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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/76—Viruses; Subviral particles; Bacteriophages
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/76—Viruses; Subviral particles; Bacteriophages
  • A61K35/761—Adenovirus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
  • C07K14/01—DNA viruses
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  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/53—Colony-stimulating factor [CSF]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/55—IL-2
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

• the present invention relates to oncolytic viruses carrying an immunostimulatory gene containing C-X-C motif chemokine ligand 10 (CXCL10) and their use in cancer treatment. More specifically, the present invention relates to oncolytic viruses carrying the CXCL10 gene, oncolytic viruses carrying the CXCL10 gene and interleukin-2 (IL-2) and/or granulocyte-macrophage colony-stimulating factor (GM-CSF) genes on the same or separate oncolytic viruses, and oncolytic viruses carrying two or three factors including CXCL10 as immunostimulatory genes, as well as cancer treatments using them, particularly treatments that are effective against invasive and metastatic intractable cancers.
• CXCL10 C-X-C motif chemokine ligand 10
• IL-2 interleukin-2
• GM-CSF granulocyte-macrophage colony-stimulating factor
• CRAs conditionally replicating adenoviruses
• These CRAs are engineered to differentiate between cancer and normal cells by modifying the E1 gene region, which is essential for adenovirus replication.
• E1 gene region which is essential for adenovirus replication.
• m-CRAs multifactorially controllable and treatable CRAs
• m-CRAs multifactorially controllable and treatable CRAs
• m-CRA Sudv.m-CRA-1
• IAP inhibitor of apoptosis
• virotherapy from chemotherapy and radiation therapy is that during the process of amplified virus destruction of cancer cells, the release of tumor-associated antigens and the adjuvant-like function of the virus induce antitumor immunity, primarily through cellular immunity, potentially exerting a systemic therapeutic effect. Specifically, during the immune system's elimination of the virus, tumor-associated antigens from destroyed cancer cells are processed and presented by antigen-presenting cells, potentially eliciting specific antitumor immunity. To maximize this antitumor immunity, oncolytic virus immunotherapy (OVI) using oncolytic viruses carrying immunostimulatory genes has been intensively investigated.
• OMI oncolytic virus immunotherapy
• Non-Patent Document 3 Using this evaluation system, they tested an m-CRA (Surv.m-CRA-2-G) containing the GM-CSF gene downstream of various promoters, demonstrating that it could be safely administered while maintaining therapeutic efficacy (Patent Document 4). Furthermore, they demonstrated that m-CRA carrying the GM-CSF gene exhibited survival benefits comparable to or greater than those of anti-PD-1 antibodies, one of the existing promising cancer immunotherapeutic agents (Patent Document 4).
• m-CRA Sudv.m-CRA-2-G
• the object of the present invention is to provide a novel OVI that not only has a cancer therapeutic effect, particularly tumor growth suppression effect in primary lesions, but also further enhances systemic anti-tumor immunity, which is important for therapeutic effect in metastatic lesions.
• the inventors have found that when the CXCL10 gene is used as an immunostimulatory gene, OVI carrying this gene can specifically damage various cancer cells more efficiently than oncolytic viruses that do not contain immune genes, and can significantly suppress tumor growth even when administered locally to a cancer-bearing hamster model.
• OVI carrying CXCL10 or GM-CSF genes alone may not exhibit significant tumor growth inhibitory effects due to differences in tumor system and dosage, the combination of OVI carrying CXCL10 genes with OVI carrying GM-CSF or IL-2 genes significantly inhibited tumor growth.
• OVI carrying IL-2 genes alone showed a tendency to inhibit tumor growth compared to oncolytic viruses without immune genes.
• the dual combination of IL-2 and CXCL10 or GM-CSF genes further enhanced this effect, and the triple combination significantly inhibited tumor growth compared to oncolytic viruses without immune genes.
• challenge tests of primary cancer cells and heterologous cancer cells at distant sites showed the induction of primary cancer cell-specific systemic antitumor immunity, and the therapeutic effect at distant metastatic sites was higher with IL-2 gene alone, followed by two-drug combination and three-drug combination, in the same order as in the primary site.
• the poor weight gain caused by the introduction of immunostimulatory genes was mild in all cases, suggesting that the combined use of two or three factors does not result in any significant side effects.
• the inventors designed and constructed a multi-factor-loaded OVI capable of expressing two factors dicistronically or three factors tricistronically, in order to increase the efficiency of co-transfection and co-expression of two or more immunostimulatory genes and reduce the viral dose.
• the triple-factor-loaded OVI suppressed primary tumor growth to a similar extent as a triple-factor cocktail of OVIs loaded with the same total dose of each gene, and surprisingly, more potently prevented distant metastasis.
• OVI loaded with three therapeutic genes derived from humans and mice suppressed both primary tumors and metastatic tumors more significantly than OVIs loaded with no therapeutic genes.
• the present invention provides the following:
• CXCL10 C-X-C motif chemokine ligand 10
• Item 2 The oncolytic virus according to Item 1, wherein the promoter is a ubiquitous promoter, a cancer cell-specific promoter, or a promoter specific to an organ from which the cancer cells are derived.
• Item 4 Item 4.
• a promoter of a nucleic acid encoding at least one factor essential for viral replication or assembly is substituted with a cancer cell-specific promoter or a promoter specific to an organ from which the cancer cells are derived.
• the cancer cell-specific promoter is a survivin promoter.
• the virus is an adenovirus.
• Item 8 The oncolytic virus according to Item 7, wherein the promoter of the nucleic acid encoding E1A is replaced with a survivin promoter, and further wherein the promoter of the nucleic acid encoding E1B ⁇ 55K is replaced with an exogenous promoter selected from a ubiquitous promoter, a cancer cell-specific promoter, and an organ-specific promoter from which the cancer cells are derived.
• Item 9 Item 9.
• the oncolytic virus according to Item 8 wherein the promoter of the nucleic acid encoding E1B ⁇ 55K is substituted with a CMV promoter.
• Item 10 The oncolytic virus according to any one of Items 1 to 9, further comprising a nucleic acid encoding interleukin-2 (IL-2) under the control of a promoter functional in cancer cells, and/or a nucleic acid encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) under the control of a promoter functional in cancer cells.
• IL-2 interleukin-2
• GM-CSF granulocyte-macrophage colony-stimulating factor
• IL-2 interleukin-2
• GM-CSF granulocyte-macrophage-colony-stimulating factor
• a nucleic acid encoding CXCL10, a nucleic acid encoding IL-2 and/or a nucleic acid encoding GM-CSF are arranged in the 5'-directed manner.
• the oncolytic virus according to Item 12 wherein the nucleotides are arranged in the order: [Section 14] Item 14.
• the oncolytic virus according to Item 13 wherein the nucleic acids are linked via a 2A sequence or an IRES sequence.
• [Section 16] A combination of the oncolytic virus according to any one of Items 1 to 9 with the following oncolytic virus (a) and/or (b), or (c): (a) an oncolytic virus comprising a nucleic acid encoding IL-2 under the control of a promoter functional in cancer cells; (b) an oncolytic virus comprising a nucleic acid encoding GM-CSF under the control of a promoter functional in cancer cells; or (c) an oncolytic virus comprising a nucleic acid encoding IL-2 under the control of a promoter functional in cancer cells and a nucleic acid encoding GM-CSF under the control of a promoter functional in cancer cells. [Item 17] Item 17.
• each of the promoters functional in the cancer cells is the same or different and is a ubiquitous promoter, a cancer cell-specific promoter, or a promoter specific to an organ from which the cancer cells are derived.
• each of the promoters functional in cancer cells is the same or different and is a CA promoter, a CMV promoter, an RSV promoter, or an E2F promoter.
• each of the promoters functional in cancer cells is the same or different and is a CA promoter, a CMV promoter, an RSV promoter, or an E2F promoter.
• the oncolytic viruses are conditionally replicating adenoviruses, and the structures of the replication control regions of the oncolytic viruses are identical.
• a cancer therapeutic agent comprising, as an active ingredient, the oncolytic virus according to any one of Items 1 to 15 or the combination according to any one of Items 16 to 19.
• a method for treating cancer comprising administering to a subject having cancer an effective amount of the oncolytic virus according to any one of Items 1 to 15 or the combination according to any one of Items 16 to 19.
• [Section 20c] Use of the oncolytic virus according to any one of Items 1 to 15 or the combination according to any one of Items 16 to 19 for the manufacture of a therapeutic agent for cancer.
• Item 21 Item 21.
• Item 20 which is administered locally to a primary cancer lesion.
• Item 22 The agent according to Item 20 or 21, which is for the treatment of invasive/metastatic cancer.
• Suction 23 Item 23. The agent according to any one of Items 20 to 22, which is administered multiple times.
• the present invention makes it possible to induce cell-mediated systemic anti-tumor immunity specific to cancer antigens released from cancer cells killed by the proliferation of oncolytic viruses to a greater extent than ever before. This not only enhances the therapeutic effect on primary lesions, but can also be an effective treatment for refractory cancers with invasive and metastatic disease.
• FIG. 1-1 is a schematic diagram of the genomic sequences of various Surv. m-CRAs having a mouse-derived CXCL10 gene unit downstream of various promoters prepared in Example 1.
