Anti-cancer-associated non-tumor cell agent comprising virus

Abstract

To provide, in one embodiment, an anti-cancer-associated non-tumor cell agent.SOLUTION: In one embodiment, the invention relates to an anti-cancer-associated non-tumor cell agent comprising an oncolytic virus comprising a p53 gene. In one embodiment, the invention relates to an anti-cancer-associated non-tumor cell agent comprising a recombinant virus comprising a first gene cassette comprising a telomerase reverse transcriptase promoter, an E1A gene, an IRES sequence and an E1B gene, and a second gene cassette comprising a promoter and a p53 gene.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an anti-cancer-related non-tumor cell agent comprising a virus, and a pharmaceutical composition comprising the anti-cancer-related non-tumor cell agent. It relates to pharmaceutical compositions, etc. [Background technology]

[0002] The tumor microenvironment is responsible for tumor growth, progression, invasion, metastasis, angiogenesis, metabolism, immunosuppression, and chemoattractant metabolism. The tumor microenvironment is thought to play an important role in cancer-related non-cancer diseases. It is composed of tumor cells and extracellular matrix, and cancer-associated non-tumor cells include tumor-associated fibroblasts. Cells (Cancer-associated fibroblasts, CAFs), tumor-associated macrophages, immune inflammatory cells , and mesenchymal stem cells, etc.

[0003] As mentioned above, the tumor microenvironment is involved in tumor growth, etc., and therefore, Agents that damage cancer-associated non-tumor cells may be useful in the treatment and / or prevention of cancer. In addition, the tumor microenvironment is also involved in drug resistance, and therefore, it is important to understand how the tumor microenvironment can damage cancer-related non-tumor cells. These drugs can also be used to enhance the effects of anticancer drugs in cancers that are drug-resistant. It is thought that this is the case.

[0004] However, no effective anti-cancer-related non-tumor cell agents are known to date. Summary of the Invention [Problem to be solved by the invention]

[0005] In one embodiment, the present invention aims to provide an anti-cancer-related non-tumor cell agent. [Means for solving the problem]

[0006] The present inventors have developed an oncolytic virus containing the p53 gene and a telomerase reverse transcriptase promoter. a first gene cassette comprising a promoter, an E1A gene, an IRES sequence, and an E1B gene; and a recombinant virus containing a promoter and a second gene cassette containing a p53 gene; The present invention was completed based on the discovery that the compound damages cancer-associated non-tumor cells.

[0007] The present invention includes the following embodiments. (1) Anti-cancer related non-tumor cell agents, including oncolytic viruses containing the p53 gene. (2) A vector containing a telomerase reverse transcriptase promoter, an E1A gene, an IRES sequence, and an E1B gene. a first gene cassette containing the a promoter and a second gene cassette containing the p53 gene An anti-cancer-related non-tumor cell agent, comprising a recombinant virus comprising: (3) The anti-cancer-related non-tumor cell according to (2), wherein the recombinant virus is an oncolytic virus. Agent. (4) The first gene cassette contains the telomerase reverse transcriptase promoter, the E1A gene, and the IRE gene. The anti-cancer-related non-tumor cell agent according to (2) or (3), which comprises an S sequence and an E1B gene in this order. . (5) The promoter in the second gene cassette is the Egr1 promoter, (4) The anti-cancer-related non-tumor cell agent according to any one of (1) to (4). (6) The cancer-associated non-tumor cells according to any one of (1) to (5), wherein the cancer-associated non-tumor cells are cancer-associated fibroblasts. Anti-cancer related non-tumor cell agents. (7) The antibody according to any one of (1) to (6), wherein the virus is a recombinant adenovirus. Cancer-associated non-tumor cell agents. (8) The virus treats and / or prevents cancer by damaging cancer-associated non-tumor cells. The anti-cancer-related non-tumor cell agent according to any one of (1) to (7). (9) The anti-cancer-related non-tumor cell agent according to (8), wherein the cancer is pancreatic cancer or gastric cancer. (10) A pharmaceutical composition comprising the anti-cancer-related non-tumor cell agent according to any one of (1) to (9). (11) A method for treating cancer-related non-cancer, comprising administering to a subject an oncolytic virus containing a p53 gene. A method for damaging tumor cells. (12) A telomerase reverse transcriptase promoter, an E1A gene, an IRES sequence, and an E1B gene a first gene cassette comprising: a promoter and a second gene cassette containing the p53 gene A method for damaging cancer-associated non-tumor cells, comprising administering to a subject a recombinant virus comprising Law. (13) The method according to (12), wherein the recombinant virus is an oncolytic virus. (14) The first gene cassette contains a telomerase reverse transcriptase promoter, an E1A gene, and an I The method according to (12) or (13), comprising a RES sequence and an E1B gene in this order. (15) The promoter in the second gene cassette is the Egr1 promoter. 2. The method according to claim 1, wherein the first step is to form a first cavity. (16) Any of (11) to (15), wherein the cancer-associated non-tumor cells are cancer-associated fibroblasts. The method described below. (17) Any of (11) to (16) above, wherein the virus is a recombinant adenovirus. How to post. (18) A virus that treats and / or prevents cancer by damaging cancer-associated non-tumor cells. The method according to any one of (11) to (17), (19) The method according to (18), wherein the cancer is pancreatic cancer or gastric cancer. (20) The method according to any one of (11) to (19), wherein the virus is administered as a pharmaceutical composition. How to post.

