Cancer gene therapy drugs

Abstract

Provided is a novel oncogene therapeutic means. More specifically, provided is a nucleic acid having a structure in which a nucleic acid (A) encoding a protein having a CD44 extracellular function, a nucleic acid (B) encoding a protein having a Notch core region function, and a nucleic acid (C) encoding a protein having an HIF-3α4 function are linked in the order of (A)-(B)-(C).

Description

[Technical Field]

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

[0002] In recent years, viral vector-based gene therapy has attracted attention as a new cancer treatment. Taking advantage of the properties of various viruses, viruses are used as gene carriers and viral vectors. One method of gene transfer is in vivo gene therapy, in which a recombinant viral vector carrying a therapeutic gene is directly administered to the body to treat the disease. Cancer gene therapy has a different mechanism of action from existing therapies such as chemotherapy: it directly suppresses genes involved in cancer development and proliferation by introducing genes into cancer cells, or directly induces cell death by introducing tumor suppressor genes into cells. Therefore, it is expected to be an effective treatment for cancers that are difficult to treat with existing chemotherapy or radiation therapy. [Prior art documents] [Non-patent literature]

[0003] [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. [Non-patent document 2] Bourguignon LY, Zhu H, Shao L, Chen YW. (2001). CD44 interaction with c-Src kinase promotes cortactin-mediated cytoskeleton function and hyaluronic acid-dependent ovarian tumor cell migration. J Biol Chem. 2001 Mar 9;276(10):7327-36. Epub 2000 Nov 17 .

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[0004] The present inventors have conducted studies with the main objective of providing a novel cancer gene therapeutic agent or method. [Means for solving the problem]

[0005] Viral vectors, such as retroviruses, lentiviruses, and Sendai viruses, are important for expressing exogenously introduced genes in cancer cells.

[0006] However, because the gene capacity that can be inserted into a viral vector is limited, it is currently necessary to design a viral vector that inserts a gene within the allowable amount to achieve its effect. Furthermore, since the cancer-suppressing effect obtained is limited by the function possessed by the inserted gene, it is necessary to find a method to limit the gene capacity inserted into the viral vector within the allowable range and achieve the effect more efficiently. In other words, creating a gene that efficiently exerts a cancer-suppressing effect within a limited capacity is a key point in improving the effectiveness of cancer therapeutic drugs using viral vectors.

[0007] Therefore, we investigated various gene modifications and combinations to attempt to create an artificial gene with particularly high cancer-suppressing effects. As a result, we found that an artificial gene fusing CD44 (particularly the extracellular portion), Notch (particularly the core region), and HIF-3α4 exhibited extremely high cancer-suppressing effects. Based on this finding, we have made further improvements.

[0008] The present disclosure includes, for example, the subject matter described in the following sections: Section 1. (A) Nucleic acid encoding a protein with CD44 extracellular function (B) Nucleic acid encoding a protein having Notch core region function (C) A nucleic acid encoding a protein having HIF-3α4 function. A nucleic acid having a structure in which (A)-(B)-(C) are linked in this order. Section 2. A nucleic acid having a structure in which a nucleic acid (A), a nucleic acid (B), and a nucleic acid (C) are linked in the order of (A)-(B)-(C), The nucleic acid (A) is (a-1): a nucleic acid consisting of the base sequence of SEQ ID NO: 1, or (a-2): A nucleic acid consisting of a base sequence in which one or more bases are deleted, substituted, or added in the base sequence of (a-1), and encoding a protein capable of binding to hyaluronic acid. and The nucleic acid (B) is (b-1): a nucleic acid consisting of the base sequence of SEQ ID NO: 2, or (b-2): A nucleic acid consisting of a base sequence in which one or more bases are deleted, substituted, or added in the base sequence of (b-1), and encoding a protein that can be cleaved by a protease. and The nucleic acid (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 in which one or more bases are deleted, substituted, or added in the base sequence of (c-1), and encoding a protein capable of binding to HIF-1α. Nucleic acid. Section 3. The nucleic acid (A) is (a-1): a nucleic acid consisting of the base sequence of SEQ ID NO: 1, or (a-2): A nucleic acid consisting of a base sequence in which one or more bases are deleted, substituted, or added in the base sequence of (a-1), and encoding a protein capable of binding to hyaluronic acid. and The nucleic acid (B) is (b-1): a nucleic acid consisting of the base sequence of SEQ ID NO: 2, or (b-2): A nucleic acid encoding a protein that can be cleaved by a protease, which is composed of a base sequence in which one or more bases are deleted, substituted, or added in the base sequence of (b-1) and has a structure in which the bases (A), (B), and (C) are linked in this order. and The nucleic acid (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 in which one or more bases are deleted, substituted, or added in the base sequence of (c-1), and encoding a protein capable of binding to HIF-1α. That is, Item 1. The nucleic acid according to Item 1. Section 4. (d-1) consists of the base sequence of SEQ ID NO: 4, (d-2) The base sequence of SEQ ID NO: 4 is a base sequence in which one or more bases are deleted, substituted, or added, and has a structure in which a portion encoding a protein capable of binding to hyaluronic acid, a portion encoding a protein that can be cleaved by a protease, and a portion encoding a protein capable of binding to HIF-1α are linked in this order, or (d-3) A base sequence in which one or more bases are deleted, substituted, or added in the base sequence of SEQ ID NO: 4, and which encodes a protein having an anticancer effect. Nucleic acid. Section 5. Item 5. The nucleic acid according to any one of Items 1 to 4, wherein the protease is an ADAM protease or γ-secretase. Section 6. A protein encoded by the nucleic acid according to any one of Items 1 to 5. Section 7. A vector incorporating the nucleic acid according to any one of Items 1 to 5 in an expressible manner. Section 8. Item 8. The vector according to Item 7, wherein the vector is an adenovirus vector. Section 9. An anti-cancer composition comprising the nucleic acid according to any one of Items 1 to 5 or the vector according to Item 7 or 8. Section 10. Item 10. The anti-cancer composition according to Item 9, which is an injection. Section 11. Item 11. The anti-cancer composition according to Item 9 or 10, which is for treating breast cancer, prostate cancer, gastric cancer, or pancreatic cancer. Section 12. Item 11. The anticancer composition according to Item 9 or 10, which is for treating triple-negative breast cancer. [Effects of the Invention]

[0009] A novel cancer gene therapy drug with extremely high anticancer effects can be provided. This drug is effective against cancer types that are not affected by existing cancer therapy drugs or are small in size, and can be preferably used for, for example, triple-negative (estrogen receptor-negative, progesterone receptor-negative, HER2-negative) breast cancer. [Brief explanation of the drawings]