• FIG. 1-2 is a schematic diagram of the genomic sequences of various Surv. m-CRAs having a human-derived CXCL10 gene unit downstream of various promoters prepared in Example 1.
• 1-3 are schematic diagrams of the genomic sequences of the various Surv.m-CRAs prepared in Example 1, including those containing three therapeutic genes.
• Figure 2-1 shows the results of ELISA quantification of mouse or human CXCL10 in the culture supernatant of HEK293 cells transfected with the P2 plasmid containing nucleic acid encoding mouse CXCL10 (A: mouse CXCL10, B: human CXCL10).
• Figure 2-2 shows the results of quantification, by ELISA, of the expression of mouse CXCL10 in HaK cells, HaP-T1 cells, or BHK-21 cells infected with CRA carrying the mouse CXCL10 gene.
• Figure 2-3 shows the results of quantifying human CXCL10 expression in HaK cells infected with CRA carrying the human CXCL10 gene, using ELISA.
• FIG. 3-2 shows representative phase-contrast images of HaK cells 3 or 5 days after infection in Example 3 at 40x (left) and 100x (right) magnifications.
• FIG. 3-3 shows representative phase-contrast images of HaP-T1 cells 3 or 5 days after infection in Example 3 at 40x (left) and 100x (right) magnifications.
• FIG. 3-4 shows representative phase-contrast images of BHK-21 cells 3 or 5 days after infection in Example 3 at 40x (left) and 100x (right) magnifications.
• FIG. 4 is a diagram outlining the process of establishing a syngenic Syrian hamster cancer model in Example 4.
• Figure 5 shows the in vivo therapeutic effect of CXCL10-loaded Surv.m-CRA on subcutaneous tumors in syngenic Syrian hamsters in Example 5.
• FIG. 6 shows the therapeutic effect of combined use of two types of cytokines on Surv.m-CRA-2 in Example 6.
• FIG. 7-1 is a diagram showing an outline of the experimental protocol in Example 7.
• FIG. 7-2 shows the therapeutic effect of three types of Surv.m-CRA-2 combined use on primary tumors in Example 7.
• FIG. 7-3 is a diagram confirming the regression of HaK tumors at distant sites by combined treatment with three types of Surv.m-CRA-2 in Example 7.
• FIG. 7-4 is a diagram confirming the regression of Hap-T1 tumor at a distant site by combined treatment with three types of Surv.m-CRA-2 in Example 7.
• FIG. 7-5 shows the changes in body weight of mice over time during treatment in Example 7.
• FIG. 8 shows the expression levels of cytokines in cells infected with Surv.m-CRA-2 carrying three types of cytokine genes.
• FIG. 9-1 shows an outline of the experimental protocol in Example 10.
• FIG. 10-1 shows an outline of the experimental protocol in Example 11.
• FIG. 10-2 shows the therapeutic effects on primary tumors of a combination of three types of Surv.m-CRA-2 and Surv.m-CRA-2 carrying three types of cytokine genes.
• FIG. 10-3 shows that the combined administration of three types of Surv.m-CRA-2 and the administration of Surv.m-CRA-2 carrying three types of cytokine genes do not affect the body weight of recipients.
• FIG. 11-1 shows an outline of the experimental protocol in Example 12.
• FIG. 11-2 shows the therapeutic effects on primary tumors of two types of Surv.m-CRA-2 carrying three types of cytokine genes derived from mouse and human, and Surv.m-CRA carrying no therapeutic gene.
• Figure 11-3 shows the therapeutic effect of two types of Surv.m-CRA-2 carrying three cytokine genes derived from mouse and human, and Surv.m-CRA carrying no therapeutic gene, on primary tumors in individual mice. The tumor formation rate 17 days after the initial virus administration is shown in the upper right corner of the graph.
• Figure 11-4 shows the therapeutic effect of two doses of Surv.m-CRA-2, which carries three cytokine genes derived from mouse and human, and Surv.m-CRA, which does not carry a therapeutic gene, on primary tumors in individual mice. The tumor formation rate 17 days after the second virus dose (35 days after the first dose) is shown in the upper right corner of the graph.
• the present invention provides an oncolytic virus carrying the CXCL10 gene as an immunostimulatory gene (hereinafter also referred to as the "oncolytic virus of the present invention").
• the oncolytic virus of the present invention is surprisingly characterized by its high tumor growth inhibitory effect, at least in primary lesions, when used alone, compared to a control oncolytic virus not carrying the CXCL10 gene.
• “high therapeutic effect” such as tumor growth inhibition, preferably means that the effect is statistically significantly (e.g., p ⁇ 0.05) higher than the control, but also encompasses cases where there is a tendency for the therapeutic effect to be increased even though there is no significant difference.
• CXCL10 is a chemokine that induces the infiltration and migration of CXCR3-positive immune cells, such as NK cells and T cells, into tumors.
• CXCR3-positive immune cells such as NK cells and T cells
• adenoviruses carrying CXCL10 increased the infiltration of immune cells into tumors, but were unable to suppress tumor growth alone (Li et al., Oncoimmunology, 2022, 11(1): 2118210). Therefore, the fact that the oncolytic viruses of the present invention can suppress tumor growth more potently than oncolytic viruses not carrying therapeutic genes alone is an exceptionally remarkable effect that even those skilled in the art would not have anticipated.
• the oncolytic virus of the present invention comprises a nucleic acid encoding CXCL10 under the control of a promoter functional in cancer cells.
• “functional in cancer cells” means that the promoter has transcriptional activity that induces the expression of CXCL10 at least in cancer cells, regardless of whether it has transcriptional activity in cells other than cancer cells.
• promoters functional in cancer cells include ubiquitous promoters, cancer cell-specific promoters, and promoters specific to the organ from which the cancer cells originate.
• cancer cell-specific and “organ-specific” are not limited to promoters that exhibit no transcriptional activity whatsoever in normal cells or other organs, but also include promoters that drive gene expression in normal cells and cells of other organs within a therapeutically acceptable range.
• Ubiquitous promoters include, for example, cytomegalovirus (CMV)-derived promoters (e.g., CMV immediate-early promoter; also referred to simply as “CMV promoter” herein), Rous sarcoma virus (RSV)-derived promoters (e.g., RSV LTR; also referred to simply as “RSV promoter” herein), chicken ⁇ -actin gene promoters with cytomegalovirus immediate-early gene enhancers (also referred to as "CA promoter” herein), human immunodeficiency virus (HIV)-derived promoters (e.g., HIV LTR), and mouse mammary tumor virus (MMTV)-derived promoters (e.g., MMTV LTR).
• CMV cytomegalovirus
• RSV LTR Rous sarcoma virus
• CA promoter also referred to simply as "RSV promoter” herein
• HAV human immunodeficiency virus
• MMTV mouse
• promoters examples include promoters derived from Moloney murine leukemia virus (MoMLV) (e.g., MoMLV LTR), promoters derived from herpes simplex virus (HSV) (e.g., HSV thymidine kinase (TK) promoter), promoters derived from SV40 (e.g., SV40 early promoter), promoters derived from Epstein-Barr virus (EBV), promoters derived from adeno-associated virus (AAV) (e.g., AAV p5 promoter), promoters derived from adenovirus (AdV) (Ad2 or Ad5 major late promoter), ⁇ -actin gene promoter, PGK gene promoter, and transferrin gene promoter.
• MoMLV LTR Moloney murine leukemia virus
• HSV herpes simplex virus
• TK HSV thymidine kinase
• SV40 e.g., SV
• Cancer cell-specific promoters include, for example, the CEA (Carcinoembryonic Antigen) promoter (Mol. Cell. Biol., 10(6), 2738-2748, 1990), the E2F promoter (Neuman, E. et al., Mol. Cell. Biol., 14(10), 6607-6615, 1994), and the OC (Osteocalcin) promoter (Morrison, N.A. et al., Science, 246, 1158-1161, 1989). ), the FLK-1 promoter specific to malignant melanoma and fibrosarcoma (Xie, B. et al., Br. J.
• hypoxia-responsive region (HRE) promoter which is specific to various cancers
• the Grp78 promoter the L-plastin promoter
• the hexokinase II promoter the survivin promoter
• the Aurora kinase A promoter the Aurora kinase B promoter.
• Organ-specific promoters from which cancer cells originate are appropriately selected depending on the organ from which the cancer to be treated originates. Examples include albumin and alpha-fetoprotein promoters, which are specific to the liver, etc.; prostate-specific antigen (PSA) promoters, which are specific to the prostate; mitochondrial creatine kinase (MCK) promoters, which are specific to various organs such as the muscle and brain; and myelin basic protein (MB), glial fibrillary acidic protein (GFAP), and neuron-specific enolase (NSE) promoters, which are specific to the nervous system such as the brain.
• PSA prostate-specific antigen
• MCK mitochondrial creatine kinase
• MB myelin basic protein
• GFAP glial fibrillary acidic protein
• NSE neuron-specific enolase
• the oncolytic virus of the present invention may contain a nucleic acid encoding CXCL10 under the control of an inducible promoter.