[0008] This specification incorporates the disclosures of Japanese Patent Application No. 2020-106251, which is the priority basis of this application. Contains. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 shows a schematic diagram of the recombinant adenoviruses used in the examples. Figure 1A shows a schematic diagram of OBP-702. OBP-702 lacks the E1 gene and contains a gene cassette containing hTERT-p, E1A, IRES, and E1B in its place. OBP-702 also lacks the E3 gene and contains a gene cassette containing the mouse Egr1 promoter (Egr1-p) and p53 in its place. Figure 1B shows a schematic diagram of OBP-301. OBP-301 lacks the E1 gene and contains a gene cassette containing hTERT-p, E1A, IRES, and E1B in its place. Figure 1C shows a schematic diagram of Ad-p53. Ad-p53 lacks the E1 and E3 genes and contains a gene cassette containing CMV-p and p53 in the place of the E1 gene. [Figure 2] Figure 2 shows the results of Western blotting (A) and immunostaining (B) in a CAF model prepared by treating fibroblasts (FEF3 or GF) with cancer-conditioned medium (CM) or TGF-β. [Figure 3] FIG. 3 shows cell viability after treatment with OBP-702 (A) and paclitaxel (PTX) (B) for normal gastric fibroblasts (GF) and CAF model cells (TGF-β-activated GF). [Figure 4] FIG. 4 shows the effects of OBP-702 on the expression of various proteins (A) and IL-6 production (B) in CAF model cells, as measured by Western blotting (A) and ELISA (B). [Figure 5] FIG. 5 shows the cell viability of CAF clinical specimens (A) and normal fibroblasts (NF) (B) after treatment with OBP-702, OBP-301, PTX, and Ad-p53. [Figure 6]Figure 6A shows the results of cell proliferation assays of CAF models obtained by treating pancreatic stellate cells (hPSC-5 or hPSC-14) with cancer-conditioned medium prepared by pancreatic cancer cells (Panc-1, MIAPaCa-2, BxPC-3, or Capan-1). Figure 6B shows the cell viability of CAF models obtained by treating pancreatic stellate cells with cancer-conditioned medium prepared by Panc-1, MIAPaCa-2, BxPC-3, or Capan-1 after treatment with OBP-301 or OBP-702. [Figure 7] Figure 7 shows the results of Western blotting to detect changes in the expression of various proteins when OBP-702 was administered to a CAF model prepared by treating pancreatic stellate cells with Panc-1 cancer-conditioned medium. [Figure 8] Figure 8A shows the results of a cell proliferation assay of a CAF model obtained by treating pancreatic stellate cells with TGF-β, and Figure 8B shows the cell viability of a CAF model obtained by treating pancreatic stellate cells with TGF-β after treatment with OBP-301 or OBP-702. [Figure 9] Figure 9A shows the cell viability of CAF models obtained by treating pancreatic stellate cells (hPSC-5 or hPSC-14) with cancer-conditioned medium prepared by pancreatic cancer cells (Panc-1, MIAPaCa-2, BxPC-3, or Capan-1) after treatment with AdDL312 or Ad CMVp53. Figure 9B shows the cell viability of CAF models obtained by treating pancreatic stellate cells with TGF-β after treatment with AdDL312 or Ad CMVp53. [Figure 10] Figure 10 shows the results of Western blotting to detect changes in the expression of various proteins when OBP-702 was administered to CAF models obtained by treating pancreatic stellate cells (hPSC-5 or hPSC-14) with TGF-β. [Figure 11] Figure 11 shows the results of Western blotting (A) and cell viability (B) in a CAF model prepared by treating pancreatic stellate cells (hPSC-5 or hPSC-14) with Panc-1 cancer-conditioned medium containing a TGF-β inhibitor. [Figure 12] FIG. 12 shows the results of observing p53 expression in 3D cultured tissue after virus administration (A), and the results of evaluating changes in the long diameter of the tumor mass (B). [Figure 13] FIG. 13 shows the observation results of TUNEL expression in 3D cultured tissues (A) and the quantification results (B). [Figure 14] Figure 14 shows a comparison of tumor volume (A), tumor photographs and tumor weight measurements (B), and tumor fold change (C) when subcutaneous tumor models were prepared using BxPC-3 mono-injection and BxPC-3 + hPSC-14 co-injection. [Figure 15] Figure 15 shows the results of comparing tumor growth in the BxPC-3+hPSC-14 co-injection model when Mock, OBP-301, or OBP-702 was administered (A), as well as photographs of the tumors and measurements of tumor weight (B). [Figure 16] FIG. 16 shows the results of observing p53 expression (A) and the quantification thereof (B) when Mock, OBP-301, or OBP-702 was administered to the BxPC-3+hPSC-14 co-injection model. DETAILED DESCRIPTION OF THE INVENTION

[0010] In one embodiment, the present invention relates to a method for the production of a p53 gene (and optionally a promoter as described herein). an anti-cancer agent comprising or consisting of an oncolytic virus (e.g., a recombinant virus) containing Related non-tumor cell agents.

[0011] In one embodiment, the present invention provides a method for producing a gene comprising a first gene cassette and a second gene cassette. The present invention relates to an anti-cancer-related non-tumor cell agent comprising or consisting of a recombinant virus.

[0012] Hereinafter, an oncolytic virus containing the p53 gene, a first gene cassette, and a second gene cassette will be described. The recombinant virus containing the progeny cassette is also referred to simply as the "virus described herein." .

[0013] <1. First gene cassette> In one embodiment, the viruses described herein contain, in addition to a second gene cassette described below: The virus contains the first gene cassette either together with or separately from the second gene cassette. Even if the gene cassette does not include the first gene cassette, the gene cassette is referred to as the "first gene cassette" for convenience. The first gene cassette contains a telomerase reverse transcriptase promoter, It contains the E1A gene, the IRES sequence, and the E1B gene.

[0014] In one embodiment, the recombinant virus described herein encodes a telomerase reverse transcriptase The promoter can drive the E1A gene, the IRES sequence, and the E1B gene. In this case, the promoter of telomerase reverse transcriptase is activated, thereby The recombinant virus can propagate, resulting in cytotoxicity due to viral proliferation in tumor cells. This results in the production of a recombinant virus carrying the telomerase reverse transcriptase promoter. can specifically kill tumor cells.

[0015] The telomerase reverse transcriptase (TERT) promoter used is derived from mammals. For example, a TERT promoter derived from a human, mouse, rat, or cow can be used. The TERT promoter of mammals, including humans, can be cloned. The sequence information is available from databases such as NCBI and GenBank. The T promoter is the human telomerase reverse transcriptase promoter (hTERT promoter ) is preferred.

[0016] Many transcription factor binding sequences have been identified in the 1.4 kbp region upstream of the 5' end of hTERT. This region is thought to be the hTERT promoter, and in particular, the 181 bp upstream of the translation initiation site The sequence is a core region important for downstream gene expression. A sequence containing the core region can be used, for example, about 378 bp upstream of the core region. The sequence of about 378 bp can be used as the hTERT promoter. It has been confirmed that the gene expression efficiency is equivalent to that of the core region of p alone. The nucleotide sequence of the 455 bp long hTERT promoter is shown in SEQ ID NO: 1. The base sequence of this region is shown in SEQ ID NO:5.

[0017] The TERT promoter includes a nucleotide sequence containing the base sequence shown in SEQ ID NO: 1 or 5. In addition, (a) a base complementary to a nucleotide containing the base sequence shown in SEQ ID NO: 1 or 5 a nucleotide that hybridizes under stringent conditions with a nucleotide comprising the sequence; (b) 80% or more, 85% or more, 90% or more, or 95% of the base sequence shown in SEQ ID NO: 1 or 5 Nucleotides containing a base sequence having a sequence identity of 98% or more, or 99% or more, (c ) In the base sequence shown in SEQ ID NO: 1 or 5, one or several bases are added, deleted or substituted. Nucleotides containing the base sequences (a) to (c) can also be used. Preferably, the tide has TERT promoter activity.