[0010] [Figure 1] The sequence of the CD44 / Notch / HIF-3α4 fusion gene thus constructed is shown. [Figure 2a] The nucleotide and amino acid sequences of human CD44 are shown. The underlined portion is the portion used to construct the CD44 / Notch / HIF-3α4 fusion gene. [Figure 2b-1] The nucleotide sequence of Notch is shown. [Figure 2b-2] The nucleotide sequence of Notch (continued) is shown. The underlined portion is the portion used to construct the CD44 / Notch / HIF-3α4 fusion gene. [Figure 2b-3] The nucleotide sequence of Notch (continued) is shown. [Figure 2b-4] The amino acid sequence of Notch is shown, with the underlined portion being the portion used to construct the CD44 / Notch / HIF-3α4 fusion gene. [Figure 2c] The nucleotide and amino acid sequences of human HIF-3α4 are shown. The underlined portion is the portion used to construct the CD44 / Notch / HIF-3α4 fusion gene. [Figure 3]The results show that cells were infected in vitro with each recombinant adenovirus vector, and whether each gene was successfully introduced was evaluated by real-time PCR. [Figure 4] Cells were infected in vitro with various recombinant adenovirus vectors, including ADX730 (a recombinant adenovirus vector incorporating the CD44 / Notch / HIF-3α4 fusion gene), and a real-time PCR test was used to compare whether the decoy function of ADX730's CD44 decoy receptor suppresses the expression of the CD44 downstream genes, Survivin and CCL2. [Figure 5] Cells were infected in vitro with each recombinant adenovirus vector containing ADX730, and a real-time PCR test was used to compare whether ADX730's inhibitory effect on HIF-1α function by HIF-3α4 suppressed the expression of HIF-1α target genes, VEGF, CyclinG2, and Bcl-xL. [Figure 6] The results show that recombinant adenoviral vectors containing ADX730 were administered intratumorally (injected) into nude mice transplanted with MDA-MB-231 human triple-negative breast cancer cells, and their tumor growth inhibitory effects were examined. The upper panel shows an outline of the administration schedule. The lower panel shows a graph showing how tumor volume changed following administration of each recombinant adenoviral vector or PBS (phosphate-buffered saline). [Figure 7] Photographs (n=5) of excised tumors are shown when each recombinant adenoviral vector containing ADX730 or PBS was administered intratumorally (injected) into nude mice transplanted with MDA-MB-231 human triple-negative breast cancer cells, according to the administration schedule shown in the upper part of Figure 6. [Figure 8a]The recombinant adenovirus vector was infected into cells in vitro, and the successful introduction of the CD44 / Notch / HIF-3α4 fusion gene was evaluated by real-time PCR using DU-145 cells. [Figure 8b] Cells were infected in vitro with a recombinant adenovirus vector, and the successful introduction of the CD44 / Notch / HIF-3α4 fusion gene was evaluated by real-time PCR using MKN45 cells. [Figure 8c] The recombinant adenovirus vector was used to infect cells in vitro, and the results of a real-time PCR test using PANC-1 cells were used to evaluate whether the CD44 / Notch / HIF-3α4 fusion gene was successfully introduced. DETAILED DESCRIPTION OF THE INVENTION

[0011] Each embodiment included in the present disclosure will be described in more detail below. The present disclosure preferably includes, for example, a specific artificial nucleic acid, a viral vector incorporating the artificial nucleic acid, an anticancer composition containing the viral vector, and a method for treating cancer using the composition, but is not limited thereto. The present disclosure includes all that is disclosed herein and that would be recognized by a person skilled in the art.

[0012] The artificial nucleic acid encompassed by the present disclosure is preferably a nucleic acid having a structure in which (A) a nucleic acid encoding a protein having CD44 extracellular function, (B) a nucleic acid encoding a protein having Notch core region function, and (C) a nucleic acid encoding a protein having HIF-3α4 function are linked in the order (A), (B), and (C). Such nucleic acid may be referred to as the "nucleic acid of the present disclosure." In the present disclosure, the nucleic acid may be DNA, RNA, PNA, etc., but DNA is particularly preferred. Furthermore, in the nucleic acid having a structure in which the nucleic acids are linked in the order (A), (B), and (C), the (A) side may be either the 3'-end or the 5'-end, but is preferably the 5'-end.

[0013] CD44 is a receptor for hyaluronan and other molecules. Upon binding to its ligand (e.g., hyaluronan), it clusters and transmits signals. This is known to result in intracellular events such as the activation of various kinases involved in cell proliferation and migration, such as c-Src, FAK, and MAPK. Furthermore, after signal transduction, the intracellular domain is cleaved by proteases, translocating to the nucleus, and the detached extracellular domain is released as soluble CD44. Due to these properties, CD44 is highly expressed in many types of cancer cells, including colon cancer, breast cancer, gastric cancer, pancreatic cancer, and prostate cancer, and is also being studied as a marker for cancer stem cells.

[0014] In the present disclosure, the extracellular function of CD44 refers to the receptor function for a ligand, and preferably includes, for example, the ability to bind to hyaluronic acid.

[0015] Thus, a protein having the extracellular function of CD44 may be, for example, a protein of the entire extracellular portion of CD44, or may include the cell membrane portion of CD44, or may be a protein of a portion of the extracellular portion of CD44, so long as it does not impair the receptor function for a ligand. Furthermore, such a protein may have one or more amino acids deleted, substituted, or added, so long as it retains the function. There are no particular limitations on the nucleic acid (A), as long as it is a nucleic acid that encodes such a polypeptide.

[0016] More specifically, preferred examples of the nucleic acid (A) include the following (a-1) and (a-2): (a-1): A nucleic acid consisting of the base sequence of SEQ ID NO: 1 (a-2): A nucleic acid comprising a base sequence in which one or more (e.g., 1 to 30, 1 to 20, 1 to 10, or 1, 2, 3, 4, or 5) bases are deleted, substituted, or added in the base sequence of (a-1), and encoding a protein capable of binding to hyaluronic acid.

[0017] Whether a protein can bind to hyaluronic acid can be determined, for example, by determining their dissociation constants.