• inducible promoters include the metallothionein-1 gene promoter.
• CXCL10 can be expressed in cancer cells by administering an inducer such as heavy metals such as gold, zinc, or cadmium, steroids such as dexamethasone, alkylating agents, chelating agents, or cytokines locally to the cancer at the desired time.
• Cytokine genes exhibit high physiological activity even at low expression levels, and their overexpression carries the risk of causing undesirable side effects such as cytokine storm.
• oncolytic viruses replicate in large quantities in cancer cells, and therefore therapeutic genes carried on viral vectors can be expressed in large quantities within cancer cells and released at high levels from destroyed cancer cells.
• a promoter that provides a CXCL10 expression level that does not cause undesirable side effects in the recipient, or that can cause tolerable side effects, within a range that suppresses tumor growth at the primary cancer site and induces cancer-specific systemic anti-tumor immunity sufficient to suppress cancer at invasive and distant metastatic sites.
• such a suitable promoter when using adenovirus as the oncolytic virus, can be selected by constructing a panel of m-CRAs in which a nucleic acid encoding CXCL10 is linked downstream of various candidate promoters using the m-CRA technology developed by the inventors, and then administering the m-CRAs to, for example, cancer cell lines or a hamster cancer model system established by the inventors that is permissive for the proliferation of adenovirus, and analyzing the expression levels, therapeutic effects, and side effects.
• a nucleic acid encoding CXCL10 can be placed under the control of a CA promoter, a CMV promoter, an RSV promoter, or an E2F promoter.
• these promoters exert high transcriptional activity in cancer cells in this order, and whichever promoter is used can kill various cancer cells in vitro with roughly equivalent efficacy.
• administration of the CMV promoter which has strong transcriptional activity, exerts a superior therapeutic effect compared to a control oncolytic virus without a therapeutic gene, without significant side effects.
• the CA promoter, CMV promoter, RSV promoter, and E2F promoter used in the present invention include nucleic acids containing the nucleotide sequences represented by SEQ ID NOs: 1, 2, 3, and 4, respectively, or nucleotide sequences that hybridize under stringent conditions to the complementary strand sequences of the respective nucleotide sequences and have cancer cell-specific transcription activity equivalent to that of promoters consisting of the respective nucleotide sequences.
• nucleic acids examples include nucleic acids containing nucleotide sequences that share at least about 80%, preferably at least about 90%, more preferably at least about 95%, particularly preferably at least about 97%, and most preferably at least about 98% identity with the nucleotide sequences represented by the respective SEQ ID NOs.
• a promoter that controls the expression of CXCL10 can be prepared by cloning genomic DNA containing the promoter region from genomic DNA extracted from cells or tissues derived from humans or other mammals using a nucleic acid consisting of a known promoter sequence (e.g., a nucleotide sequence represented by any of SEQ ID NOS: 1 to 4) as a probe, cleaving the genomic DNA with a DNase, for example, an appropriate restriction enzyme, into DNA fragments containing the desired partial promoter sequence, separating the fragments by gel electrophoresis, recovering the desired band, and purifying the DNA.
• a known promoter sequence e.g., a nucleotide sequence represented by any of SEQ ID NOS: 1 to 4
• a DNase for example, an appropriate restriction enzyme
• CXCL10-encoding nucleic acids used in the present invention include nucleic acids that contain the nucleotide sequence represented by SEQ ID NO: 5 (corresponding to the nucleotide sequence (CDS) from nucleotides 67 to 360 of the mRNA sequence of human CXCL10 registered in GenBank under accession number NM_001565), or a nucleotide sequence that hybridizes under stringent conditions with its complementary strand sequence, and encode a protein with activity equivalent to that of CXCL10 (e.g., the ability to induce immune cell infiltration and migration into tumors).
• SEQ ID NO: 5 corresponding to the nucleotide sequence (CDS) from nucleotides 67 to 360 of the mRNA sequence of human CXCL10 registered in GenBank under accession number NM_001565
• CDS nucleotide sequence from nucleotides 67 to 360 of the mRNA sequence of human CXCL10 registered in GenBank under accession number NM_001565
• nucleic acids that hybridize under stringent conditions with the complementary strand sequence of the nucleotide sequence represented by SEQ ID NO: 5 include nucleic acids containing a nucleotide sequence that shares at least about 60%, preferably at least about 70%, more preferably at least about 80%, particularly preferably at least about 90%, and most preferably at least about 95% identity with the nucleotide sequence represented by SEQ ID NO: 5.
• the nucleic acid encodes an amino acid sequence that has at least about 90% identity, preferably at least about 95%, more preferably at least about 97%, and particularly preferably at least about 98% identity to the amino acid sequence represented by SEQ ID NO: 6, and that causes a protein containing the amino acid sequence to have substantially the same activity (e.g., the activity of inducing immune cell infiltration and migration into tumors) as a protein containing the amino acid sequence represented by SEQ ID NO: 6.
• the nucleic acid encoding CXCL10 may be an ortholog in a non-human mammal of a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 5 (for example, a nucleic acid encoding mouse CXCL10 consists of the nucleotide sequence represented by SEQ ID NO: 7 (corresponding to the nucleotide sequence (CDS) from nucleotides 76 to 369 of the mRNA sequence of mouse CXCL10 registered in GenBank under accession number NM_021274)).
• CDS nucleotide sequence
• the nucleic acid encoding CXCL10 is a nucleic acid encoding human CXCL10 (i.e., a protein consisting of the amino acid sequence represented by SEQ ID NO: 6).
• a nucleic acid encoding CXCL10 can be cloned, for example, by PCR amplification using synthetic DNA primers containing a portion of the nucleotide sequence of the CDS region of the CXCL10 gene, or by hybridizing DNA incorporated into an appropriate expression vector with a labeled DNA fragment or synthetic DNA containing the nucleotide sequence of the CDS region of the CXCL10 gene. Hybridization can be performed, for example, according to the method described in Molecular Cloning, 2nd ed. (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989).
• the nucleotide sequence of the DNA can be converted using known kits such as MutanTM-super Express Km (Takara Shuzo Co., Ltd.) and MutanTM-K (Takara Shuzo Co., Ltd.) according to known methods such as the ODA-LA PCR method, the Gappedduplex method, the Kunkel method, or methods similar thereto.
• the cloned DNA can be used as is, depending on the purpose, or after digestion with restriction enzymes or the addition of linkers, as desired.
• the DNA may have a translation initiation codon ATG at its 5' end and a translation termination codon TAA, TGA, or TAG at its 3' end. These translation initiation and termination codons can be added using an appropriate synthetic DNA adapter.
• An expression vector containing a nucleic acid encoding CXCL10 can be produced, for example, by excising a desired fragment from a nucleic acid encoding the CDS region of the CXCL10 gene and ligating the fragment downstream of the promoter in the expression vector described above.
• the expression vector preferably contains a transcription termination signal, i.e., a terminator region, downstream of the nucleic acid encoding CXCL10.
• it may further contain a selection marker gene for selecting transformed cells (such as a gene that confers resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, or phosphinothricin, or a gene that complements an auxotrophic mutation).
• the oncolytic virus of the present invention is not particularly limited in type, as long as it replicates specifically in cancer cells, killing (lysing) those cancer cells, and the daughter viruses re-infect nearby cancer cells one after another, thereby exerting a tumor-suppressing effect. It may be conferred cancer selectivity by any mechanism, but it must be capable of carrying at least a CXCL10 expression cassette as an immunostimulatory gene.
• Oncolytic viruses that have been marketed or advanced to clinical trials include adenovirus, herpes simplex virus (HSV), vaccinia virus, measles virus, reovirus, Newcastle disease virus, coxsackievirus, and parvovirus.
• HSV its pathogenicity in normal cells is abolished by deleting genes for DNA polymerases such as ribonucleotide reductase and thymidine kinase, allowing it to replicate only in cancer cells in which cell proliferation is activated.
• the ⁇ 34.5 gene product prevents viral growth from being inhibited by host protein kinase R (PKR); deleting this gene reduces pathogenicity in normal cells and allows the virus to grow only in cancer cells that express Ras, which inhibits PKR.
• PSR host protein kinase R
• inactivating the thymidine kinase gene allows the virus to grow in a manner dependent on thymidine kinase in host cancer cells.
• measles virus the virus can grow in various cancer cells that highly express CD46, the receptor used by the measles vaccine strain.
• the oncolytic virus of the present invention has a promoter for a nucleic acid encoding at least one factor essential for viral replication or assembly substituted with a cancer cell-specific promoter or a promoter specific to the organ from which the cancer cells are derived.
• Vectors essential for viral replication or assembly means any protein essential for viral replication or assembly, such as viral proteins required for the transcription of various viral genes (for example, in adenovirus, transcription of the early genes E1A, E1B, E2, and E4 is required before transcription of viral structural proteins; E1A in particular is the first protein to be transcribed and translated after infection, and transcription of this E1A protein is essential for the initiation of subsequent transcription of various viral proteins), or viral structural proteins (for example, in adenovirus, the late gene products L1, L2, L3, L4, L5, etc.).