[0018] As used herein, "stringent conditions" include, for example, 1xSSC to 2xSSC, Examples of suitable conditions include 0.1% to 0.5% SDS and 42°C to 68°C, and more specifically, 1xSSC to 2xSSC, 0 Prehybridization was performed in 1% to 0.5% SDS at 60 to 68°C for at least 30 minutes, followed by 2x SSC. The hybridization conditions include washing 4 to 6 times in 0.1% SDS at room temperature for 5 to 15 minutes each. Detailed procedures for the cloning method are known, and are described, for example, in "Molecular Cloning, A Laboratory Manu al 4th ed. (Cold Spring Harbor Laboratory (2012)).

[0019] As used herein, the "sequence identity" of bases refers to the difference between the two sequences when they are aligned. and introduce gaps as necessary to maximize the degree of agreement between the two. This refers to the percentage of identical bases between two sequences.

[0020] In the present specification, "several" means, for example, 2 to 10, 2 to 7, 2 to 5, 2 to 3, or 2. Taste.

[0021] The E1A and E1B genes are genes contained in the adenovirus E1 gene. The genes are the early (E) and late (lat) genes involved in DNA replication of the virus. e:L) and is a protein involved in the control of transcription of the viral genome. The E1A protein encoded by the E1A gene is the protein that makes the virus capable of infecting the host. It activates the transcription of genes (E1B, E2, E4, etc.) necessary for cell production. The E1B proteins are transduced by the late gene (L gene) mRNA, which is then transduced into the cytoplasm of the infected host cell. It promotes viral replication by helping the virus accumulate in the host cell and inhibiting protein synthesis in the host cell. The nucleotide sequences of the E1A gene and the E1B gene are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively. The E1A gene and the E1B gene are selected from the group consisting of nucleotide sequences including the base sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3. In addition to the above, (a) for nucleotides containing the base sequence shown in SEQ ID NO: 2 or 3, respectively Nucleotides that hybridize under stringent conditions with nucleotides containing complementary base sequences (b) nucleotides that are 80% or more and 85% of the nucleotide sequences shown in SEQ ID NO: 2 or 3, respectively or more, 90% or more, 95% or more, 98% or more, or 99% or more sequence identity. (c) a nucleotide containing one or several nucleotides in the base sequence shown in SEQ ID NO: 2 or 3, respectively; Nucleotides containing a base sequence in which one or more bases are added, deleted or substituted are also included. nucleotides (a) to (c) encode a protein having E1A activity or E1B activity; is preferred.

[0022] IRES (Internal Ribosome Entry Site) is a protein specific to picornaviruses. It is a protein synthesis initiation signal and has a sequence complementary to the 3' end of 18S ribosomal RNA. It is thought to act as a cytoplasmic membrane-binding site. It is known that the derived mRNA is translated via this sequence. The translation efficiency from the IRES sequence is The cap structure is highly efficient, and protein synthesis can occur even from the middle of mRNA in a cap-independent manner. In the virus described in

[2004] , the E1A and E1B genes are transfected by the human telomerase promoter. Both are translated independently. When an IRES is used, the TERT promoter controls the expression of the E1A gene. TERT is used to target either the E1A or E1B gene, so that the E1A gene can be independently targeted to the E1B gene. Compared to when the virus is controlled by a promoter, the proliferation of the virus is suppressed by the cells with telomerase activity. The IRES sequence is shown in SEQ ID NO: 4. nucleotides containing the base sequence shown in SEQ ID NO: 4, as well as (a) the base sequence shown in SEQ ID NO: 4 Nucleotides containing a base sequence complementary to the nucleotide containing the base sequence and stringency (b) a nucleotide that hybridizes under optimal conditions with respect to the base sequence shown in SEQ ID NO: 4; and 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more sequence identity. (c) a nucleotide comprising a base sequence having one or more of the following in the base sequence shown in SEQ ID NO: 4: The above also includes nucleotides containing a base sequence in which several bases are added, deleted or substituted. The nucleotides (a) to (c) preferably have IRES activity.

[0023] The order of the telomerase reverse transcriptase promoter, E1A gene, IRES sequence, and E1B gene is The E1A and E1B genes are expressed under the control of the telomerase reverse transcriptase promoter. For example, the viruses described herein may be those that have a 5' to 3' sequence of nucleotides. It contains the ERJ reverse transcriptase promoter, the E1A gene, the IRES sequence, and the E1B gene in this order. Alternatively, from the 5' to the 3' end, the telomerase reverse transcriptase promoter, E1 It may contain the B gene, an IRES sequence and the E1A gene in that order.

[0024] <2. Second gene cassette> In one embodiment, the virus described herein comprises, in addition to the first gene cassette, The virus contains a second gene cassette in addition to or separate from the first gene cassette. Even if the gene cassette does not include the second gene cassette, the gene cassette is referred to as the "second gene cassette" for convenience. The second gene cassette contains a promoter and the p53 gene.

[0025] The promoter is the promoter that drives the p53 gene in cancer-related non-tumor cells. Any promoter can be used as long as it is capable of producing a gene that is induced by radiation or viruses. A promoter that is induced by cellular stress such as bacterial infection, and a promoter that is constitutively expressed in cells Examples of promoters include Egr1 (Early growth response e protein 1 promoter, cytomegalovirus (CMV) promoter, telomerase Reverse transcriptase promoter, SV40 late promoter, MMTV LTR promoter, RSV LTR promoter The Egr1 promoter can be a promoter such as a promoter of the SRα gene, a promoter of the Egr1 gene, or a promoter of the SRα gene. Those derived from mammals can be used, for example, human, mouse, rat, or The Egr1 promoter derived from E. coli or the like can be used. The sequences of each promoter are known. For example, the mouse Egr1 promoter is a nucleotide sequence containing the nucleotide sequence shown in SEQ ID NO:6. In addition to the nucleotide, (a) a salt complementary to the nucleotide containing the base sequence shown in SEQ ID NO: 6 Nucleotides that hybridize under stringent conditions with nucleotides containing the base sequence (b) 80% or more, 85% or more, 90% or more, or 95% or more of the base sequence shown in SEQ ID NO: 6 (c) a nucleotide sequence having 98% or more, or 99% or more sequence identity; A salt in which one or several bases are added, deleted or substituted in the base sequence shown in column number 6. Nucleotides containing the base sequence can also be used. It is preferable that it has Egr1 promoter activity.