[0018] Notch is a receptor expressed on the cell surface, and the Notch signaling pathway is one of the major signaling pathways responsible for intercellular signaling. In most cases, the mechanism of intercellular signaling is a soluble ligand produced and released by the signal-sender cell, which binds to a receptor on the cell surface of the receiver cell. In the receiver cell, downstream signaling pathways, including phosphorylation cascades, are activated, resulting in changes in the activity of specific transcription factors, thereby regulating gene expression. However, the Notch signaling pathway is characterized by signal transduction through direct interactions between neighboring cells. The Notch receptor binds to its ligands, Delta or Serrate (Jagged in mammals), on the cell surface. Further conformational changes due to physical forces induce a release of the Notch intracellular domain through the action of protease enzymes, such as ADAM protease or γ-secretase. The released Notch intracellular domain then translocates to the nucleus, where it interacts with transcription factors and coactivators upstream of target genes to regulate their transcription.

[0019] In the present disclosure, the Notch core region is a region that contains a site that is cleaved by the action of a protease, and the Notch core region function is the function of being able to be cleaved (preferably, be separated by restriction) by a protease that can cleave the Notch core region.

[0020] Thus, a protein having the function of a Notch core region may be, for example, a protein containing only the portion of the Notch core region necessary for protease cleavage, or a protein containing the portion necessary for protease cleavage together with one or more (e.g., 1 to 30, 1 to 20, 1 to 10, or 1, 2, 3, 4, or 5) amino acids before and after the portion necessary for protease cleavage, as long as it is cleaved by the action of a protease. Furthermore, as long as it is cleaved by the action of a protease, such a protein may have one or more (e.g., 1 to 30, 1 to 20, 1 to 10, or 1, 2, 3, 4, or 5) amino acids deleted, substituted, or added. Nucleic acid (B) is not particularly limited, as long as it is a nucleic acid that encodes such a protein.

[0021] More specifically, preferred examples of the nucleic acid (B) include the following (b-1) and (b-2): (b-1): a nucleic acid consisting of the base sequence of SEQ ID NO: 2 (b-2): A nucleic acid consisting of a base sequence in which one or more bases are deleted, substituted, or added in the base sequence of (b-1), and encoding a protein that can be cleaved by a protease.

[0022] As mentioned above, preferred proteases include ADAM protease and γ-secretase, and it is more preferable to encode a polypeptide that is cleaved by either or both of these proteases. ADAM protease is a proteolytic enzyme that belongs to the group known as the disintegrin and metalloprotease family.

[0023] Whether a protein can be cleaved by a protease can be confirmed by treating the protein with a protease and then subjecting the protein to electrophoresis (for example, SDS-PAGE).

[0024] Furthermore, (b-2) is more preferably a nucleic acid that encodes a protein that can be cleaved by an intracellular protease when a ligand binds to the protein encoded by nucleic acid (A), in a protein encoded by a nucleic acid having a structure in which one or more bases are deleted, substituted, or added in the base sequence of (b-2'):(b-1), and in which the nucleic acid has a structure in which the bases (A), (B), and (C) are linked in that order.

[0025] HIF (Hypoxia Inducible Factor) is a transcription factor activated by intracellular hypoxia. It is a heterodimer consisting of HIF-1α and HIF-1β. HIF-1α is not only degraded by PHDs under normal oxygen levels, but also suppressed by a transcription factor called IPAS (Inhibitory PAS domain protein), which was discovered in mice. IPAS was identified as a splicing variant of HIF-3α, a type of HIF. While IPAS does not exhibit transcriptional activity itself, it inhibits HIF-1α's DNA binding by interacting with it, thereby suppressing its function. In humans, HIF-3α4, a splicing variant of HIF-3α, has been identified and is shown to function similarly to IPAS.

[0026] Therefore, in the present disclosure, the HIF-3α4 function is the function of being able to suppress HIF-1α, and more specifically, the function of interacting with (binding to) HIF-1α.

[0027] Therefore, a protein having HIF-3α4 function may be, for example, HIF-3α4 itself, or a protein in which one or more (e.g., 1 to 30, 1 to 20, 1 to 10, or 1, 2, 3, 4, or 5) amino acids have been further added to HIF-3α4, so long as it is capable of suppressing HIF-1α. Furthermore, such a protein may have one or more (e.g., 1 to 30, 1 to 20, 1 to 10, or 1, 2, 3, 4, or 5) amino acids deleted, substituted, or added, so long as it is capable of suppressing HIF-1α. Nucleic acid (C) is not particularly limited, as long as it is a nucleic acid that encodes such a protein.

[0028] More specifically, preferred examples of the nucleic acid (C) include the following (c-1) and (c-2): (c-1): a nucleic acid consisting of the base sequence of SEQ ID NO: 3 (c-2): A nucleic acid consisting of a base sequence in which one or more bases are deleted, substituted, or added in the base sequence of (c-1), and encoding a protein capable of binding to HIF-1α.

[0029] Whether a protein can bind to HIF-1α can be examined by co-immunoprecipitation or the like using the target protein, HIF-1α, and antibodies that recognize them.

[0030] Furthermore, in linking nucleic acid (A), nucleic acid (B), and nucleic acid (C), these nucleic acids may be linked directly, or each nucleic acid may be linked via a linker. The linker is not particularly limited as long as it does not impair the effects of the nucleic acid of the present disclosure, but is preferably, for example, a nucleic acid consisting of one or more bases.

[0031] The base length of the nucleic acid of the present disclosure (nucleic acid having a structure in which (A)-(B)-(C) are linked in this order) is preferably, for example, 8000 bp or less, and more preferably 7500, 7000, 6500, 6000, 5500, 5000, 4500, 4000, 3500, or 3000 bp or less.

[0032] A preferred embodiment of the nucleic acid of the present disclosure includes (d-1) a nucleic acid consisting of the base sequence of SEQ ID NO: 4. Another preferred embodiment of the nucleic acid of the present disclosure includes (d-2) a nucleic acid consisting of the base sequence of SEQ ID NO: 4 in which one or more bases have been deleted, substituted, or added, and in which a portion encoding a protein capable of binding to hyaluronic acid, a portion encoding a protein that can be cleaved by protease, and a portion encoding a protein capable of binding to HIF-1α are linked in this order. A further example includes (d-3) a nucleic acid consisting of the base sequence of SEQ ID NO: 4 in which one or more bases have been deleted, substituted, or added, and encoding a protein having anticancer activity. Whether a protein encoded by a nucleic acid has anticancer activity can be examined by expressibly incorporating the nucleic acid into an adenovirus vector and administering it to cancer cells.

[0033] In addition, in (a-2), (b-2), (b-2'), (c-2), (d-2), or (d-3), the number of bases to be deleted, substituted, or added, or the number of bases constituting the linker, is, for example, preferably 1 to 100 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 1 , 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). Furthermore, the type of base in the present disclosure is not particularly limited, but is preferably A (adenithine), T (thymine), G (guanine), C (cytosine), or U (uracil).