• viral proteins required for the transcription of various viral genes for example, in adenovirus, transcription of the early genes E1A, E1B, E2, and E4 is required before transcription of viral structural proteins; E1A in particular is the first protein to be transcribed and translated after infection, and transcription of this E1A protein is essential for the initiation of subsequent transcription of various viral proteins
• viral structural proteins for example, in adenovirus, the late gene products L1, L2, L
• Such factors vary depending on the virus species used, but for example, in the case of adenovirus, they include E1A, E1B, E2, and E4, preferably E1A and E1B, and more preferably E1A; in the case of adeno-associated virus, they include Rep78 and Rep68 under the control of the p5 promoter, and Rep52 and Rep40 under the control of the p19 promoter; in the case of herpes simplex virus, they include early gene products such as ICP0, ICP4, ICP22, and ICP27, and thymidine kinase; and in the case of Sendai virus, they include N protein, P protein, and L protein.
• Factors essential for viral replication may be those that are essential for inducing the cellular environment necessary for viral replication in normal cells, but that lack regions that are not necessary for viral replication in cancer cells.
• viral replication in normal cells requires the inactivation of Rb and p53 to initiate the cell cycle, but cancer cells are already in a cell cycle state, so in the case of adenovirus, the Rb-binding region of E1A and the p53-binding region of E1B are not required for viral replication in cancer cells.
• conditionally replicating adenovirus (CRA) of the present invention can achieve cancer cell-specific viral replication by deleting the E1A 24KDa region (E1A ⁇ 24), the E1B 55KDa region (E1B ⁇ 55K), or the E1B 19KDa region (E1B ⁇ 19).
• At least one endogenous promoter of a gene encoding a factor essential for the replication or assembly of the virus is replaced with a cancer cell-specific promoter or a promoter specific to the organ from which the cancer cells are derived.
• the "cancer cell-specific promoter” and “cancer cell-derived organ-specific promoter” may similarly be preferably one of those exemplified as promoters that control the expression of CXCL10.
• Preferred examples of cancer cell-specific promoters include the survivin promoter, Aurora kinase A promoter, or Aurora kinase B promoter, with the survivin promoter being more preferred.
• the survivin promoter used in the oncolytic viruses of the present invention is the promoter of the human survivin gene or its orthologous gene in other mammals (e.g., monkeys, cows, horses, pigs, dogs, cats, sheep, goats, rabbits, mice, rats, etc.), preferably the promoter of the human or mouse-derived survivin gene (comprising the nucleotide sequences set forth in SEQ ID NOS: 9 and 10, respectively, or a partial sequence thereof).
• a homologous survivin promoter it is preferable to use a homologous survivin promoter; however, a heterologous promoter may also be used as long as it can exert promoter activity sufficient to infect cancer cells efficiently and achieve killing effects.
• an oncolytic virus containing the mouse survivin gene promoter can be used as a vector for treating human cancer.
• the Aurora kinase promoter used in the oncolytic virus of the present invention is not particularly limited as long as it is derived from a gene belonging to the Aurora kinase family, but examples include mammalian (e.g., human, monkey, cow, horse, pig, dog, cat, sheep, goat, rabbit, mouse, rat, etc.) orthologs of the Drosophila Aurora-A, -B, and -C genes.
• a promoter of the Aurora kinase A gene or Aurora kinase B gene derived from human or other mammals is preferred, and a human Aurora kinase A or human Aurora kinase B promoter is more preferred.
• an Aurora kinase promoter of the same species; however, a heterologous promoter may also be used as long as it can exert promoter activity sufficient to provide sufficient infection efficiency and killing effect on cancer cells.
• the nucleotide sequence length of the survivin promoter and Aurora kinase promoter is not particularly limited, as long as they are specific to target cancer cells and can activate the transcription of the gene linked downstream to an extent that exerts sufficient therapeutic activity against cancer.
• sequences having the sequence lengths described in WO 2019/093435 can be used.
• the desired specificity and transcriptional activity can be achieved by including the nucleotide sequence from positions -173 to -19, with the translation start point at +1 (the nucleotide sequence from positions 1124 to 1278 in the nucleotide sequence shown in SEQ ID NO: 10), and in the case of a human survivin promoter, the nucleotide sequence from positions -173 to -1, with the translation start point at +1 (the nucleotide sequence from positions 1296 to 1468 in the nucleotide sequence shown in SEQ ID NO: 9).
• the survivin promoter used in the present invention preferably comprises at least a partial nucleotide sequence from positions 1124 to 1278 of the nucleotide sequence shown in SEQ ID NO: 10, or at least a partial nucleotide sequence from positions 1296 to 1468 of the nucleotide sequence shown in SEQ ID NO: 9, and in a preferred embodiment, the survivin promoter essentially consists of such a partial nucleotide sequence.
• WO 2019/093435 may also be referenced for suitable nucleotide sequences of Aurora kinase promoters.
• conditionally replicating virus When a conditionally replicating virus (CRV) is introduced into a cell and is dependent on a cancer cell-specific or organ-specific promoter from which the cancer cells are derived, it cannot replicate in an environment where the promoter is not activated (e.g., normal cells), and therefore the cell is not harmed.
• a cancer cell-specific or organ-specific promoter-dependent CRV enters an environment where the promoter is activated (e.g., cancer cells)
• the virus replicates there and damages the cell due to the cytotoxicity of the viral proteins.
• the virus released from the lysed cell successively infects surrounding cells that have not been transfected with the vector, and the same process is repeated. In this way, theoretically, the CRV can ultimately be introduced into all cancer cells within the lesion.
• nucleic acids encoding factors essential for viral replication or assembly is under the control of a cancer cell-specific or organ-specific promoter, viral growth or assembly is limited to an environment in which the promoter is activated, nucleic acids encoding other factors essential for viral replication or assembly may be under the control of any exogenous promoter different from the promoter.
• the ubiquitous promoter, cancer cell-specific promoter, cancer cell-derived organ-specific promoter, inducible promoter, etc. exemplified above as promoters controlling the expression of CXCL10, can be similarly preferably used.
• nucleic acids encoding factors essential for the replication or assembly of two or more viruses when placed under the control of the same cancer cell-specific or organ-specific promoter, they may be arranged polycistronically under the control of a single promoter, or monocistronically under the control of separate promoters.
• nucleic acid encoding a factor essential for viral replication that is controlled by a promoter other than a cancer cell-specific or organ-specific promoter
• a nucleic acid encoding the above-mentioned mutant viral protein e.g., E1A ⁇ 24, E1B ⁇ 55K
• E1A ⁇ 24, E1B ⁇ 55K a nucleic acid encoding the above-mentioned mutant viral protein that has been deleted for a region that is essential for inducing the cellular environment necessary for viral proliferation in normal cells but is not necessary for viral proliferation in target cancer cells
• the CRA of the present invention has the promoter of the nucleic acid encoding E1A replaced with a survivin promoter, and the promoter of the nucleic acid encoding E1B ⁇ 55K replaced with an exogenous promoter selected from a ubiquitous promoter, a cancer cell-specific promoter, and a promoter specific to the organ from which the cancer cells are derived.
• the exogenous promoter is a CMV promoter.
• the multifactorial cancer-specific growth-regulated recombinant adenovirus system (m-CRA; JP 2005-046101 A and WO 2005/012536 A) developed by the present inventors is used.
• m-CRA multifactorial cancer-specific growth-regulated recombinant adenovirus system
• Examples of plasmid vectors suitable for use in constructing m-CRA are illustrated in the above-mentioned patent documents.
• the survivin promoter or the like is used as promoter A and/or promoter B in plasmid vector P1
• any of the promoters controlling the expression of CXCL10 can be used as promoter C in plasmid vector P2.
• a plasmid vector P1 comprising an E1A gene (which may lack the 24 KDa region) operably linked to a survivin promoter and an E1B gene (which may lack the 19 KDa or 55 KDa region) operably linked to a ubiquitous promoter (such as a CMV promoter); a plasmid vector P2 comprising a nucleic acid encoding CXCL10 operably linked to a ubiquitous promoter (such as a CA promoter, CMV promoter, or RSV promoter) or a cancer cell-specific promoter (such as an E2F promoter); and a backbone plasmid P3 comprising an adenovirus genome lacking the E1 region (which may have a target cell-specific mutation in the fiber gene).
• a ubiquitous promoter such as a CMV promoter
• a plasmid vector P2 comprising a nucleic acid encoding CXCL10 operably linked to a ubiquitous promoter (such as a CA promoter, CMV promote
• CRA cancer cell-specific adenovirus
• the present invention also provides an oncolytic virus that combines the CXCL10 gene with an IL-2 and/or GM-CSF gene as an immunostimulatory gene.
• the nucleic acid encoding IL-2 and/or the nucleic acid encoding GM-CSF may be contained in a single oncolytic virus together with the nucleic acid encoding CXCL10, or may be contained in an oncolytic virus separate from the nucleic acid encoding CXCL10.
• the promoters functional in cancer cells in the oncolytic viruses (a) and (b) above may each independently be a ubiquitous promoter, a cancer cell-specific promoter, or a promoter specific to the organ from which the cancer cells are derived.
• the ubiquitous promoters, cancer cell-specific promoters, and organ-specific promoters from which the cancer cells are derived can also be preferably used as these promoters.