[0026] The p53 gene is involved in the activation of DNA repair proteins when DNA is damaged, the regulation of the cell cycle, and the It is involved in the induction of apoptosis when NA is irreparably damaged. By expressing it using a recombinant virus, it is possible to damage cancer-associated non-tumor cells. The present inventors have first discovered that the p53 gene can be expressed by the p53 gene expressed in the p53 gene sequence shown in SEQ ID NO: 7. In addition to nucleotides containing the base sequence, (a) a nucleotide containing the base sequence shown in SEQ ID NO: 7 It hybridizes under stringent conditions with a nucleotide containing a base sequence complementary to the nucleotide. (b) a nucleotide sequence that is 80% or more, 85% or more of the nucleotide sequence shown in SEQ ID NO: 7 Nucleotide sequences with 90% or more, 95% or more, 98% or more, or 99% or more sequence identity (c) a nucleotide sequence shown in SEQ ID NO: 7 in which one or several nucleotides are added or deleted Nucleotides containing deleted or substituted base sequences can also be used. Preferably, the nucleotides encode a protein having p53 activity.

[0027] The second gene cassette further contains a poly(A) sequence downstream of the promoter and p53 gene. It is preferable to include a poly(A) sequence, which can protect the mRNA from degradation in the cytoplasm. In addition, at least one of the functions of transcription termination, nuclear export, and translation may be assisted.

[0028] <3. Other components of the virus> In this specification, each gene contained in a gene cassette can be produced by conventional genetic engineering techniques. For example, DNA synthesis, which is commonly used as a genetic engineering technique, can be used. Nucleic acid synthesis using a synthesis device can be used. Alternatively, after synthesis, specific primers for each gene are designed and used in a PCR machine. The gene sequence is amplified using PCR or a gene amplification method using a cloning vector. The above method can be performed as described in "Molecular Cloning, A Laboratory Manual 4th ed." ( This can be easily done by a person skilled in the art according to the method described above.

[0029] Then, the genes obtained as described above can be linked together. Each of the above genes is digested with a known restriction enzyme or the like, and the digested DNA fragments of the gene are then subjected to a known vector. The vector is inserted and ligated into a vector according to a known method. The pIRES vector can be used as an IRES vector for encephalomyocarditis virus (ECMV). It contains two open reading frames (ORFs) from one type of mRNA. pIRES vectors are vectors that can be translated. By sequentially inserting the required genes into the first gene cassette, a polynucleotide containing the first gene cassette is obtained. It is possible to create nucleotides. DNA ligase can be used to join DNA. Expression of the E1 gene required for adenovirus replication under the control of the hTERT promoter. This allows the virus to grow specifically in cancer cells. By incorporating the gene cassette into a virus, a recombinant virus can be produced. can.

[0030] The recombinant viruses (e.g., adenoviruses) described herein contain the E1 region of the virus itself. The adenovirus E1 region is involved in viral replication, so By deleting this region, unintended viral replication can be prevented. In one embodiment, the recombinant adenovirus described herein is E1 region deleted, and The E1 region may be deleted in its entirety, and its function may be maintained. It may be partially deleted so that the

[0031] The recombinant adenoviruses described herein may lack the E3 region of the virus itself. The adenovirus E3 region contains an 11.6 kDa adenovirus death protein (ADP), which DP has the function of promoting cell damage and viral spread, but it also inhibits the proliferation of adenovirus. The deletion of the E3 region reduces the genome sequence of the recombinant virus. This reduces the genome size and allows for a larger gene to be introduced. In one embodiment, the recombinant adenovirus described herein lacks the E3 region. The E3 region may be deleted in its entirety, and It may be partially deleted so that the function of the gene is lost.

[0032] The types of viruses described herein include, but are not limited to, adenoviruses, adenoviruses, and the like. Associated viruses (AAV), herpesviruses, retroviruses, lentiviruses, vaccinia Examples of viruses include reoviruses, poxviruses, and picornaviruses.

[0033] The viruses described herein can be readily prepared by one skilled in the art. Viruses containing the genes shown in Figure 1 can be produced using a gene recombination kit such as In-fusion. The E1 and E3 regions of the adenovirus were transformed into the genes shown in Figure 1 (E1:hTERT-p-E1A-IRE). It can be easily produced by substituting S-E1B, E3:Egr1-p-p53-polyA).

[0034] In one embodiment, the viruses described herein are capable of damaging cancer-associated non-tumor cells. In one embodiment, the viruses described herein are and for treating and / or preventing cancer by damaging cancer-associated non-tumor cells. That's fine.

[0035] The tumor microenvironment that constitutes the tumor is also involved in drug resistance, etc., and therefore, Viruses are also used to enhance the effectiveness of other anticancer drugs in cancers that are drug-resistant. Other anti-cancer drugs include, but are not limited to, alkylating agents, platinum agents, topoisomerase inhibitors, and the like. Isomerase inhibitors, metabolic antagonists, microtubule inhibitors, antibiotics, anticancer drugs, molecular targeted drugs (small molecules) compounds and antibody drugs), cell therapy, oncolytic viruses, immunotherapy (immune checkpoint inhibitors), (including steroid inhibitors)

[0036] In the present specification, the type of cancer is not limited, but examples thereof include stomach, pancreas, colon, lung, liver, and prostate cancer. Effective against solid cancers in the gland, esophagus, bladder, gallbladder / bile duct, breast, uterus, thyroid, ovary, etc. Among these, pancreatic cancer, lung cancer, gastric cancer, esophageal cancer, and colon cancer are known to contain abundant CAFs in tumors. Cancer is preferred.

[0037] As used herein, the term "cancer-associated non-tumor cells" refers to tumor-associated fibroblasts (cancer-associated non-tumor cells). associated fibroblasts, CAFs), immune cells (tumor-associated macrophages, T cells, neutrophils, etc.), and mesenchymal stem cells, preferably CAFs.

[0038] In one embodiment, the virus described herein is an oncolytic virus. In antiviral therapy, the presence of cancer-associated non-tumor cells allows the virus to be transmitted from cancer cells to cancer cells. Therefore, in one embodiment, the diffusion of The virus damages cancer-related non-tumor cells, and therefore has excellent viral diffusion inhibition properties and / or It has excellent antitumor effects.

[0039] 4. Pharmaceutical Compositions and Methods In one embodiment, the present invention provides an anti-cancer-related non-tumor cell agent described herein (hereinafter, "the present invention"). The pharmaceutical composition of the present invention relates to a pharmaceutical composition comprising the compound (also referred to as "the damaging agent described in the specification"). In addition to the damaging agents described herein, known pharmaceutical additives such as excipients, fillers, binders, lubricants, etc. Acceptable carriers, known additives (buffers, isotonicity agents, chelating agents, coloring agents, preservatives, fragrances) The composition may contain one or more ingredients selected from the group consisting of: flavoring agents, sweetening agents, and the like.

[0040] In one embodiment, the pharmaceutical compositions described herein are used in combination with other anti-cancer agents. In one embodiment, the pharmaceutical compositions described herein further comprise other anti-cancer agents. Cancer drugs are as described above.