[0034] The nucleic acids of the present disclosure can be produced by known methods or methods that can be easily derived from known methods. For example, they can be produced by genetic engineering techniques. For example, they can be produced by extracting DNA or RNA encoding CD44, Notch, and HIF-3α4 from a human sample, artificially mutating them as necessary, and then ligating them, or by chemical synthesis.

[0035] Without wishing to be bound by theory, it is believed that the anti-cancer effect of the protein encoded by the nucleic acid of the present disclosure is due to the following mechanism of action. The fusion protein encoded by the nucleic acid of the present disclosure includes (i) a protein portion having CD44 extracellular function, (ii) a protein portion having Notch core region function, and (iii) a protein portion having HIF-3α4 function. Therefore, (i) can function as a decoy receptor and transmit a signal to (iii) via (ii). This is more effective when (i) is fused upstream of (ii). Furthermore, by fusing (iii) downstream of (ii), HIF-1α activated in tumors can be suppressed. This is believed to enable the gene inserted into the viral vector to be kept within an acceptable range and to exert an anti-tumor effect consisting of multiple mechanisms of action.

[0036] A vector incorporating the nucleic acid of the present disclosure in an expressible manner is useful for cancer treatment or for increasing the production of the nucleic acid of the present disclosure. Examples of vectors include plasmid vectors, cosmid vectors, fosmid vectors, and viral vectors. When using the nucleic acid of the present disclosure for cancer treatment, viral vectors are particularly suitable. Examples of viral vectors include adenoviral vectors, retroviral vectors, lentiviral vectors, and Sendai viral vectors. Among these, adenoviral vectors are preferred. Both replicative and non-replicative viral vectors can be used. In particular, replicative or non-replicative adenoviral vectors are preferred. The nucleic acid of the present disclosure can be incorporated into a vector by known methods or methods that can be easily derived from known methods.

[0037] An anti-cancer composition comprising a vector incorporating a nucleic acid of the present disclosure in an expressible manner exhibits an extremely excellent anti-cancer effect (particularly an effect in cancer treatment). The administration form of the anti-cancer composition is not particularly limited as long as the anti-cancer effect is exhibited. For example, intratumoral administration is generally preferred, but depending on the vector, intravenous administration may also be used. Furthermore, the dosage form of the anti-cancer composition is not particularly limited as long as the anti-cancer effect is exhibited. For example, an injection is preferred.

[0038] The anti-cancer composition may contain other components besides the vector as needed, such as pharmaceutically acceptable carriers (e.g., water), and an appropriate carrier may be selected depending on the treatment site and the dosage form of the anti-cancer composition.

[0039] Such anti-cancer compositions can also be prepared based on known methods.

[0040] The types of cancers that can be treated with the anticancer composition are not particularly limited as long as they exhibit anticancer effects. Examples include solid tumors and hematological tumors. More specifically, examples include, but are not limited to, brain tumors, maxillary cancer, nasopharyngeal cancer, lung cancer, esophageal cancer, rectal cancer, colon cancer, liver cancer, stomach cancer, gallbladder cancer, pancreatic cancer, skin cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, kidney cancer, bladder cancer, thyroid cancer, multiple myeloma, lymphoma, acute myeloid leukemia, and chronic myeloid leukemia. The anticancer composition is particularly effective against breast cancer, prostate cancer, stomach cancer, and pancreatic cancer. Because the anticancer effect of the anticancer composition is so high, it can be used for cancers that are ineffective or only minimally affected by existing cancer treatments, which is preferable. For example, it can be preferably used for triple-negative (estrogen receptor-negative, progesterone receptor-negative, HER2-negative) breast cancer. The type of anticancer effect of the anticancer composition is also not particularly limited. For example, those capable of suppressing tumor growth and / or invasion are preferred.

[0041] In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." Furthermore, the present disclosure encompasses all arbitrary combinations of the constituent elements described in this specification.

[0042] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein. [Example]

[0043] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0044] <Experimental Method> Construction of insert DNA (CD44 / Notch / HIF-3α4 fusion gene) A nucleic acid having a structure in which (A) a nucleic acid encoding a protein having CD44 extracellular function, (B) a nucleic acid encoding a protein having Notch core domain function, and (C) a nucleic acid encoding a protein having HIF-3α4 function are linked in the order (A), (B), and (C) was prepared as follows. Note that a preferred embodiment of the nucleic acid actually prepared in this example is also referred to as a "CD44 / Notch / HIF-3α4 fusion gene."

[0045] Construction of the CD44 / Notch / HIF-3α4 fusion gene was outsourced to GENEWIZ Solid Science Superior Service. DNA fragments from each of these three human genes were digested with the restriction enzyme Swal (TaKaRa, Siga, Japan), electrophoresed, and purified before use as insert DNA. Each domain of the fusion gene was designed with reference to published literature (particularly the above-mentioned Non-Patent Documents 9, 10, and 12), the transmembrane domain search tool TMHMM (http: / / www.cbs.dtu.dk / services / TMHMM / ), and the signal peptide sequence search tool SignalP (http: / / www.cbs.dtu.dk / services / SignalP / ). The sequence of the constructed CD44 / Notch / HIF-3α4 fusion gene is shown in Figure 1. The nucleotide sequences of the three human genes used (CD44, Notch, and HIF-3α4) are shown in Figures 2a to 2c, respectively.

[0046] Preparation of vector DNA The vector DNA used to integrate the insert DNA was the pAxCAwtit2 cosmid vector included in the Adenovirus Dual Expression Kit (TaKaRa, Siga, Japan). As with the insert DNA, the cosmid vector was digested with a restriction enzyme at the SwaI sequence in the cloning site and purified by phenol-chloroform extraction.