• the promoter functional in cancer cells in the oncolytic viruses (a) and/or (b) may be the same as or different from the promoter controlling the expression of CXCL10 in the oncolytic viruses of the present invention.
• the promoter controlling the expression of IL-2 and the promoter controlling the expression of GM-CSF are each independently a CA promoter, a CMV promoter, an RSV promoter, or an E2F promoter.
• the oncolytic viruses (a) and (b) can be oncolytic viruses having the same structure as the oncolytic viruses containing the nucleic acid encoding CXCL10, except that the nucleic acid encoding CXCL10 is a nucleic acid encoding IL-2 or GM-CSF. Therefore, in a particularly preferred embodiment, the oncolytic viruses (a) and (b) are CRAs, like the oncolytic viruses of the present invention, and the structure of the proliferation control region of each oncolytic virus is also identical to that of the oncolytic viruses of the present invention.
• the promoter of the nucleic acid encoding E1A is replaced with a survivin promoter, and further that the promoter of the nucleic acid encoding E1B ⁇ 55K is replaced with an exogenous promoter selected from a ubiquitous promoter, a cancer cell-specific promoter, and an organ-specific promoter from which the cancer cells are derived, preferably a CMV promoter.
• nucleic acid encoding IL-2 used in the present invention includes a nucleic acid that contains a nucleotide sequence represented by SEQ ID NO: 11 (corresponding to the nucleotide sequence (CDS) from positions 286 to 744 of the human IL-2 mRNA sequence registered in GenBank under accession number NM_000586) or a nucleotide sequence that hybridizes under stringent conditions with its complementary sequence, and encodes a protein with activity equivalent to that of IL-2 (e.g., T cell stimulating activity).
• SEQ ID NO: 11 corresponding to the nucleotide sequence (CDS) from positions 286 to 744 of the human IL-2 mRNA sequence registered in GenBank under accession number NM_000586) or a nucleotide sequence that hybridizes under stringent conditions with its complementary sequence, and encodes a protein with activity equivalent to that of IL-2 (e.g., T cell stimulating activity).
• nucleic acids that hybridize under stringent conditions with the complementary sequence of the nucleotide sequence represented by SEQ ID NO: 11 include nucleic acids containing a nucleotide sequence that shares at least about 60%, preferably at least about 70%, more preferably at least about 80%, particularly preferably at least about 90%, and most preferably at least about 95% identity with the nucleotide sequence represented by SEQ ID NO: 11.
• the nucleic acid encodes an amino acid sequence that has at least about 90% identity, preferably at least about 95%, more preferably at least about 97%, and particularly preferably at least about 98% identity to the amino acid sequence represented by SEQ ID NO: 12, such that a protein comprising the amino acid sequence has substantially the same activity (e.g., T cell stimulating activity) as a protein comprising the amino acid sequence represented by SEQ ID NO: 12.
• the nucleic acid encoding IL-2 may be an ortholog in a non-human mammal of a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 11 (for example, a nucleic acid encoding mouse IL-2 consists of the nucleotide sequence represented by SEQ ID NO: 13 (corresponding to the nucleotide sequence (CDS) from positions 49 to 555 of the mRNA sequence of mouse IL-2 registered in GenBank under accession number NM_008366)).
• CDS nucleotide sequence
• the nucleic acid encoding IL-2 is a nucleic acid encoding human IL-2 (i.e., a protein consisting of the amino acid sequence represented by SEQ ID NO: 12).
• nucleic acid encoding GM-CSF used in the present invention includes a nucleic acid that contains a nucleotide sequence represented by SEQ ID NO: 15 (corresponding to the nucleotide sequence (CDS) from positions 36 to 467 of the mRNA sequence of human GM-CSF registered in GenBank under accession number NM_000758), or a nucleotide sequence that hybridizes under stringent conditions with its complementary strand sequence, and encodes a protein with activity equivalent to that of GM-CSF (e.g., antigen presentation enhancing activity).
• SEQ ID NO: 15 corresponding to the nucleotide sequence (CDS) from positions 36 to 467 of the mRNA sequence of human GM-CSF registered in GenBank under accession number NM_000758
• CDS nucleotide sequence from positions 36 to 467 of the mRNA sequence of human GM-CSF registered in GenBank under accession number NM_000758
• nucleic acids that hybridize under stringent conditions with the complementary strand sequence of the nucleotide sequence represented by SEQ ID NO: 15 include nucleic acids containing a nucleotide sequence that has about 60% or more identity to the nucleotide sequence represented by SEQ ID NO: 15, preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more identity.
• the nucleic acid encodes an amino acid sequence that has at least about 90% identity, preferably at least about 95%, more preferably at least about 97%, and particularly preferably at least about 98% identity with the amino acid sequence represented by SEQ ID NO: 16, and that causes a protein containing the amino acid sequence to have substantially the same activity (e.g., antigen presentation enhancing activity) as a protein containing the amino acid sequence represented by SEQ ID NO: 16.
• the nucleic acid encoding GM-CSF may be an ortholog in a non-human mammal of a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 15 (for example, a nucleic acid encoding mouse GM-CSF consists of the nucleotide sequence represented by SEQ ID NO: 17 (corresponding to the nucleotide sequence (CDS) from positions 290 to 712 of the mRNA sequence of mouse GM-CSF registered in GenBank under accession number NM_009969)).
• CDS nucleotide sequence
• the nucleic acid encoding GM-CSF is a nucleic acid encoding human GM-CSF (i.e., a protein consisting of the amino acid sequence represented by SEQ ID NO: 18).
• Nucleic acids encoding IL-2 or GM-CSF can be cloned, for example, by amplifying them by PCR using synthetic DNA primers containing a portion of the nucleotide sequence of the CDS region of the IL-2 or GM-CSF gene, or by hybridizing the DNA incorporated into an appropriate expression vector with a labeled DNA fragment or synthetic DNA containing the nucleotide sequence of the CDS region of the IL-2 or GM-CSF gene. Hybridization can be performed, for example, according to the method described in Molecular Cloning, 2nd ed. (see above).
• the nucleotide sequence of the DNA can be converted using known kits such as MutanTM-super Express Km (Takara Shuzo Co., Ltd.) and MutanTM-K (Takara Shuzo Co., Ltd.) according to known methods such as the ODA-LA PCR method, the Gappedduplex method, the Kunkel method, or methods similar thereto.
• the cloned DNA can be used as is, depending on the purpose, or after digestion with restriction enzymes or the addition of linkers, as desired.
• the DNA may have a translation initiation codon ATG at its 5' end and a translation termination codon TAA, TGA, or TAG at its 3' end. These translation initiation and termination codons can be added using an appropriate synthetic DNA adapter.
• An expression vector containing a nucleic acid encoding IL-2 or GM-CSF can be produced, for example, by excising the desired fragment from the nucleic acid encoding the CDS region of the IL-2 or GM-CSF gene and ligating the fragment downstream of the promoter in the expression vector described above.
• the expression vector preferably contains a transcription termination signal, i.e., a terminator region, downstream of the nucleic acid encoding IL-2 or GM-CSF.
• a selection marker gene for selecting transformed cells such as a gene that confers resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, or phosphinothricin, or a gene that complements an auxotrophic mutation.
• the oncolytic virus of the present invention comprises a nucleic acid encoding IL-2 and/or a nucleic acid encoding GM-CSF together with a nucleic acid encoding CXCL10 in a single oncolytic virus. Accordingly, the present invention also provides the oncolytic virus of the present invention, further comprising a nucleic acid encoding IL-2 under the control of a promoter functional in cancer cells and/or a nucleic acid encoding GM-CSF under the control of a promoter functional in cancer cells.
• the promoter controlling the expression of CXCL10 and the promoter controlling the expression of IL-2 and/or the promoter controlling the expression of GM-CSF may be a single promoter or separate promoters.
• the promoter controlling the expression of CXCL10 and the promoter controlling the expression of IL-2 and/or the promoter controlling the expression of GM-CSF are a single promoter, the nucleic acid encoding CXCL10 and the nucleic acid encoding IL-2 and/or the nucleic acid encoding GM-CSF are linked via a sequence enabling polycistronic expression (e.g., an IRES sequence, a 2A sequence (P2A, T2A, E2A, F2A)).
• the oncolytic virus of the present invention is an adenovirus
• the size of the foreign gene that can be carried is limited. Therefore, it is advantageous to place them under the control of a single promoter, particularly when carrying a nucleic acid encoding IL-2 and a nucleic acid encoding GM-CSF in addition to a nucleic acid encoding CXCL10.
• the oncolytic virus used allows for the insertion of larger foreign genes, it may be preferable to place each nucleic acid under the control of a separate promoter, as this allows for high-level control of the expression of each immunostimulatory gene.
• each promoter When each nucleic acid is placed under the control of a separate promoter, each promoter may be the same or different, and may be an exogenous promoter selected from a ubiquitous promoter, a cancer cell-specific promoter, and a promoter specific to the organ from which the cancer cells are derived. Polycistronic expression may result in differences in the expression levels of each gene, so each nucleic acid may be linked downstream of the same separate promoter to ensure equivalent expression levels for each gene.