[0041] In one embodiment, the pharmaceutical compositions described herein are used to treat and / or prevent cancer. The types of cancer are as described above. In one embodiment, The pharmaceutical composition is intended to treat and / or prevent cancer by damaging cancer-associated non-tumor cells. It is used for this purpose.

[0042] The route of administration of the damaging agent or pharmaceutical composition described herein is not limited, but may be oral or parenteral. Administration may be by oral administration, for example, intravenous, intramuscular, intraperitoneal, intratumoral or subcutaneous injection; nasal Inhalation through the cavity, oral cavity, or lungs; or through suppositories, external applications, etc. It can also be administered.

[0043] The form of the agent or pharmaceutical composition described herein is not limited, and may be in the form of a tablet, capsule, powder, or the like. The pharmaceutical composition may be in the form of granules, pills, liquids, syrups, injections, external preparations, suppositories, or eye drops.

[0044] The dosage of the agent or pharmaceutical composition described herein depends on the type of active ingredient, the route of administration, and the dosage of the agent or pharmaceutical composition. The daily dose is selected according to the patient's age, weight, sex, symptoms, and other conditions. As a result, the amount of the virus of the present invention, which is usually an active ingredient, is 10 6 ~10 13 About PFU, e.g., 10 9 ~10 1 2 The number of doses can be about PFU. There is no limit to the number of doses, and it can be administered once a day or several times a day. It can also be administered in divided doses.

[0045] The subject to which the damaging agent or pharmaceutical composition described herein is administered is, for example, a mammal, such as a human. primates, laboratory animals such as rats, mice, and brown rats, pigs, cattle, horses, and sheep Examples of the animal include domestic animals such as pigs, and preferably humans.

[0046] In one embodiment, the present invention provides a method for treating a viral infection, a viral infection, or a pharmaceutical composition described herein. and / or administering to a subject a method for damaging cancer-associated non-tumor cells or for treating and / or preventing cancer. The viruses, damaging agents or pharmaceutical compositions described herein are directed to methods of preventing cancer. The compound may be administered in a therapeutically effective amount to a subject suffering from the disease. [Example]

[0047] <Example 1: Preparation of CAF model> Materials and Methods The cells used were fibroblasts (FEF3: human fetal esophageal fibroblasts (obtained from Wister Institute) ), or GF (gastric fibroblast): fibroblasts isolated from gastric tissue (obtained from clinical specimens) GF was obtained by culturing 6-7 mm cubes of tissue that had been finely minced (see details). Tsuyoshi Hasegawa et al., International Journal of Cancer, 134, pp. 1785~1795 , 2014). FEF3 or GF was cultured at a concentration of 50,000 cells / mL for 10 cm 2 DMEM seeding in a dish The cells were cultured in a medium containing 10 ng / ml of TGF-β at 37°C under 5% CO2 for 12 to 24 hours. The medium was replaced with DMEM containing 100% methylprednisolone (MMP), or cancer-conditioned medium (CM) collected from cancer cell cultures. CM is 28.5x10 4 MKN7 cells were seeded into a 175T flask at a concentration of 175 cells / mL and cultured in serum-free DMEM medium. After culturing the cells at 37°C under 5% CO2 for 72 hours, the cells were centrifuged to remove the cellular components. 72-96 hours after cell seeding, CAFs (cancer-associated fibroblasts) The expression of αSMA or FAP, which are markers of t), in cells was confirmed by Western blotting. First, 20 μg of protein was separated by SDS-PAGE. Proteins were detected using anti-αSMA antibody (CST) or anti-FAP antibody (abcam).

[0048] (result) The results of Western blotting are shown in Figure 2A, and the results of immunostaining are shown in Figure 2B. As shown above, by applying CM or TGF-β to fibroblasts FEF3 and GF, the expression of αSMA and FAP was enhanced. Furthermore, as shown in Figure 2B, the expression of αSMA was increased by applying TGF-β to GF. rose.

[0049] These results suggest that fibroblasts can be transformed into CAFs by applying CM or TGF-β to the cells. This shows that...

[0050] Example 2: Damaging effect of OBP-702 on CAFs and normal fibroblasts Materials and Methods Preparation of OBP-702 The concentration of OBP-702 (obtained from Oncolys BioPharma) was adjusted using DMEM medium. The sequence of the amplification cassette (hTERTp-E1A-IRES-E1B (including spacer)) contained in -702 was Number 8 shows the full-length sequence of OBP-702 as SEQ ID NO:9. XTT assay Normal fibroblasts (GF) (obtained from clinical specimens) and CAF model cells (TGF-β-activated GF) ( Normal fibroblasts were stimulated with TGF-β as described in Example 1) for 96 hours. The cells were seeded onto a plate at a density of 10,000 cells / mL and cultured in DMEM medium at 37°C under 5% CO2 for 24 hours. After 1 day, OBP-702 was added at concentrations of 1-100 MOI, or after 3 days, 5-250 nM of plaque was added. Taxel (obtained from Nippon Kayaku) was added. Four days after cell seeding, the cells were analyzed using a SpectraMax i3. The injury rate was confirmed by XTT assay.

[0051] (result) The results for OBP-702 are shown in Figure 3A, and the results for paclitaxel (PTX) are shown in Figure 3B. As shown, OBP-702 causes minimal damage to normal cells, but dose-dependently damages CAF model cells. On the other hand, as shown in Figure 3B, PTX caused little damage to CAF model cells. It damages normal cells.

[0052] These results suggest that OBP-702 effectively inhibits CAFs while suppressing damage to normal cells compared to PTX. This indicates that it can cause injury.

[0053] Example 3: Effect of OBP-702 on the expression of various proteins in CAF model cells Materials and Methods Fibroblasts (obtained from clinical specimens) were cultured at 10cm 2 The cells were seeded at a density of 50,000 cells / mL onto a dish and cultured in DMEM medium. The cells were cultured at 37°C under 5% CO2 for 24 hours. After one day, TGF-β was added at a concentration of 10 ng / ml. Four days later, OBP-702 was added at concentrations of 0-100 MOI. Four days after OBP-702 administration, the wells were cultured as follows: The expression of various proteins was confirmed by stain blotting. Proteins were separated by SDS-PAGE. Then, anti-αSMA antibody (CST) and anti-FAP antibody were used as primary antibodies. (abcam), anti-p53 antibody (CST), anti-E1a antibody (BD Pharmingen), anti-PARP antibody (CST), anti-p62 antibody ( Protein was detected using an IL-6 ELISA kit (R&D Systems). I used it.

[0054] (result) The results are shown in Figures 4A and 4B. As shown in Figure 4A, administration of OBP-702 increased the expression of p53 and E1a. This indicates that OBP-702 has proliferated. It has been shown that autophagy is promoted. Increased expression of PARP indicated the progression of apoptosis. As shown in Figure 4B, administration of OBP-702 suppressed IL-6 production.