[0047] Construction of recombinant cosmid vectors and transformation into E. coli To ligate the insert DNA and the pAxCAwtit2 cosmid vector prepared above, a ligation reaction (16°C, 30 minutes) was performed using Ligation Mix (TaKaRa, Siga, Japan). Following the reaction, the reaction mixture was used to transform E. coli HST08 Premium Competent Cells (TaKaRa, Siga, Japan) according to the kit's instructions. The transformed E. coli HST08 Premium Competent Cells were plated on LB agar medium (Nacalai Tesque, Kyoto, Japan) supplemented with 100 μg / ml ampicillin and cultured overnight at 37°C. After incubation, insert-check PCR was performed using KOD FX Neo (TOYOBO CO., LTD., Osaka, Japan) with colonies grown on the LB agar medium as templates, and successful transformation was confirmed by agarose gel electrophoresis. After confirmation, suitable colonies were subcultured in LB liquid medium (Nacalai Tesque, Kyoto, Japan) supplemented with 100 μg / ml ampicillin, and the cosmid vector was extracted from the culture using the Genopure Plasmid Maxi Kit (Roche). Because the extracted cosmid vector exceeded 40 kbp in size, further restriction enzyme digestion with NruI (New England BioLabs) and ligation were performed to delete most of the adenovirus genome and reduce the cosmid size. The sequence of the reduced cosmid vector extracted in the same manner was confirmed by DNA sequencing.

[0048] Construction of recombinant adenovirus vectors by restriction enzyme digestion and phenol-chloroform extraction The recombinant cosmid vector DNA prepared and sequence-confirmed as described above was digested with the restriction enzyme BspT104I (TaKaRa, Siga, Japan). After digestion, the digest was purified by phenol-chloroform extraction and ethanol precipitation and dissolved in 30 μl of sterile purified water. Agarose gel electrophoresis was performed using 1 μl of the digested DNA to confirm digestion with the restriction enzyme BspT104I. After this, HEK293 cells (National Institutes of Biomedical Innovation, Health and Nutrition) cultured to confluence in 60 mm cell culture petri dishes (TPP) were lipofected with 10 μg of the BspT104I-digested cosmid using Lipofectamine LTX (Invitrogen, Waltham, MA). The cultured cells were then harvested and seeded onto Biocoat Collagen I Cellware 96-well plates (Corning, NY, USA). Five and ten days after seeding, 50 μl of Dulbecco's modified Eagle's medium (D-MEM; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing 10% fetal bovine serum (FBS; Sigma Aldrich, St. Louis, MO) was added to each well, and the cells were further cultured until complete cell denaturation. Eighteen days after seeding, the cells were collected from four wells where complete cell denaturation had occurred, transferred to a 1.5 ml Eppendorf tube, and subjected to six cycles of freezing in liquid nitrogen and thawing in a 37°C water bath. After the final freeze-thaw cycle, the cells were centrifuged (5,000 rpm, 5 minutes, 4°C), and the collected supernatant was stored as the primary virus solution. All of these processes were performed according to the Adenovirus Dual Expression Kit (TaKaRa, Siga, Japan) manual.

[0049] The adenoviral vector into which the CD44 / Notch / HIF-3α4 fusion gene has been incorporated in an expressible manner is referred to as ADX730.

[0050] Purification of high-titer recombinant adenoviral vectors HEK293 and A549 cells (National Institutes of Biomedical Innovation, Health and Nutrition) were cultured in Biocoat Collagen I Cellware 24-well plates (CORNING, NY, USA) until they reached 70–100% confluence. After incubation, the medium was removed, and 0.1 ml of 5% FBS-D-MEM and 10 μl of the primary virus solution prepared above were added per well to infect each cell. During infection, the plate was gently shaken several times in an incubator (37°C, 5% CO2) every 15 minutes for four cycles. After 1 hour of infection, an additional 0.4 ml of 5% FBS-D-MEM was added to each well and the cells were cultured for three days. After incubation, one clone was selected that showed no degeneration of A549 cells but complete degeneration of HEK293 cells, and the cells were harvested along with the culture medium. The collected cells were frozen in liquid nitrogen and thawed in a 37°C water bath six times, in the same manner as the primary virus solution. After the final freeze-thaw cycle, the cells were centrifuged (5,000 rpm, 5 minutes, 4°C), and the collected supernatant was stored as the secondary virus solution.

[0051] Next, Collagen Type I-Coated 25cm 2 HEK293 cells were cultured in a flask (IWAKI) until they reached 70-100% confluence. 0.5 ml of 5% FBS-D-MEM and 15 μl of the secondary virus solution prepared above were gently added to the plate for infection. During infection, the plate was gently shaken several times in an incubator (37°C, 5% CO2) every 15 minutes, four times. After 1 hour of infection, an additional 4.5 ml of 5% FBS-D-MEM was added to each well and the cells were cultured for three days. After culturing, it was confirmed that all cells had denatured, and the cells were harvested along with the culture medium. The harvested cells were frozen in liquid nitrogen and thawed in a 37°C water bath six times, similar to the primary virus solution. After the final freeze-thaw cycle, the cells were centrifuged (3,000 rpm, 10 minutes, 4°C), and the collected supernatant was stored as the tertiary virus solution. Furthermore, the cells were frozen in a Collagen Type I-coated 75cm 2HEK293 cells were cultured in a flask (IWAKI) until they reached 70–100% confluence. 2 ml of 5% FBS-D-MEM and 50 μl of the tertiary virus solution prepared above were gently added to the cells for infection. During infection, the plate was gently shaken several times in an incubator (37°C, 5% CO2) every 15 minutes for four cycles. After 1 hour of infection, an additional 13 ml of 5% FBS-D-MEM was added to each well and the cells were cultured for three days. After incubation, all cells were confirmed to have denatured, and the cells were harvested along with the culture medium. The harvested cells were frozen in liquid nitrogen and thawed six times in a 37°C water bath, similar to the primary virus solution. After the final freeze-thaw cycle, the cells were centrifuged (3,000 rpm, 10 minutes, 4°C), and the collected supernatant was stored as the quaternary virus solution. All of these steps were carried out according to the instructions in the Adenovirus Dual Expression Kit (TaKaRa, Siga, Japan).

[0052] Confirmation of the structure of recombinant adenovirus vector DNA When preparing the quaternary virus solution and storing it in aliquots as described above, one sample was centrifuged (5,000 rpm, 5 minutes, 4°C) after the final freeze-thaw cycle, and the entire supernatant was removed to collect and store only the cells (cell pack). The following reagents were added to the cell pack to make the total volume 400 μl.

[0053] 10x TNE Buffer 40 μl Proteinase K (20mg / ml) 4μl Sterile purified water up to 400μl

[0054] The prepared cell pack was thoroughly mixed by vortexing, then 4 μl of 10% SDS was added and further mixed by vortexing. After incubation at 50°C for 1 hour, phenol-chloroform extraction and chloroform extraction were performed twice, followed by ethanol precipitation. After ethanol precipitation, the sample was dissolved in 50 μl of TE buffer containing RNase A, and 15 μl of the solution was used for restriction enzyme digestion with the restriction enzyme XhoI. After restriction enzyme digestion, the electrophoretic pattern of the resulting product was confirmed by agarose gel electrophoresis. All of these steps were performed according to the manual for the Adenovirus Dual Expression Kit (TaKaRa, Siga, Japan).