• nucleic acid encoding CXCL10 and a nucleic acid encoding IL-2 and/or GM-CSF in a single oncolytic virus is improved infection and expression efficiency and reduced dosage. If each gene were included in a separate oncolytic virus, they would simultaneously infect cancer cells, which could reduce the efficiency of expression, and require two or three times the dosage to introduce the same number of copies.
• An oncolytic virus comprising a nucleic acid encoding CXCL10 and a nucleic acid encoding IL-2 and/or a nucleic acid encoding GM-CSF in a polycistronic expression arrangement, (i) CXCL10-IL-2; (ii) CXCL10-GM-CSF; (iii) IL-2-CXCL10; (iv) GM-CSF-CXCL10; (v) CXCL10-IL-2-GM-CSF; (vi) CXCL10-GM-CSF-IL-2; (vii) IL-2-CXCL10-GM-CSF; (viii) IL-2-GM-CSF-CXCL10; (ix) GM-CSF-CXCL10-IL-2; or (x) GM-CSF-IL-2-CXCL10 Examples of such viruses include oncolytic viruses, which are arranged in the order of
• an oncolytic virus comprising a nucleic acid encoding CXCL10, a nucleic acid encoding IL-2, and a nucleic acid encoding GM-CSF in a polycistronic expression-enabling arrangement
• the first nucleic acid and the second nucleic acid are linked from the 5' side via a P2A sequence
• the second nucleic acid and the third nucleic acid are linked via a T2A sequence.
• the present invention provides oncolytic virus immunotherapy (OVI) that uses two immunostimulatory genes in combination: the CXCL10 gene and either the IL-2 gene or the GM-CSF gene; or OVI that uses three immunostimulatory genes in combination: the CXCL10 gene, the IL-2 gene, and the GM-CSF gene. While the combination of two factors enhances the therapeutic effect compared to the CXCL10 gene alone, the combination of three factors can further significantly enhance the therapeutic effect.
• OVI oncolytic virus immunotherapy
• an oncolytic virus of the present invention containing only a nucleic acid encoding CXCL10 as an immunostimulatory gene, and (c)
• a combination with an oncolytic virus is provided, which includes a nucleic acid encoding IL-2 under the control of a promoter functional in cancer cells and a nucleic acid encoding GM-CSF under the control of a promoter functional in cancer cells.
• the oncolytic virus (c) above can be prepared in the same manner as the oncolytic virus of the present invention containing two or more immunostimulatory genes.
• the present invention also provides an oncolytic virus immunotherapy agent (OVI), i.e., a cancer therapeutic agent (hereinafter also referred to as “the OVI of the present invention” or “the therapeutic agent of the present invention”), which contains any of the oncolytic viruses of the present invention or any combination of the oncolytic viruses as an active ingredient.
• OVI oncolytic virus immunotherapy agent
• the cancers targeted by the therapeutic agent of the present invention are not particularly limited, and examples thereof include renal cell carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, and liver cancer.
• cholangiocarcinoma bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, acute lymphocytic leukemia, acute myeloid leukemia, chronic leukemia, polycythemia vera, lymphoma, and multiple myeloma.
• Preferred target cancers include refractory invasive and metastatic cancers that are ineffective against conventional OVI and other existing cancer
• the oncolytic virus which is the active ingredient
• a pharmacologically acceptable carrier used here is a variety of organic or inorganic carrier substances commonly used as pharmaceutical ingredients, and is incorporated as an excipient, lubricant, binder, or disintegrant in solid formulations; or as a solvent, solubilizer, suspending agent, isotonicity agent, buffer, or soothing agent in liquid formulations.
• formulation additives such as preservatives, antioxidants, colorants, and sweeteners can also be used as needed.
• excipients include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropyl cellulose, sodium carboxymethylcellulose, gum arabic, pullulan, light anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminometasilicate.
• Suitable examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica, and the like.
• binders include pregelatinized starch, sucrose, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropyl cellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone.
• disintegrants include lactose, sucrose, starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, carboxymethylstarch sodium, light anhydrous silicic acid, and low-substituted hydroxypropylcellulose.
• the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil, and the like.
• Suitable examples of the solubilizing agent include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate.
• suspending agents include surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glycerin monostearate; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; polysorbates; and polyoxyethylene hydrogenated castor oil.
• Suitable examples of the isotonic agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, and the like.
• Suitable examples of the buffering agent include buffer solutions such as phosphate, acetate, carbonate, and citrate.
• Suitable examples of soothing agents include benzyl alcohol.
• Suitable examples of the preservative include parahydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid.
• Suitable examples of antioxidants include sulfites and ascorbic acid salts.
• Suitable examples of coloring agents include water-soluble food tar dyes (e.g., food dyes such as Food Red Nos. 2 and 3, Food Yellow Nos. 4 and 5, and Food Blue Nos.
• water-insoluble lake dyes e.g., aluminum salts of the above-mentioned water-soluble food tar dyes
• natural dyes e.g., ⁇ -carotene, chlorophyll, red iron oxide, etc.
• sweeteners include saccharin sodium, dipotassium glycyrrhizinate, aspartame, stevia, and the like.
• the therapeutic agent of the present invention can be administered either by the ex vivo method, in which the cancer cells of the animal being treated are removed from the body, cultured, and then introduced back into the body (or transplanted), or by the in vivo method, in which the vector is administered directly into the body of the recipient; however, the in vivo method is preferred.
• the vector can be introduced into the target cells by microinjection, calcium phosphate co-precipitation, PEG, electroporation, etc.
• the formulation can be administered by, for example, injection, catheter, balloon catheter, local injection, or implantation of an implant incorporating the oncolytic virus of the present invention into the lesion.
• the dosage of the therapeutic agent of the present invention varies depending on the type of oncolytic virus, promoter activity in target cancer cells, type of immunostimulatory gene to be combined, administration route, severity of disease, target animal species, drug tolerance, body weight, age, etc.; however, for example, when a cancer-specific replication-promoting adenovirus is used as the oncolytic virus, safety has been confirmed in conventional clinical trials of cancer gene therapy using viral particles (vp) of 1 x 10 to 10 vp/tumor, and this amount serves as a guide for administration (Molecular Therapy, 18: 429-434, 2010).
• vp viral particles
• the therapeutic agent of the present invention can be administered in a single dose or multiple doses.
• the number of doses and the interval between doses can be selected appropriately.
• the therapeutic agent of the present invention can be used in combination with other cancer therapeutic agents or treatment methods.
• other cancer therapeutic agents or treatment methods include immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, etc.), CAR-T cell therapy, chemotherapy agents (e.g., 5-fluorouracil, cisplatin, etc.), and radiation therapy.
• immune checkpoint inhibitors are known to be less effective against cancers with low immune cell infiltration
• the therapeutic agent of the present invention activates immune cells and promotes their infiltration into cancer tissue, which is expected to produce a strong synergistic effect.
• CAR-T cell therapy has yet to achieve sufficient therapeutic efficacy against solid cancers.
• Possible reasons for this include the difficulty of CAR-T cells infiltrating into solid cancer tissue, the presence of cancer cells that do not express the target cancer antigen, and the immunosuppressive environment within cancer tissue, which inhibits CAR-T cell function.
• Administration of the OVI of the present invention is expected to induce the expression of cytokines and chemokines, promote tumor infiltration of CAR-T cells, and improve the immunosuppressive environment within cancer tissue, preventing the suppression of CAR-T cell function.
• cancer therapeutic agents or treatment methods used in combination with the therapeutic agent of the present invention can be administered or carried out in accordance with the dosage and administration method used when the therapeutic agent or treatment method is used alone.
• the Syrian hamster renal cancer cell line (HaK) was provided by Dr. William S.M. Wold (Saint Louis University School of Medicine) [Thomas, MA, et al., Syrian hamster as a permissive immunocompetent animal model for the study of oncolytic adenovirus vectors. Cancer Res, 2006. 66(3): pp. 1270-1276.].
• HaP-T1 (Syrian hamster pancreatic adenocarcinoma) cells were obtained from the Riken Cell Bank (Ibaraki, Japan), and BHK-21 (Baby hamster kidney) cells were obtained from the JCRB Cell Bank (Osaka, Japan).
• HaK cells were cultured in Dulbecco's modified Eagle's medium (Nacalai Tesque, Kyoto, Japan).
• HaP-T1 cells were cultured in MEM containing 1% non-essential amino acids (Sigma-Aldrich, St Louis, MO) and 1 mM sodium pyruvate (Thermo Fisher Scientific, Waltham, MA).
• BHK-21 cells were cultured in MEM supplemented with 1% NEAA. All media were supplemented with 10% fetal bovine serum (FBS, Biowest, Nuaille, France), 100 units/mL penicillin, and 100 ⁇ g/mL streptomycin (Thermo Fisher Scientific).
• HaK parental
• HaP-T1 heterologous cancer cells
• the hamsters were inoculated with HaK and HaP-T1 cells (1 ⁇ 10 cells per group) in the left and right dorsal regions, respectively. Animals were observed macroscopically for the presence of tumor nodules after various viral treatments for an additional 74 days.