[0055] <Example 4: Confirmation of damaging effects using CAF clinical specimens and normal fibroblasts (NF)> Materials and Methods NF: fibroblasts isolated from gastric tissue (obtained from clinical specimens) and CAFs obtained from gastric cancer patients Clinical specimens (6-7 mm square tissues) were obtained by finely mincing and culturing. For details, see Tsuyos (see Hasegawa et al. (supra)) were seeded in a 96-well plate at a concentration of 10,000 cells / mL and cultured in DM The cells were cultured in EM medium at 37°C under 5% CO2 for 24 hours. After one day, OBP-702, Patent No. 3867968 The infectious recombinant adenoviruses TRAD (OBP-301) and PTX (Nippon Kayaku) were After adding Ad-p53 (obtained from Introgen Therapeutics) at the specified concentrations, SpectraMatrix The injury rate was confirmed by XTT assay using x i3.

[0056] (result) The results for CAF clinical specimens are shown in Figure 5A, and the results for NF specimens are shown in Figure 5B. The proliferation cassette (hTERT-p) similar to OBP-702 was expressed in the CAFs, but the CAFs were injured in a dose-dependent manner. OBP containing a gene cassette containing E1A, IRES, and E1B) but not an expression cassette for p53 In the case of Ad-p53, which contains the p53 expression cassette but not the proliferation cassette, both NF and CAF were injured. On the other hand, PTX damages CAF but more than that, it damages NF.

[0057] These results indicate that OBP-702 is more effective than other drugs such as PTX while suppressing damage to normal cells. This indicates that CAFs can be effectively damaged.

[0058] Example 5: Damaging effect on pancreatic stellate cells Materials and Methods Preparation of cancer conditioned medium (CM) Panc-1, MIAPaCa-2, BxPC-3, and Capan-1 were cultured in a 175T flask at 2.0 × 10 6 Seed at a concentration of cells / mL Panc-1 and MIAPaCa-2 were cultured in DMEM medium, BxPC-3 in RPMI medium, and Capan-1 in IMDM medium at 37°C in 5% CO₂. The cells were cultured under CO2 conditions. When all cells except Capan-1 reached 90-95% confluence, Capan-1 was cultured for 60-95% confluence. When the cells reached 70% confluence, the medium was changed to serum-free PSC medium (Thermo Fischer Scientific). After 48 hours, the culture supernatant was obtained as cancer-conditioned medium (CM). Cell viability assay Fibroblasts (pancreatic stellate cells, hPSC-5 or hPSC-14) were plated in a 96-well plate at 3000 cells / well (hPS C-5), or 4000 cells / well (hPSC-14), and incubated in PSC medium at 37°C under 5% CO2. After 24 hours, the medium was replaced with one of the four types of CM prepared as described above, and the cells were cultured for 72 hours. Afterwards, a cell proliferation assay was performed using the XTT assay with SpectraMax i3. Separately, 24 hours after cell seeding, the medium was changed to the same medium as above containing OBP-301 or 702 at an MOI of 1-200. After 72 hours, the cells were replaced with one of the four types of CM prepared in the same manner, and then subjected to XTT assay using SpectraMax i3. A cell viability assay was performed.

[0059] (result) The results of the cell proliferation assay are shown in Figure 6A, and the results of the cell survival assay are shown in Figure 6B. As shown in Figure 6B, cells activated with CM proliferated more than those in the control group. Administration of OBP-702 produced more CM than cells treated with the control (serum-free medium). The cells treated with thrombin showed significantly stronger cytotoxicity.

[0060] Example 6: Changes in the expression of various proteins in CAF model cells by administration of OBP-702 Materials and Methods Fibroblasts (pancreatic stellate cells, 5 × 10 5 hPSC-5 or 7 x 10 cells 5 Cells (hPSC-14) were cultured in a 10cm dish. After 24 hours, the medium was replaced with the PSC medium prepared in Example 5. The medium was replaced with nc-1 CM, and OBP-702 was administered at an MOI of 25-100. After 72 hours, Western blotting was performed. Western blotting was performed using 10% to 15% SDS-PAGE. The same procedure as in Example 3 was carried out using acrylamide gel.

[0061] (result) The results are shown in Figure 7. Administration of OBP-702 promoted the expression and phosphorylation of P53 and the expression of PARP. This suggests that OBP-702 induces cell death through apoptosis and autophagy. This indicates that

[0062] Example 7: Damaging effect of TGF-β on pancreatic stellate cells in a CAF model Materials and Methods Fibroblasts (pancreatic stellate cells, hPSC-5 or hPSC-14) were plated in a 96-well plate at 3000 cells / well (hPS C-5), or 4000 cells / well (hPSC-14), and incubated in PSC medium at 37°C under 5% CO2. After 24 hours, 2.5-10 ng / mL of TGF-β was added, and after 72 hours, the cells were analyzed by SpectraMax i3. A cell proliferation assay was performed using the XTT assay. After 24 hours, 2.5-10 ng / mL of TGF-β and 1-200 MOI of OBP-301 or 702 were applied, and after 72 hours Cell viability assays were performed using the XTT assay with SpectraMax i3.

[0063] (result) The results of the cell proliferation assay are shown in Figure 8A, and the results of the cell survival assay are shown in Figure 8B. As shown in Figure 8B, TGF-β-activated cells proliferated more than the control cells. As shown in Fig. 1, administration of OBP-702 resulted in a greater increase in CM than cells treated with the control (serum-free medium). The cells exposed to the α-glucan showed significantly stronger cytotoxicity.

[0064] Example 8: CAF-damaging effect of AdCMVp53 Materials and Methods Fibroblasts (pancreatic stellate cells, hPSC-5 or hPSC-14) were plated in a 96-well plate at 3000 cells / well (hPS C-5), or 4000 cells / well (hPSC-14), and incubated in PSC medium at 37°C under 5% CO2. 24 hours after cell seeding, the medium was replaced with 1-2000 MOI of E1A-deleted type 5 cells, which were growth-restricted. The adenoviral vector dDL312 (Patricia C Ryan et al., 2004, Cancer Gene Therapy erapy, volume 11, pages 555-569, and Young CSH et al, 1984, The adenoviruses. pp. 125-172) or AdCMVp53 (same as Ad-p53 described in Example 4), The CM was replaced with one of the four prepared CMs, and after 72 hours, the cells were analyzed by XTT assay using SpectraMax i3. A survival assay was performed. In addition to the four types of CM prepared in Example 5, 24 hours after cell seeding, 2.5 ng / mL TGF-β and 1-2000 MOI of AdDL312 or AdCMVp53 were applied, and after 72 hours, SpectraMa Cell viability assay was performed using XTT assay with x i3.