[0055] Additionally, PCR reactions were performed using pre-designed and prepared insert DNA-specific primers (Table 1) with the cell pack as a template. The resulting PCR products were subjected to agarose gel electrophoresis to confirm the migration pattern of the insert DNA. At the same time, primers specific to the cosmid vector DNA and primers for sequence analysis were designed (Table 2), and PCR reactions were performed using the cell pack as a template. The resulting PCR products were subjected to agarose gel electrophoresis and purification, and the sequences were confirmed by DNA sequence analysis using the resulting PCR products as templates.

[0056] [Table 1]

[0057] [Table 2]

[0058] Large-scale cultivation and purification of recombinant adenovirus vectors The recombinant adenovirus vector constructed above was cultured in large quantities and purified for use in subsequent experiments. 2HEK293 cells were seeded onto five flasks (CORNING, NY, USA) at 70–100% confluence. 15 ml of 5% FBS-D-MEM and 150 μl of the tertiary virus solution prepared above were gently added to the plates for virus infection. During infection, the plates were gently shaken several times in an incubator (37°C, 5% CO2) every 15 minutes for four cycles. After 1 hour of infection, an additional 35 ml of 5% FBS-D-MEM was added to each well and the plates were cultured for three days. After incubation, all cells were confirmed to be denatured, and the cells were harvested along with the culture medium. After harvesting, the cells were centrifuged (3000 rpm, 10 minutes, 4°C), the supernatant was removed, and 5 ml of fresh 10% FBS-D-MEM was added. The cell pellet was then resuspended in 5 ml of fresh 10% FBS-D-MEM. The cells were then frozen in liquid nitrogen and thawed in a 37°C water bath six times. After the final freeze-thaw cycle, cells were again collected by centrifugation (3000 rpm, 10 min, 4°C). The supernatant was collected, and 5 ml of Benzonase Nuclease (25 U / μl) (TaKaRa, Siga, Japan) was added and incubated (37°C, 30 min). An equal volume of 1× Dilution Buffer (TaKaRa, Siga, Japan) was then added, and the lysate was filtered through a 0.45 μm Syringe-tip Pre-filter (TaKaRa, Siga, Japan) using a 20 ml syringe (TaKaRa, Siga, Japan). At the same time, 5 ml of 1× Equilibration Buffer (TaKaRa, Siga, Japan) was added dropwise to the filter of an Adeno-X Maxi Purification Assembly (TaKaRa, Siga, Japan) at a flow rate of 3 ml / min (~1 drop / sec) to equilibrate the filter. The filtered lysate was passed through an equilibrated filter to trap the virus, and the filter was then washed with 20 ml of 1× Wash Buffer (TaKaRa, Siga, Japan).After washing, the filter was removed and attached to a 5 ml syringe (TaKaRa, Siga, Japan) containing 3 ml of 1x Elution Buffer (TaKaRa, Siga, Japan), and 1 ml of 1x Elution Buffer was extruded to recover the virus. After recovery, the filter was incubated once (room temperature, 5 minutes), and then the remaining 2 ml of 1x Elution Buffer was extruded to elute the virus. All of these steps were performed according to the Adeno-X Maxi Purification Kit (TaKaRa, Siga, Japan) manual.

[0059] Titering recombinant adenoviral vectors The titer of the recombinant adenovirus vector prepared above was measured as follows. HEK293 cells were seeded into 12-well flat-bottom cell culture plates (CORNING, NY, USA). Ten-fold diluted virus solutions, ranging from 1:100 to 1:10,000,000, were added dropwise to each well and cultured for two days. After incubation, the medium was removed, the cells were allowed to dry slightly, and 1 ml of chilled methanol (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was added dropwise and incubated (-20°C, 10 minutes). The methanol was then removed, and the cells were washed three times with Dulbecco's PBS (-) "Nissui" (PBS; NISSUI PHARMACEUTICAL CO., LTD., Tokyo, Japan) containing 1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO). After washing, 0.5 ml of Mouse Anti-Hexon Antibody (TaKaRa, Siga, Japan) diluted 1:1000 in PBS containing 1% BSA was added dropwise and incubated with shaking on a shaker (37°C, 1 hour). After the reaction, the plate was washed three times with PBS containing 1% BSA. 0.5 ml of HRP-conjugated Rat Anti-Mouse Antibody (TaKaRa, Siga, Japan) diluted 5:500 in PBS containing 1% BSA was added dropwise and incubated with shaking on a shaker (37°C, 1 hour). After the reaction, the plate was washed three times with PBS containing 1% BSA. 0.5 ml of 10x DAB Substrate (TaKaRa, Siga, Japan) diluted 1:10 in 1x Stable Peroxidase Buffer (TaKaRa, Siga, Japan) was added dropwise and incubated (room temperature, 10 minutes). After the reaction, the reaction mixture was removed and 1 ml of PBS was added. The titer was determined by observing the degenerated cells under a microscope. All of these steps were performed according to the manual for the Adeno-X Rapid Titer Kit (TaKaRa, Siga, Japan).

[0060] Cells and media HEK293 cells were cultured in D-MEM containing 10% FBS and 1% 100 U / ml penicillin and 100 mg / ml streptomycin (P / S; Nacalai Tesque, Kyoto, Japan). A549 cells were cultured in Ham's F-12K medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing 10% FBS and 1% P / S at 37°C under 5% CO2. MDA-MB-231 cells (The European Collection of Cell Cultures) were cultured in Leibovitz's L-15 medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing 15% FBS and 1% P / S at 37°C under conditions without CO2 equilibration.

[0061] Various recombinant viruses The ADX730, recombinant adenovirus vector carrying the SOCS3 gene (rAd-SOCS3), recombinant adenovirus vector carrying the p53 gene (rAd-p53), and recombinant adenovirus vector carrying the LacZ gene (rAd-LacZ) used in the experiments were cultured in large quantities and purified. Afterwards, the buffer was exchanged using a Slide-A-Lyzer Dialysis Cassette (Extra Strength) (Thermo Fisher Scientific, Waltham, MA) into a dialysis buffer containing 10% glycerol (Nacalai Tesque, Kyoto, Japan) and 1% 1 mol / L Tris-HCl Buffer Solution (Nacalai Tesque, Kyoto, Japan). Appropriate aliquots of each were stored at −80°C.

