Oncolytic adenoviruses and topoisomerase I inhibitors for use in cancer treatment

Abstract

This disclosure relates, in particular, to combination therapies of oncolytic adenoviruses with topoisomerase I inhibitors or their prodrugs, such as topotecan or SN-38, or topoisomerase inhibitor prodrugs like irinotecan, for the treatment or prevention of ovarian cancer, cervical cancer, lung cancer, colon cancer or colorectal cancer, and / or pancreatic cancer.

Description

[Technical Field]

[0001] This disclosure relates, in particular, to combination therapies with the oncolytic adenoviruses topotecan, irinotecan, or SN-38 for use in certain tumors.

[0002] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 584,003, filed on 20 September 2023, and U.S. Provisional Application No. 63 / 624,896, filed on 25 January 2024, the entire contents of these applications being incorporated herein by reference.

[0003] Description of the electronically submitted XML file This application includes a sequence listing submitted electronically in XML file format, the entirety of which is incorporated herein by reference. The XML file, created on September 19, 2024, is named "VCN-007_Sequence_Listing.xml" and is 53,098 bytes in size. [Background technology]

[0004] Combination therapy is a central trend in cancer treatment. Combining anticancer drugs may synergistically target key pathways, potentially enhancing efficacy compared to monotherapy approaches. This approach may reduce resistance while also offering therapeutic anticancer benefits such as reduced tumor growth and limited tumor metastasis.

[0005] There is a need for new treatment strategies that offer efficient and effective results at a low cost, especially those that can be used in conjunction with current therapies. [Overview of the Initiative]

[0006] Accordingly, in some embodiments, the present disclosure provides a method for treating or preventing pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, and / or colon or colorectal cancer in patients in need of treatment or prevention, comprising co-administering to a patient (i) an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and optionally a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug of a topoisomerase inhibitor to a patient.

[0007] In the embodiments, oncolytic adenoviruses are administered, for example, by intravenous, intra-arterial, intratumoral, and / or intraperitoneal injection. In the embodiments, topoisomerase I inhibitors or their prodrugs are administered, for example, by intraperitoneal, intravenous, intra-arterial, or intratumoral injection and / or oral administration.

[0008] In one embodiment, the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug are formulated in separate compositions. In another embodiment, the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug are formulated in a single composition. In yet another embodiment, the separate compositions are administered simultaneously or concurrently.

[0009] In one embodiment, the oncolytic adenovirus is administered first, and the topoisomerase I inhibitor or its prodrug is administered within approximately 60 minutes of the administration of the oncolytic adenovirus. In another embodiment, the topoisomerase I inhibitor or its prodrug is administered within approximately 30 minutes, 20 minutes, 10 minutes, 5 minutes, or 1 minute of the administration of the oncolytic adenovirus. In yet another embodiment, the topoisomerase I inhibitor or its prodrug is administered first, and the oncolytic adenovirus is administered within approximately 60 minutes of the administration of the topoisomerase I inhibitor or its prodrug. In yet another embodiment, the oncolytic adenovirus is administered within approximately 30 minutes, 20 minutes, 10 minutes, 5 minutes, or 1 minute of the administration of the topoisomerase I inhibitor or its prodrug.

[0010] In the embodiment, the oncolytic adenovirus is administered first, and the topoisomerase I inhibitor or its prodrug is administered within approximately one day of the administration of the oncolytic adenovirus. In the embodiment, the topoisomerase I inhibitor or its prodrug is administered within approximately 14 days, approximately 10 days, approximately 7 days, approximately 5 days, or approximately one day of the administration of the oncolytic adenovirus. In the embodiment, the topoisomerase I inhibitor or its prodrug is administered first, and the oncolytic adenovirus is administered within approximately one day of the administration of the topoisomerase I inhibitor or its prodrug. In the embodiment, the oncolytic adenovirus is administered within approximately 14 days, approximately 10 days, approximately 7 days, approximately 5 days, or approximately one day of the administration of the topoisomerase I inhibitor or its prodrug.

[0011] In this embodiment, the topoisomerase I inhibitor is topotecan, or SN-38, or irinotecan, an SN-38 prodrug that is converted to SN-38 in vivo.

[0012] In the embodiments, the oncolytic adenovirus optionally includes a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome. In the embodiments, the hyaluronidase enzyme is human hyaluronidase enzyme PH20. In the embodiments, the sequence encoding the hyaluronidase enzyme is Sequence ID No. 9 or a sequence having at least 75%, at least about 80%, or at least about 85%, or at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto.