[0065] (result) The results for CAFs activated with four types of CM are shown in Figure 9A, and the results for CAFs activated with TGF-β are shown in Figure 9B. AdDL312 stimulated cells in a control (serum-free medium) and cells activated with four types of CM. AdCMVp53 had no cytotoxic effect on any of the cells, but the control (serum-free medium) The cytotoxic effect was significantly stronger in CM-activated cells than in unactivated cells. These results indicate that the damaging effects of CM-activated CAFs are mediated by p53. On the other hand, neither AdDL312 nor AdCMVp53 had a cytotoxic effect on TGF-β-activated CAFs. Did not show.

[0066] Example 9: Involvement of TGF-β signaling in p53 expression Materials and Methods Fibroblasts (pancreatic stellate cells, 5 × 10 5 hPSC-5 or 7 x 10 cells 5 Cells (hPSC-14) were cultured in a 10cm dish. After 24 hours, the cells were cultured in PSC medium at 37°C under 5% CO2 conditions. The PSC medium was changed to OBP-702 at an MOI of 25-100. After 72 hours, Western blot analysis was performed. Western blotting was performed using 10% to 15% SDS-PAGE. The same procedure as in Example 3 was carried out using acrylamide gel.

[0067] Next, fibroblasts (pancreatic stellate cells, 5 × 10 5 hPSC-5 cells were seeded onto a 10cm dish and cultured in PSC medium After 24 hours, the cells were cultured in a 5% CO2 atmosphere at 37°C. After 24 hours, the cells were treated with 100MOI of OBP-702 and 0.5-2.0 μg / ml of TGF- The plate was replaced with Panc-1 CM containing β-blocker (R&D Systems), and after 72 hours, the plate was resuspended in Western blotting medium in the same manner as in Example 3. The expression of p53 was compared by immunoblotting. hPSC-5 or hPSC-14) were plated in a 96-well plate at 3000 cells / well (hPSC-5) or 4000 cells / well (hPSC-14). ) and cultured in PSC medium at 37°C under 5% CO2. After 24 hours, OBP-7 cells were cultured at 100 MOI. The medium was replaced with Panc-1 CM or BxPC-3 CM containing 0.25-2.0 μg / mL of TGF-β inhibitor and incubated for 7 days. After 2 hours, a cell proliferation assay was performed using the XTT assay on a SpectraMax i3.

[0068] (result) The results are shown in Figure 10. In the TGF-β-added group, the increase in p53 expression caused by OBP-702 was increased (Figure 10). 10) This is because OBP-702 increases p53 expression even in cells transformed into CAFs by TGF-β. This indicates that the enhancement of cytotoxic activity by OBP-702 shown in Example 7 is due to the increase in p53 expression. This indicates that potentiation is involved.

[0069] On the other hand, when TGF-β contained in Panc-1 CM was inhibited with an inhibitor, the effect of OBP-702 on p53 expression was not observed. The increase in the cytotoxic activity of the tumor cells was attenuated, and the cytotoxic activity was also slightly attenuated (Fig. 11). These results suggest that TGF-β, which is contained in the α-glucan, affects p53 expression and CAF-damaging effects of OBP-702. Furthermore, in Example 8, AdCMVp53 did not have cytotoxicity against CAFs activated by TGF-β. These results suggest that OBP-702 inhibits the proliferation of important cytokines in the tumor microenvironment compared to AdCMVp53. The p53 gene was introduced more strongly into CAFs affected by TGF-β, a cytotoxic factor. This shows that it demonstrates the following.

[0070] Example 10: Effect on tumor mass in 3D cultured tissue Materials and Methods Pancreatic stellate cells (hPSC-5, hPSC-14) were cultured in 0.04 mg / ml fibronectin (Sigma Aldrich) and 0.04 mg / ml The chamber was then immersed in 150 mM isotonic Tris solution (total 1 ml) containing 1 ml gelatin (Fujifilm Wako Pure Chemical Industries, Ltd.) for 30 minutes. The mixture was shaken at room temperature and centrifuged (2000 rpm, 2 minutes). Chin coated 2.5 x 10 5 cells pancreatic stellate cells (hPSC-5, hPSC-14) and 5000 cells of Cap an-2 was placed in a 24-well culture insert (0.4 μm polyethylene terephthalate transparent membrane) The 3D culture was performed by seeding the cells onto a plate (Corning, 3D Imaging Laboratory, Tokyo, Japan) and co-culturing them in PSC medium at 37°C under 5% CO2 conditions. The effect of OBP-702 on the tissue was evaluated (for details of the 3D culture method, see Hiroyoshi Y Tanaka). (See et al., Biomaterials, 2020, 30; 251: 120077). 96 hours after cell seeding, the virus The mice were then treated with either OBP-301 or OBP-702, and 3D imaging was performed 72 hours after virus administration (120 hours after cell seeding). The tissue was fixed with 4% paraformaldehyde and then permeabilized with 0.2% (v / v) Triton X-100 / PBS. After blocking with Blocking One (Nacalai Tesque), the primary antibody was added. After washing the 3D tissue with PBS, the secondary antibody was added and incubated overnight at 4°C. The primary antibodies were Pan-Keratin (C11) Mouse mAb #4545 (CST) and p53 (7F5) Rab. The primary antibody was Alexa Fluor 488 (anti-mouse, Invitrogen), and the secondary antibody was Alexa Fluor 488 (anti-mouse, Invitrogen). Multiple staining was performed using Fluor 568 (anti-rabbit, Invitrogen). The 3D tissue was washed with PBS. After washing, Hoechst 33342 was added and incubated overnight at 4℃ for nuclear staining. -Cut out the membrane from the insert, mount it on a slide, and cover it with mounting medium (Dako / Agile The cells were mounted in a plastic lid (Santa Clara, CA) and observed under a confocal laser microscope. The size of the intracellular tumor nest is determined by the concentration of green fluorescently labeled pan-keratin-positive cells. The change in nest size due to virus administration was evaluated by the long diameter. The dosage of the virus is: Low: 1.0 x 10 6 PFU, Middle: 5.0 x 10 6 PFU and High: 1.0 x 10 7 PFU and (In Figure 12B, 301-L indicates administration of OBP-301 at Low dose, 301-M indicates administration of OBP-301 at Middle dose, and 301- H means high dose of OBP-301. The same applies to OBP-702.

[0071] (result) The results are shown in Figure 12. In the 3D cultured tissue, OBP-702 reduced the size of the tumor mass more than OBP-301. The malignant potential of pancreatic cancer cells generally increases when co-cultured with CAFs. These results indicate that OBP-702 is also potent in co-cultured cancer cells through the expression of p53. In addition, in a model administered with OBP-702, In the pan-keratin negative area (stromal area), very strong p53 expression was visualized. These results indicate that OBP-702 induces p53 expression in stromal tissues. .