• Example 1 Preparation of Surv.m-CRA-2 Carrying a Cytokine Gene.
• An E1-deleted replication-deficient adenovirus (Ad.dE1.3) was prepared according to a previously published report [Murofushi, Y., et al., Cell cycle-specific changes in hTERT promoter activity in normal and cancerous cells in adenoviral gene therapy: a promising implication of telomerase-dependent targeted cancer gene therapy. Int J Oncol, 2006. 29(3): pp. 681-8; Kamizono, J., et al., Survivin-responsive conditionally replicating adenovirus exhibits cancer-specific and efficient viral replication. Cancer Res, 2005. 65(12): pp.
• Ad.dE1.3 does not contain a transgene.
• Suv.m-CRA which contains wild-type E1A downstream of the Survivin promoter, E1B ⁇ 55K downstream of the CMV promoter, and a therapeutic gene downstream of a specific promoter, was constructed using a previously reported m-CRA construction method [Watanabe, M., et al., Adenovirus Biology, Recombinant Adenovirus, and Adenovirus Usage in Gene Therapy. Viruses, 2021. 13(12)., Kamizono, J., et al., Survivin-responsive conditionally replicating adenovirus exhibits cancer-specific and efficient viral replication. Cancer Res, 2005. 65(12): p.
• CRAs carrying nucleic acids encoding CXCL10, GM-CSF, and/or IL-2 as therapeutic genes Specifically, we constructed the following 16 types of CRAs: (1) Surv.m-CRA/E2Fp-mCXCL10 (2) Surv.m-CRA/RSVp-mCXCL10 (3) Surv.m-CRA/CMVp-mCXCL10 (4) Surv.m-CRA/CAp-mCXCL10 (5) Surv.m-CRA/E2Fp-hCXCL10 (6) Surv.m-CRA/RSVp-hCXCL10 (7) Surv.m-CRA/CMVp-hCXCL10 (8) Surv.m-CRA/CAp-hCXCL10 (9) Surv.m-CRA/E2Fp-mGM-CSF (10) Surv.m-CRA/RSVp-mIL-2 (11) Surv.m-CRA/CMV-mCXCL10-mIL2-mGM (12) Surv.m-CRA-CRA
• E2Fp stands for the E2F-1 promoter
• RSVp stands for the Rous sarcoma virus long terminal repeat (RSV) promoter
• CMVp stands for the human cytomegalovirus (CMV) immediate early gene enhancer/promoter
• Cap stands for the CMV enhancer and beta-actin (CA) promoter.
• the "m” or "h” before each cytokine name indicates that it is of mouse or human origin, respectively.
• Surv.m-CRA No transgene
• Surv.m-CRA/CMVp-EGFP expresses the enhanced green fluorescent protein (EGFP) gene under the CMV promoter.
• Figure 1-1 the gene sequences of CRAs carrying only mouse and human CXCL10 are shown in Figure 1-1 (mouse) and Figure 1-2 (human). Additionally, the gene sequences of CRAs carrying three types of therapeutic genes are shown in Figure 1-3 (mouse and human).
• P2A represents the 2A sequence derived from porcine teschovirus-1
• T2A represents the 2A sequence derived from Thosea asigna virus.
• Example 2 In vitro functional verification of the prepared Surv.m-CRA-2 Among the various CRAs prepared in Example 1, the protein expression of CXCL10 in cells was verified. Specifically, HEK293 cells seeded on 6-well plates were transfected with various P2 plasmids expressing the CXCL10 gene. Additionally, various Surv.m-CRAs expressing the CXCL10 gene were infected into HaK, HaP-T1, or BHK-21 cells at an MOI of 1, 10, or 100 (only HaK cells were infected with CRAs carrying the human CXCL10 gene). All cells were cultured for 48 hours, after which the supernatants were collected and frozen at -80°C.
• CXCL10 protein expression analysis in HEK293 cells and in HaK, HaP-T1, or BHK-21 cells was performed as follows.
• Mouse CXCL10 was assayed using the Mouse CXCL10 DuoSet ELISA (DY466, R&D Systems, Minneapolis, MN), and human CXCL10 was assayed using the Human CXCL10 Quantikine ELISA Kit (DIP100, R&D Systems) according to the manufacturer's protocol.
• the total protein concentration of each sample was determined by Bradford assay (Bio-Rad, Hercules, CA).
• Figure 2-1 A: Expression of mouse CXCL10 in HEK293 cells transfected with the P2 plasmid containing nucleic acid encoding mouse CXCL10; B: Expression of human CXCL10 in HEK293 cells transfected with the P2 plasmid containing nucleic acid encoding human CXCL10), Figure 2-2 (Expression of mouse CXCL10 in HaK cells, HaP-T1 cells, or BHK-21 cells infected with CRA carrying the mouse CXCL10 gene), and Figure 2-3 (Expression of human CXCL10 in HaK cells infected with CRA carrying the human CXCL10 gene).
• mouse CXCL10 protein was not detected after infection with Surv.m-CRA (no transgene) or in uninfected cells.
• infection with Surv.m-CRA/E2Fp-mCXCL10, Surv.m-CRA/RSVp-mCXCL10, Surv.m-CRA/CMVp-mCXCL10, or Surv.m-CRA/CAp-mCXCL10 resulted in a virus dose-dependent increase in mouse CXCL10 protein secretion in all cell lines at low, mild, moderate, or high levels (approximately 1-fold, approximately 10-fold, and approximately 100-fold differences, respectively).
• Example 3 Examination of in vitro cytotoxicity after virus infection (WST assay) The cytotoxic effect of Surv.m-CRA carrying the mouse CXCL10 gene prepared in Example 1 after viral infection was examined in vitro. Specifically, the experiment was carried out as follows: HaK cells (800 cells/well), HaP-T1 cells, or BHK-21 cells (500 cells/well) seeded in a 24-well plate were infected with Surv.m-CRA or Ad.dE1.3 at an MOI of 0.3.
• the viruses used were Ad.dE1.3, Surv.m-CRA (no transgene), Surv.m-CRA/CMV-EGFP, Surv.m-CRA/E2F-mCXCL10, Surv.m-CRA/RSV-mCXCL10, Surv.m-CRA/CMV-mCXCL10, and Surv.m-CRA/CA-mCXCL10 (MOI: 0.3).
• Hak cells were seeded in a 24-well plate at 800 cells/well, and Hap-T1 cells or BHK-21 cells were seeded at 500 cells/well.
• Culture for 24 hours. Virus infection and incubation (3 to 5 days).
• Replace the medium with 500 ⁇ l of medium containing 50 ⁇ l of Cell Count Reagent SF.
• Figure 3-1 The results are shown in Figure 3-1.
• Figures 3-2 to 3-4 show representative phase-contrast images of HaK cells, HaP-T1 cells, and BHK-21 cells 3 and 5 days after virus infection, at 40x (left) and 100x (right) magnifications, respectively.
• Example 4 Establishment of a syngenic Syrian hamster cancer model To fully investigate in vivo therapeutic effects, such as replicating virus-dependent cytotoxicity and cytokine-induced immune responses, a syngenic hamster cancer model was established by subcutaneously transplanting HaK cells into Syrian hamsters that are permissive for human serotype 5 adenovirus replication.
• Figure 4 shows an overview of the establishment process.
• Human adenovirus does not grow in mice, but can partially grow in Syrian hamsters, suggesting that this model could be used as a refractory cancer model for Surv.m-CRA treatment.
• Example 5 Examination of the therapeutic effect of CXCL10-loaded Surv.m-CRA-2 Using the Syrian hamster subcutaneous tumor model established in Example 4, the therapeutic effect of CXCL10-loaded Surv.m-CRA-2 was examined.
• Subcutaneous tumor models in hamsters were established by implanting 2 ⁇ 10 HaK cells suspended in 200 ⁇ L of DMEM containing 50% Matrigel (BD Biosciences, Franklin Lakes, NJ) into the dorsal flank of 5- to 6-week-old female Syrian hamsters (Japan SLC, Shizuoka, Japan). After tumor volumes reached 200–580 mm, the hamsters were randomly divided into 5–6 groups.
• one hamster from each group received a single intratumoral injection of 1.0 ⁇ 10 pfu (plaque-forming units) in 100 ⁇ L of buffer (10 mmol/L Tris-HCl pH 7.4, 1 mmol/L MgCl2, 10% glycerol, and 20 ⁇ g/ml hexadimethrine bromide) on day 0.
• buffer 10 mmol/L Tris-HCl pH 7.4, 1 mmol/L MgCl2, 10% glycerol, and 20 ⁇ g/ml hexadimethrine bromide
• Body weight was monitored twice weekly using a digital balance. Histopathological analysis was performed as previously reported [Kamizono, J., et al., Survivin-responsive conditionally replicating adenovirus exhibits cancer-specific and efficient viral replication. Cancer Res, 2005. 65(12): pp. 5284-91; Chen, SH, et al., Combination gene therapy for liver metastasis of colon carcinoma in vivo. Proc Natl Acad Sci U S A, 1995. 92(7): pp.