[0013] In this embodiment, the oncolytic adenovirus is produced from human adenovirus serotype 5.

[0014] In embodiments, replication of the oncolytic adenovirus occurs in tumor cells (e.g., solid tumors) that have an abnormal Rb-E2F pathway, and does not occur in healthy, non-neoplastic, or normal cells. In embodiments, this oncolytic adenovirus is engineered to delete the Rb binding domain from the sequence encoding the E1a protein, and further insert four binding sites for E2F-1 and one binding site for Sp1 into the endogenous promoter of E1a, so as to control the expression of E1a, replicate only in tumor cells, and not replicate in healthy cells, non-tumor cells, or normal cells.

[0015] In embodiments, replication of the oncolytic adenovirus occurs in tumor cells (e.g., solid tumors) that have an abnormal Rb-E2F pathway, and does not occur in healthy, non-neoplastic, or normal cells. In embodiments, this oncolytic adenovirus is engineered to delete Δ24 from the sequence encoding the E1a protein, and further insert four binding sites for E2F-1 and one binding site for Sp1 into the endogenous promoter of E1a, so as to control the expression of E1a, replicate only in tumor cells, and not replicate in healthy cells, non-tumor cells, or normal cells.

[0016] In embodiments, the oncolytic adenovirus has the heparan sulfate binding domain present in the adenovirus fiber 91 KKTK 94 has a capsid modified such that (SEQ ID NO: 6) is substituted.

[0017] In embodiments, the oncolytic adenovirus has the heparan sulfate binding domain present in the adenovirus fiber 91 KKTK 94 (SEQ ID NO: 6) is substituted by the domain 91 RGDK 94 has a capsid modified such that it is substituted by (SEQ ID NO: 7).

[0018] In embodiments, the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3), or a functional variant thereof.

[0019] In the embodiment, the patient is a human patient. In the embodiment, the human patient is a female human patient.

[0020] In this embodiment, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, and / or colon cancer or colorectal cancer are resistant to conventional chemotherapy and / or radiotherapy.

[0021] In the embodiments, the method improves and / or enhances and / or enhances the antitumor effect compared to treatment with a topoisomerase I inhibitor or its prodrug that is not associated with oncolytic adenovirus. In the embodiments, the method results in a reduction or maintenance of tumor size and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with ovarian cancer compared to treatment with a topoisomerase I inhibitor or its prodrug that is not associated with oncolytic adenovirus. In the embodiments, the method results in a reduction or maintenance of tumor size and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with cervical cancer compared to treatment with a topoisomerase I inhibitor or its prodrug that is not associated with oncolytic adenovirus. In the embodiments, the method results in a reduction or maintenance of tumor size and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with lung cancer compared to treatment with a topoisomerase I inhibitor or its prodrug that is not associated with oncolytic adenovirus. In the embodiment, the method results in a reduction or maintenance of tumor size and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with colon cancer or colorectal cancer, compared to treatment with a topoisomerase I inhibitor or its prodrug that does not involve oncolytic adenovirus. In the embodiment, the method results in a reduction or maintenance of tumor size and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with pancreatic cancer, compared to treatment with a topoisomerase I inhibitor or its prodrug that does not involve oncolytic adenovirus.

[0022] In some embodiments, the Disclosure provides a method for treating or preventing pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, and / or colon or colorectal cancer in a patient in need of treatment or prevention, comprising administering to the patient an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and optionally a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is or has been treated with a topoisomerase I inhibitor or a prodrug thereof.

[0023] In the embodiments, oncolytic adenoviruses are administered, for example, by intravenous, intra-arterial, intratumoral, and / or intraperitoneal injection. In the embodiments, topoisomerase I inhibitors or their prodrugs are administered by intraperitoneal, intravenous, intra-arterial, intratumoral, or oral administration.

[0024] In this embodiment, the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug are formulated in separate compositions.

[0025] In this embodiment, the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug are formulated in a single composition.

[0026] In one embodiment, an oncolytic adenovirus is administered first, and a topoisomerase I inhibitor or its prodrug is administered within approximately 12 weeks of the administration of the oncolytic adenovirus. In another embodiment, the topoisomerase I inhibitor or its prodrug is administered within approximately 60 minutes, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, approximately 11 days, approximately 12 days, approximately 13 days, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks, approximately 8 weeks, approximately 9 weeks, approximately 10 weeks, or approximately 11 weeks of the administration of the oncolytic adenovirus.