[0072] Example 11: Effect on CAFs in 3D cultured tissue Materials and Methods Using 3D models of hPSC5+Capan-2 and hPSC-5+BxPC-3 prepared in the same manner as in Example 10, Virus was administered 96 hours after cell seeding, and 72 hours after virus administration (120 hours after cell seeding). The amount of apoptotic cells in the TUNEL (TdT-mediated dUTP Nick End Labeling) assay was The TUNEL assay was performed using Click-iT TM TUNEL Alexa Fluor TM 594 Obtain the Imaging Assay for microscopy & HCS and follow the Invitrogen protocol. After the TUNEL reaction, multiple staining of the 3D tissue was performed using the same steps as in Example 10.

[0073] (result) The results are shown in Figure 13. In both 3D models, the pan cytokeratin-negative area (interstitial area) When TUNEL expression in CAFs was quantified, OBP-702 was significantly higher than Mock and OBP-301. This indicates that OBP-702 significantly increased TUNEL expression in CAFs, not just tumor cells. These results also indicate that p53 expression mediates apoptosis.

[0074] <Example 12: Preparation of an in vivo tumor cell + stromal cell (CAF) mixed subcutaneous tumor model> Materials and Methods 1.0×10 5 BxPC-3 mono-i cells were subcutaneously administered to nude mice (BALB / c-nu / nu). injection subcutaneous tumor model, 1.0 × 10 5 cells BxPC-3 and 9.0 × 10 5 hPSC-14 cells were cultured in nude mice. We created a subcutaneous tumor model by co-injecting BxPC-3 and hPSC-14 into BALB / c-nu / nu mice. The tumor volumes and weights were compared.

[0075] (result) The results are shown in Figure 14. The number of tumor cells administered was the same, but the co-injection model was mono-injected. Compared with the injectable method, significantly larger tumors were formed and the tumor growth rate was also significantly increased. The results indicate that PDAC-CAF interactions result in more aggressive tumor behavior. There are.

[0076] Example 13: Effect on in vivo subcutaneous tumor model containing mixed tumor cells and stromal cells (CAFs) Materials and Methods Cell transfer was performed on the BxPC-3+hPSC-14 co-injection model prepared according to Example 12. From 11 days after transplantation, Mock, OBP-301 (1.0 × 10 8 PFUs), OBP-702 (1.0 × 10 8 PFUs) administered three times The tumor volume was measured, and the tumor was excised 22 days after the final administration and the tumor weight was compared. did.

[0077] (result) The results are shown in Figure 15. Both OBP-301 and OBP-702 significantly suppressed tumor growth compared to Mock. Furthermore, OBP-702 showed significantly stronger tumor growth inhibitory effects than OBP-301. Our data show that OBP-301 and OBP-702 show similar efficacy in the BxPC-3 mono-injection subcutaneous tumor model. These results suggest that the oncolytic virus-mediated targeting of the ERK Signaling Pathway Inhibits Invasive Propensity in Human Pancreatic Ca Cancer. Mol Ther Oncolytics. 2020 Mar 31;17:107-117.) Therefore, the results of this experiment are In tumors that become highly malignant in the presence of abundant stroma, OBP-702 suppresses stroma and promotes tumor growth. These results indicate that the compound exerts a strong tumor growth inhibitory effect.

[0078] Example 14: Effect on in vivo tumor cell + stromal cell (CAF) mixed subcutaneous tumor model 2 Materials and Methods Cell transfer was performed on the BxPC-3+hPSC-14 co-injection model prepared according to Example 12. From 21 days after transplantation, Mock, OBP-301 (1.0 × 10 8 PFUs), OBP-702 (1.0 × 10 8 PFUs) were administered. The tumor was excised two days after the final administration. The excised tumor was fixed in paraffin and the tumor tissue was analyzed. Pan-cytokeratin and p53 were labeled with green and red fluorescence, respectively, and observed under a fluorescence microscope. . Primary antibodies are Pan-Keratin (C11) Mouse mAb #4545(CST), p53 (7F5) Rabbit mAb #2527 (CST), and the secondary antibodies were Alexa Fluor 488 (anti-mouse, Invitrogen) and Alexa Fluor 568 (a Multiplex staining was performed using primary and secondary antibodies (anti rabbit, Invitrogen). Each was one hour.

[0079] (result) The results are shown in Figure 16. In the OBP-702 administration group, strong localized expression of p53 was observed. By fluorescently labeling p53, tumor cells and stromal areas can be distinguished, and the p53 expression intensity in the stromal area can be measured. Compared with the OBP-301 and OBP-702 groups, the OBP-702 group showed a significant increase in p53 expression in the interstitium. These results suggest that OBP-702 acts not only on tumor cells but also on stromal cells in vivo. These results suggest that the effect of p53 expression on the oxidative stress response is mediated by p53 expression.

[0080] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. shall be incorporated.

Claims

1. Anti-cancer related non-tumor cell agents, including oncolytic viruses containing the p53 gene.

2. A telomerase reverse transcriptase promoter, an E1A gene, an IRES sequence, and an E1B gene are included in the first nucleotide sequence. one gene cassette, and a promoter and a second gene cassette containing the p53 gene An anti-cancer-related non-tumor cell agent, comprising a recombinant virus comprising:

3. The anti-cancer-related non-tumor cell agent according to claim 2, wherein the recombinant virus is an oncolytic virus. 。

4. The first gene cassette contains the telomerase reverse transcriptase promoter, the E1A gene, and an IRES sequence.

4. The anti-cancer-related non-tumor cell agent according to claim 2 or 3, comprising the E1B gene and the E1C gene in this order.

5. Claims 2 to 4, wherein the promoter in the second gene cassette is the Egr1 promoter. The anti-cancer-related non-tumor cell agent according to any one of the preceding claims.

6. The method according to any one of claims 1 to 5, wherein the cancer-associated non-tumor cells are cancer-associated fibroblasts. Anti-cancer related non-tumor cell agents.

7. The anti-cancer agent according to any one of claims 1 to 6, wherein the virus is a recombinant adenovirus. Related non-tumor cell agents.

8. The virus treats and / or prevents cancer by damaging cancer-associated non-tumor cells. The anti-cancer-related non-tumor cell agent according to any one of claims 1 to 7.

9. The anti-cancer-related non-tumor cell agent according to claim 8, wherein the cancer is pancreatic cancer or gastric cancer.

10. A pharmaceutical composition comprising the anti-cancer-related non-tumor cell agent according to any one of claims 1 to 9.

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