• Example 7 Examination of the therapeutic effect of Surv.m-CRA-2 by the combination of three cytokines. We investigated whether the combination of three cytokines enhances the therapeutic effect. In this example, we also examined the therapeutic effect on the primary tumor and the induction of primary tumor-specific systemic antitumor immunity by a distant site challenge test. An overview of the experiment is shown in Figure 7-1.
• the hamsters were inoculated with HaK cells and HaP-T1 cells (1 ⁇ 10 cells per group) in the left and right dorsal regions, respectively. Treated animals were observed macroscopically for the presence of tumor nodules after various viral treatments for an additional 74 days, and body weight was monitored as an indicator of side effects of these treatments.
• Figure 7-2 therapeutic effect of the combination of three types of Surv.m-CRA-2 on the primary tumor
• Figure 7-3 regression of a distant HaK tumor by combined treatment with three types of Surv.m-CRA-2 (challenge test)
• Figure 7-4 regression of a distant Hap-T1 tumor by combined treatment with three types of Surv.m-CRA-2 (challenge test)
• Figure 7-5 changes in body weight over time
• Example 8 Confirmation of expression of each gene in P2 plasmid carrying three therapeutic genes.
• the three therapeutic genes prepared in Example 1 were carried by Surv.m-CRA-2 (i.e., (12) Surv.m-CRA/CMV-mCXCL10-mIL2-mGM (hereinafter, sometimes referred to as "Surv.m-CRA/CMVp-mCIG"), (13) Surv.m-CRA/CMV-mCXCL10-mGM-mIL2 (hereinafter, sometimes referred to as "Surv.m-CRA/CMVp-mCGI"), (14) Surv.m-CRA/CMV-hCXCL10-hIL2-hGM (hereinafter, sometimes referred to as "Surv.m-CRA/CMVp-hCIG”), and (15) Each P2 plasmid used to construct Surv.m-CRA/CMV-hCXCL10-hGM-hIL2 (hereinafter sometimes referred to as "Surv.m-CRA
• MILLIPLEX® Mouse Cytokine/Chemokine Magnetic Bead Panel MCYTOMAG-70K, Millipore, Burlington, MA
• MILLIPLEX® Human Cytokine/Chemokine/Growth Factor Panel A HCYTA-60K, Millipore
• transfection of the P2 plasmid containing nucleic acids encoding mouse or human CXCL10, IL-2, and GM-CSF confirmed the expression of mouse or human CXCL10, IL-2, and GM-CSF.
• Example 9 Confirmation of the expression of each gene in Surv.m-CRA-2 carrying three types of therapeutic genes Surv.m -CRA/CMVp-mCIG, Surv.m-CRA/CMVp-mCGI, Surv.m-CRA/CMVp-hCIG, and Surv.m-CRA/CMVp-hCGI carrying the three types of therapeutic genes prepared in Example 1 were infected into HEK293 cells, and the expression level of the protein encoded by each therapeutic gene was confirmed using ELISA. Culture supernatants were collected 48 hours after virus infection, and expression levels of mouse genes were examined using the MILLIPLEX® Mouse Cytokine/Chemokine panel.
• HEK293 cells were transfected with the P2 plasmids used to construct each virus (pUni/CMVp-mCIG, pUni/CMVp-mCGI, pUni/CMVp-hCIG, and pUni/CMVp-hCGI), and the supernatants collected 96 hours later were used as positive controls.
• Example 10 Examination of the in vitro cytotoxicity of Surv.m-CRA-2 carrying three types of therapeutic genes (WST assay) The cytotoxicity of hamster-derived cancer cells (HaK and HaP-T1) and normal cells (BHK-21) infected with Surv.m-CRA/CMVp-mCGI or Surv.m-CRA/CMVp-mCIG was examined by viable cell count assay. Each cell line was seeded at 5 ⁇ 10 cells/well into a 96-well plate the day before.
• WST assay The cytotoxicity of hamster-derived cancer cells (HaK and HaP-T1) and normal cells (BHK-21) infected with Surv.m-CRA/CMVp-mCGI or Surv.m-CRA/CMVp-mCIG was examined by viable cell count assay. Each cell line was seeded at 5 ⁇ 10 cells/well into a 96-well plate the day before.
• Example 11 Examination of the therapeutic effect of Surv.m-CRA-2 carrying three therapeutic genes. This example examines the in vivo therapeutic effect of Surv.m-CRA-2 carrying three therapeutic genes. Using the same method as in Example 7 above, except for using Surv.m-CRA-2 carrying three therapeutic genes, we examine the therapeutic effect on the primary tumor in a Syrian hamster subcutaneous tumor model and the induction of primary tumor-specific systemic antitumor immunity in a distant site challenge test. When three types of OVIs carrying different genes are administered, it is not guaranteed that all three genes will be fully introduced into a single cell. In contrast, when one type of OVI carrying all three genes is administered, all three genes are reliably introduced into a single cell.
• administering one virus carrying all three genes may enhance the therapeutic effect compared to administering three different types of OVIs. Furthermore, administering one virus carrying all three genes simultaneously allows for a lower dosage compared to administering three types of OVIs, which is expected to improve safety.
• Example 9 confirmed that Surv.m-CRA-2 carrying all three genes correctly expressed the proteins encoded by each therapeutic gene, and Example 10 confirmed its in vitro cytotoxic effect on cancer cells. Therefore, treatment with Surv.m-CRA-2 carrying all three genes simultaneously is expected to achieve therapeutic efficacy and safety comparable to or superior to that shown in Example 7.
• HaK cells were subcutaneously transplanted into a single site on the dorsum of 6-week-old female Syrian hamsters.
• the HaK cells were prepared in Coring® Matrigel basement membrane matrix (Coring) at a concentration of 1 ⁇ 10 cells/200 ⁇ l in 50% Matrigel.
• the experimental protocol is shown in Figure 10-1. Tumor size was measured twice weekly using digital calipers. Tumor volume was calculated using the long axis (mm) ⁇ short axis (mm) ⁇ short axis (mm) ⁇ 0.5 ( mm3 ). Data are presented as mean ⁇ standard error, and statistical significance between groups was tested using Student's T-test.
• Surv.m-CRA/CAp-mCXCL10 + Surv.m-CRA/E2Fp-mGM-CSF + Surv.m-CRA/RSVp-mIL-2 and Surv.m-CRA/CMVp-mCGI showed significantly enhanced therapeutic effects (tumor suppression) against treated primary tumor cells (nodules) compared to Surv.m-CRA (No transgene), which does not carry a therapeutic gene (#, P ⁇ 0.05 vs. Surv.m-CRA (No transgene)) (Figure 10-2).
• Example 12 Examination of the therapeutic efficacy of Surv.m-CRA-2 carrying three therapeutic genes derived from mice or humans (1) Inhibitory effect on tumor growth at primary tumors.
• Six-week-old female Syrian hamsters were subcutaneously implanted with 1 x 10 HaK cells at a single site on the dorsum.
• HaK cells were prepared in Coring® Matrigel basement membrane matrix (Coring) at a concentration of 1 x 10 cells/200 ⁇ l in 50% Matrigel.
• Ad.dE1.3 control
• Surv.m-CRA no transgene
• Surv.m-CRA/CMVp-mCGI Surv.m-CRA/CMVp-hCGI viruses
• the changes in tumor diameter over time were evaluated. Seventeen days after the first administration, the same dose of virus was administered.
• the experimental protocol is shown in Figure 11-1. Tumor size was measured twice weekly using digital calipers. Tumor volume was calculated using the long axis (mm) ⁇ short axis (mm) ⁇ short axis (mm) ⁇ 0.5 ( mm3 ). Data are presented as mean ⁇ standard error, and statistical significance between groups was tested using Student's T-test.
• Surv.m-CRA No transgene
• Surv.m-CRA/CMVp-mCGI No transgene
• Surv.m-CRA/CMVp-hCGI all showed significant therapeutic effects (tumor-suppressing effects) against the treated primary cancer cells (nodules) compared to the control (Ad.dE1.3) (*, p ⁇ 0.05 vs. Ad.dE1.3 (Control)) ( Figure 11-2).
• the two types, Surv.m-CRA/CMVp-mCGI and Surv.m-CRA/CMVp-hCGI showed a tendency for further enhanced therapeutic effects (tumor-suppressing effects) compared to Surv.m-CRA (No transgene), which does not carry a therapeutic gene.
• Surv.m-CRA Compared with Surv.m-CRA without a therapeutic gene, Surv.m-CRA carrying three therapeutic genes derived from mouse or human demonstrated significantly enhanced therapeutic efficacy (Figure 11-4). Without being bound by any particular theory, one possible reason why Surv.m-CRA, which carries mouse genes, had a greater therapeutic effect than human genes is that there is greater homology between mice and hamsters than between humans and hamsters, especially with regard to GM-CSF.
• the OVI of the present invention not only has an enhanced tumor growth inhibitory effect in primary lesions compared to conventional OVI, but also has a significantly enhanced ability to induce systemic primary cancer-specific anti-tumor immunity, making it possible for it to exert excellent therapeutic effects even against refractory invasive and metastatic cancers that are ineffective against conventional cancer viral therapies, including OVI, and other existing cancer treatments. Therefore, the present invention is extremely useful.

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