[0062] rAd-SOCS3 and rAd-p53 are adenovirus vectors that are known candidates for cancer gene therapy drugs.

[0063] Flow cytometry ADX730 was actually transfected into cells in vitro, and the increase or decrease in the expression level of the CD44 region of the fusion gene incorporated into AD730 was evaluated using flow cytemetry. 1 × 10 cells were transfected into a 6-well flat bottom cell culture plate (CORNING, NY, USA). 6 MDA-MB-231 cells were seeded at 100 cells / well and cultured overnight at 37°C without CO2 equilibration. After incubation, cells were infected with ADX730, rAd-SOCS3, rAd-p53, or rAd-LacZ at a multiplicity of infection (MOI) of 40 and cultured for an additional 48 hours. Cells were then washed with PBS and harvested. Blocking was performed for 10 minutes at room temperature using Blocking One Histo (Nacalai Tesque, Kyoto, Japan). After blocking, cells were washed again with PBS and incubated with 200x diluted FITC anti-mouse / human CD44 clone IM7 (BioLegend, San Diego, CA) or 100x diluted FITC Rat IgG2a, κ isotype Ctrl clone RTK2758 (BioLegend, San Diego, CA) for 30 minutes on ice, protected from light. After incubation, the cells were washed again with PBS, and 100x diluted BD Pharmingen 7-AAD (BD Biosciences, San Diego, CA) was added dropwise and incubated on ice for 5 minutes in the dark. After incubation, the cells were washed with PBS and analyzed using a Guava easyCyte (Merck Millipore, Burlington, MA). Data analysis was performed using the accompanying InCyte software.

[0064] Real-time PCR Real-time PCR was used to confirm gene transfer by each recombinant adenoviral vector, including ADX730, and the effects of its function. 6 MDA-MB-231 cells were seeded at 1000 cells / well and incubated at 37°C, 5% CO2, 1% or less O2 in Anaeropack Kenki 5% (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan) and sodium hyaluronate (40–80 kDa) (PG Research, Tokyo, Japan). 2、 The cells were cultured overnight under 0.04 mg / ml conditions. After incubation, they were infected with ADX730, rAd-SOCS3, rAd-p53, or rAd-LacZ at an MOI of 40 and cultured for an additional 48 hours. The cells were then harvested and total RNA was extracted using NucleoSpin RNA (TaKaRa, Siga, Japan). cDNA was synthesized from the extracted RNA using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Siga, Japan). PCR and comparative C were performed using the primers (Table 3), TB Green Premix Ex Taq II (TaKaRa, Siga, Japan), and the Thermal Cycler Dice Real Time System (TaKaRa, Siga, Japan). t method (ΔΔC t Analysis was performed using the FTIR method.

[0065] [Table 3]

[0066] Mice and specimen collection To compare the antitumor effects of ADX730 with those of other cancer gene therapy drugs, an in vivo experiment was conducted using mice. Six-week-old female BALB / cAJcl-nu / nu mice (CLEA Japan, Inc., Tokyo, Japan) were treated with 1 × 10 MDA-MB-231 cells. 6 A mixture of 70 μl of cells and 70 μl of Matrigel Matrix Basement Membrane HC (CORNING, NY, USA) was subcutaneously inoculated into the right lumbar region of mice, and tumors were transplanted into five mice per group (n = 5). 14 days after transplantation, when tumor engraftment was confirmed, ADX730 (1 × 10 9 PFU / 50 μl), rAd-SOCS3 (1 × 10 9 PFU / 50 μl), rAd-p53 (1 × 10 9 PFU / 50 μl), rAd-LacZ (1 × 10 9 The tumors were intratumorally administered 50 μl of either 50 μl of 1000 PFU / 50 μl or 50 μl of PBS every other day for a total of eight times (Days 14, 16, 18, 20, 22, 24, 26, and 28). Tumor diameter measurements were performed twice a week for a total of five times from the day of virus administration. After measurements were completed, the tumors were collected and fixed with 4% Paraformaldehyde Phosphate Buffer Solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and stored at 4°C or -80°C. The tumor volume was determined by measuring the long diameter (L) and short diameter (W). 2 × L) / 2.

[0067] <Result> Flow cytometry Comparing the mean fluorescence intensity of each sample for CD44, it was found that the values ​​were higher when infected with ADX730 than when infected with other cancer gene therapy drugs rAd-SOCS3, rAd-p53, or the negative control rAd-LacZ (Table 4).

[0068] [Table 4]

[0069] Real-time PCR [Confirmation of transfection of each recombinant adenovirus vector] Cells were infected in vitro with each recombinant adenoviral vector, including ADX730, and the successful introduction of each gene was evaluated by real-time PCR. The TBP (TATA-Box binding protein) gene was used as an endogenous control gene (forward primer: 5'-GCCAGCTTCGGAGAGTTCTGGGATT-3', reverse primer: 5'-CGGGCACGAAGTGCAATGGTCTTTA-3'). The relative expression ratios compared to the control gene are shown in Figure 3. In study (a), we used primers targeting HIF-3α4 to confirm and compare the transfection of the CD44 / Notch / HIF-3α4 fusion gene with ADX730, demonstrating a significant increase in expression of the CD44 / Notch / HIF-3α4 fusion gene compared with other vectors. In study (b), we used primers targeting SOCS3 to confirm and compare the transfection of the SOCS3 gene with rAd-SOCS3, demonstrating a significant increase in expression of the SOCS3 gene compared with other vectors. In study (c), we used primers targeting p53 to confirm and compare the transfection of the p53 gene with rAd-p53, demonstrating a significant increase in expression of the p53 gene compared with other vectors.

[0070] [ADX730 suppresses CD44 downstream genes] Cells were infected in vitro with each recombinant adenovirus vector containing ADX730, and a real-time PCR test was used to compare whether the decoy function of ADX730's CD44 decoy receptor suppressed CD44 downstream genes (Figure 4). In test (a), primers targeting Survivin, a downstream gene of CD44, were used to compare their expression, and the suppression by ADX730 was confirmed. D44 / Notch / HIF-3α4 fusion gene In study (b), the expression of the Survivin gene was compared using primers targeting CCL2 downstream of CD44, and the effect of ADX730 on the expression of the Survivin gene was confirmed. D44 / Notch / HIF-3α4 fusion gene It was confirmed that the introduction of CCL2 significantly reduced the expression of the CCL2 gene.

