24 hours after administration, and CD44 expression was investigated by immunohistochemical staining. Figure 48 The results are displayed. For example... Figure 48 As shown in the lower part, a significant increase in CD44 expression was observed on the cell membrane of tumors treated with CRAd-synNotch or ADX730 compared to tumors treated with CRAd-GFP or PBS.

[0565] 3. Discussion

[0566] The above results demonstrate that CRAd-synNotch possesses antitumor effects. The results also indicate that the antitumor effect can be achieved through the decoy function of the extracellular portion of CD44 contained in CRAd-synNotch, which inhibits the expression of downstream genes of CD44, and through the inhibition of the expression of downstream genes of HIF-1α by the HIF-3α4 portion contained in CRAd-synNotch. Furthermore, the results show that the HIF-3α4 portion is cleaved from the fusion protein after expression.

Claims

1. An oncolytic adenovirus vector comprising nucleic acid, wherein the nucleic acid comprises: A base sequence (I) having a structure in which the following base sequences are linked in the order (A)-(B)-(C): (A) Base sequence encoding a protein with extracellular function of CD44. (B) The base sequence encoding a protein that functions in the Notch core region, and (C) The base sequence encoding a protein with HIF-3α4 function; and The base sequence (II) contains: (D) Base sequence containing the COX-2 promoter; (E) The base sequence encoding the E1A protein, placed under the control of the COX-2 promoter; and (F) The base sequence encoding the E1B protein, placed under the control of the COX-2 promoter. The base sequence (I) and the base sequence (II) are aligned in the same direction.

2. An oncolytic adenovirus vector containing nucleic acid, wherein the nucleic acid comprises: A base sequence (I) having a structure in which base sequences (A), (B), and (C) are linked in the order (A)-(B)-(C); and The base sequence (II) contains: (D) Base sequence containing the COX-2 promoter; (E) The base sequence encoding the E1A protein, placed under the control of the COX-2 promoter; and (F) The base sequence encoding the E1B protein, placed under the control of the COX-2 promoter. The base sequence (I) and the base sequence (II) are aligned in the same direction. 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 more than 85% sequence identity with (a-1) and encoding a protein that can bind 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 consisting of more than 85% sequence identity with (b-1) and encoding a protein that can be cleaved by proteases. 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 consisting of more than 85% sequence identity with (c-1) and encoding a protein that can bind to HIF-1α. The base sequence (D) is: (d-1): A base sequence consisting of the base sequence of SEQ ID NO: 4, or (d-2): A sequence of bases that has more than 85% sequence identity with (d-1) and encodes the promoter sequence that is induced to express COX-2 under conditions that induce COX-2 expression. 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 consisting of more than 85% sequence identity with (e-1) and encoding a protein with E1A function. 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 consisting of more than 85% sequence identity with the base sequence of (f-1) and encoding a protein with E1B function.

3. The oncolytic adenovirus vector according to claim 1, in, 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 more than 85% sequence identity with the base sequence of (a-1) and encoding a protein that can bind 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 nucleic acid that is composed of a base sequence having more than 85% sequence identity with the base sequence of (b-1) and encodes a protein that can be cleaved by a protease in a nucleic acid having a structure in which (A), (B) and (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 that shares more than 85% sequence identity with (c-1) and encodes a protein that can bind to HIF-1α. The base sequence (D) is: (d-1): A base sequence consisting of the base sequence of SEQ ID NO: 4, or (d-2): A sequence of bases that has more than 85% sequence identity with (d-1) and encodes the promoter sequence that is induced to express COX-2 under conditions that induce COX-2 expression. 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 consisting of more than 85% sequence identity with (e-1) and encoding a protein with E1A function. 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 consisting of more than 85% sequence identity with the base sequence of (f-1) and encoding a protein with E1B function.

4. An oncolytic adenovirus vector comprising nucleic acid, wherein the nucleic acid comprises: The base sequence (III) is as follows: (g-1) is a base sequence consisting of the base sequence of SEQ ID NO:

7. (g-2) is a base sequence consisting of more than 85% sequence identity with the base sequence of SEQ ID NO: 7, and having a structure in which the portions encoding a protein that can bind to hyaluronic acid, a portion encoding a protein that can be cleaved by a protease, and a portion encoding a protein that can bind to HIF-1α are linked in this order, or (g-3) is a base sequence consisting of more than 85% sequence identity with the base sequence of SEQ ID NO: 7, and encoding a protein with anticancer activity; and The base sequence (II) contains: (D) Base sequence containing the COX-2 promoter; (E) The base sequence encoding the E1A protein, controlled by the COX-2 promoter; and (F) The base sequence encoding the E1B protein, placed under the control of the COX-2 promoter. The base sequence (III) and the base sequence (II) are aligned in the same direction. The base sequence (D) is: (d-1): A base sequence consisting of the base sequence of SEQ ID NO: 4, or (d-2): A sequence of bases that has more than 85% sequence identity with (d-1) and encodes the promoter sequence that is induced to express COX-2 under conditions that induce COX-2 expression. 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 consisting of more than 85% sequence identity with (e-1) and encoding a protein with E1A function. 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 consisting of more than 85% sequence identity with the base sequence of (f-1) and encoding a protein with E1B function.

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

6. The oncolytic adenovirus vector according to claim 5, The base sequence (H) is as follows: (h-1): A base sequence consisting of the base sequence of SEQ ID NO: 8, or (h-2): A base sequence consisting of more than 85% sequence identity with the base sequence of (h-1) and encoding a protein that can bind to CD46.

7. The oncolytic adenovirus vector according to any one of claims 2 to 4 and 6, wherein, The protease is either ADAM protease or γ-secretase.

8. An anticancer composition comprising an oncolytic adenovirus vector according to any one of claims 1 to 4 and 6.

9. The anticancer composition according to claim 8, wherein it is an injectable preparation.

10. The anticancer composition according to claim 8, for the treatment of COX-2 positive cancers.

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

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