[0071] [Suppression of HIF-3α4 target genes by ADX730] Cells were infected in vitro with each recombinant adenoviral vector containing ADX730, and real-time PCR was used to compare whether ADX730's suppression of HIF-3α4 by HIF-1α function resulted in suppression of target gene expression (Figure 5). In study (a), expression was compared using primers targeting VEGF, a HIF-1α target gene, and ADX730 transfection confirmed that expression was significantly reduced. In study (b), expression was compared using primers targeting CyclinG2, a HIF-1α target gene, and ADX730 transfection confirmed that expression was significantly reduced compared to rAd-SOCS3 and rAd-p53. In study (c), expression was compared using primers targeting Bcl-xL, a HIF-1α target gene, and ADX730 transfection confirmed that expression was significantly reduced compared to rAd-SOCS3.

[0072] In vivo testing Recombinant adenoviral vectors containing ADX730 (which can also be referred to as cancer gene therapy drugs in this study) were administered intratumorally (injected) into nude mice implanted with MDA-MB-231 human triple-negative breast cancer cells, and the mice were monitored for progression. Administration of each therapeutic drug began 14 days after cancer cell implantation into the nude mice, with eight doses administered every other day. Twenty-eight days after cancer cell implantation, tumor growth was significantly suppressed in the ADX730-treated group compared with the other therapeutic drug and control groups (Figure 6). After administration was completed, all tumors were removed and compared (Figure 7).

[0073] These results demonstrate that ADX730 exhibits a high therapeutic effect on cancer, even in cases where existing therapeutic drugs are difficult to treat, such as triple-negative breast cancer. Furthermore, in the above-mentioned Real-time PCR study, breast cancer-derived MDA-MB-231 cells were used, but the cells used were changed to prostate cancer-derived DU-145 cells, gastric cancer-derived MKN45 cells, or pancreatic cancer-derived PANC-1 cells, and gene transfer by each recombinant adenoviral vector containing ADX730 and the effects obtained by its function were confirmed by Real-time PCR. The number of cells seeded was 5 × 10 5 Using a primer set targeting HIF-3α4 (Table 3), we confirmed and compared the transfection of the CD44 / Notch / HIF-3α4 fusion gene with ADX730. The results are shown in Figure 8a (DU-145 cells), Figure 8b (MKN45 cells), and Figure 8c (PANC-1 cells). Regardless of the cell type, we confirmed that the expression of the CD44 / Notch / HIF-3α4 fusion gene was significantly increased.

Claims

1. (A) a nucleic acid encoding a protein derived from the CD44 extracellular domain and capable of binding to hyaluronic acid; (B) a nucleic acid encoding a protein derived from the Notch core region and capable of being cleaved by a protease (C) a nucleic acid encoding a protein derived from HIF-3α4 and capable of binding to HIF-1α; A nucleic acid having a structure in which (A)-(B)-(C) are linked in this order.

2. A nucleic acid having a structure in which a nucleic acid (A), a nucleic acid (B), and a nucleic acid (C) are linked in the order of (A)-(B)-(C), The nucleic acid (A) is (a-1): a nucleic acid consisting of the base sequence of SEQ ID NO: 1, or (a-2): A nucleic acid consisting of a base sequence in which 1 to 73 bases are deleted, substituted, or added in the base sequence of (a-1), and encoding a protein capable of binding to hyaluronic acid. and The nucleic acid (B) is (b-1): a nucleic acid consisting of the base sequence of SEQ ID NO: 2, or (b-2): A nucleic acid consisting of a base sequence in which 1 to 100 bases are deleted, substituted, or added in the base sequence of (b-1), and encoding a protein that can be cleaved by a protease. and The nucleic acid (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 in which 1 to 100 bases are deleted, substituted, or added in the base sequence of (c-1), and encoding a protein capable of binding to HIF-1α. Nucleic acid.

3. The nucleic acid (A) is (a-1): a nucleic acid consisting of the base sequence of SEQ ID NO: 1, or (a-2): A nucleic acid consisting of a base sequence in which 1 to 73 bases are deleted, substituted, or added in the base sequence of (a-1), and encoding a protein capable of binding to hyaluronic acid. and The nucleic acid (B) is (b-1): a nucleic acid consisting of the base sequence of SEQ ID NO: 2, or (b-2): A nucleic acid encoding a protein that can be cleaved by a protease, which is composed of a base sequence in which 1 to 100 bases are deleted, substituted, or added in the base sequence of (b-1) and has a structure in which the bases (A), (B), and (C) are linked in this order. and The nucleic acid (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 in which 1 to 100 bases are deleted, substituted, or added in the base sequence of (c-1), and encoding a protein capable of binding to HIF-1α. That is, The nucleic acid of claim 1.

4. (d-1) consists of the base sequence of SEQ ID NO: 4; (d-2) The base sequence of SEQ ID NO: 4 is a base sequence in which 1 to 100 bases are deleted, substituted, or added, and has a structure in which a portion encoding a protein capable of binding to hyaluronic acid, a portion encoding a protein that can be cleaved by a protease, and a portion encoding a protein capable of binding to HIF-1α are linked in this order, or (d-3) A base sequence in which 1 to 100 bases are deleted, substituted, or added in the base sequence of SEQ ID NO: 4, and which encodes a protein having an anticancer effect. Nucleic acid.

5. The nucleic acid according to any one of claims 1 to 4, wherein the protease is an ADAM protease or γ-secretase.

6. A nucleic acid having a structure in which the nucleic acid (A), the nucleic acid (B), and the nucleic acid (C) are linked in the order of (A)-(B)-(C) according to any one of claims 1 to 3 and 5, or a protein encoded by the nucleic acid according to claim 4.

7. A nucleic acid having a structure in which the nucleic acid (A), the nucleic acid (B), and the nucleic acid (C) according to any one of claims 1 to 3 and 5 are linked in the order of (A)-(B)-(C), or a vector into which the nucleic acid according to claim 4 has been incorporated in an expressible manner.

8. The vector of claim 7 , wherein the vector is an adenovirus vector.

9. An anti-cancer composition comprising the nucleic acid according to any one of claims 1 to 3 and 5, having a structure in which the nucleic acid (A), the nucleic acid (B), and the nucleic acid (C) are linked in the order of (A)-(B)-(C), or the nucleic acid according to claim 4, or the vector according to claim 7 or 8.

10. The anticancer composition according to claim 9, which is an injectable preparation.

11. The anti-cancer composition according to claim 9 or 10, which is for treating breast cancer, prostate cancer, gastric cancer, or pancreatic cancer.

12. The anticancer composition according to claim 9 or 10, which is for treating triple-negative breast cancer.

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