[0027] In one embodiment, a topoisomerase I inhibitor or its prodrug is administered first, and the oncolytic adenovirus is administered within approximately 12 weeks of the administration of the topoisomerase I inhibitor or its prodrug. In another embodiment, the oncolytic adenovirus is administered within approximately 60 minutes, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, approximately 11 days, approximately 12 days, approximately 13 days, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks, approximately 8 weeks, approximately 9 weeks, approximately 10 weeks, or approximately 11 weeks of the administration of the topoisomerase I inhibitor or its prodrug.

[0028] In this embodiment, the topoisomerase I inhibitor or its prodrug is topotecan, SN-38, and / or irinotecan.

[0029] In the embodiment, the hyaluronidase enzyme is human hyaluronidase enzyme PH20. In the embodiment, the sequence encoding the hyaluronidase enzyme is sequence number 9 or a sequence having at least 75%, at least about 80%, or at least about 85%, or at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto.

[0030] In this embodiment, the oncolytic adenovirus is produced from human adenovirus serotype 5.

[0031] In the embodiment, replication of the oncolytic adenovirus occurs in tumor cells with an abnormal Rb-E2F pathway, but not in healthy, non-tumorous, or normal cells. In the embodiment, the oncolytic adenovirus is engineered to replicate only in tumor cells and not in healthy, non-tumor, or normal cells by deleting the Rb-binding domain or Δ24 from the sequence encoding the E1a protein, and by inserting four E2F-1 binding sites and one Sp1 binding site into the endogenous promoter of E1a.

[0032] In an embodiment, the oncolytic adenovirus comprises the heparan sulfate binding domain present in the adenovirus fiber 91 KKTK 94 (SEQ ID NO: 6) has a capsid modified such that it is substituted. In an embodiment, the oncolytic adenovirus comprises the heparan sulfate binding domain present in the adenovirus fiber 91 KKTK 94 (SEQ ID NO: 6) is a domain 91 RGDK 94 (SEQ ID NO: 7) has a capsid modified such that it is substituted.

[0033] In an embodiment, the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3), or a functional variant thereof.

[0034] In an embodiment, the patient is a human patient. In an embodiment, the human patient is a female human patient.

[0035] In an embodiment, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, and / or colon cancer or colorectal cancer is resistant to treatment with conventional chemotherapy and / or radiation therapy.

[0036] In the embodiments, the method improves and / or enhances and / or enhances the antitumor effect compared to treatment with a topoisomerase I inhibitor or its prodrug that is not associated with oncolytic adenovirus. In the embodiments, the method results in a reduction or maintenance of tumor size and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with ovarian cancer compared to treatment with a topoisomerase I inhibitor or its prodrug that is not associated with oncolytic adenovirus. In the embodiments, the method results in a reduction or maintenance of tumor size and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with cervical cancer compared to treatment with a topoisomerase I inhibitor or its prodrug that is not associated with oncolytic adenovirus. In the embodiments, the method results in a reduction or maintenance of tumor size and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with lung cancer compared to treatment with a topoisomerase I inhibitor or its prodrug that is not associated with oncolytic adenovirus. In the embodiment, the method results in a reduction or maintenance of tumor size and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with colon cancer or colorectal cancer, compared to treatment with a topoisomerase I inhibitor or its prodrug that does not involve oncolytic adenovirus. In the embodiment, the method results in a reduction or maintenance of tumor size and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with pancreatic cancer, compared to treatment with a topoisomerase I inhibitor or its prodrug that does not involve oncolytic adenovirus.

[0037] In the embodiment, the method results in a reduction or maintenance of tumor size and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with colon cancer or colorectal cancer, compared to treatment with a topoisomerase I inhibitor or its prodrug that does not involve oncolytic adenovirus. In the embodiment, the method results in a reduction or maintenance of tumor size and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with pancreatic cancer, compared to treatment with a topoisomerase I inhibitor or its prodrug that does not involve oncolytic adenovirus.

[0038] In this embodiment, ovarian cancer is epithelial ovarian cancer, germ cell tumor, and / or stromal cell tumor. In this embodiment, cervical cancer is squamous cell carcinoma, adenocarcinoma, and / or adenosquamous carcinoma (mixed type). In this embodiment, lung cancer is small cell lung cancer (also called oat cell carcinoma), non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer, mesothelioma, and / or lung carcinoid tumor. In this embodiment, colon cancer or colorectal cancer is adenocarcinoma, gastrointestinal stromal tumor (GIST), squamous cell carcinoma, carcinoid tumor, lymphoma, or colon sarcoma. In this embodiment, pancreatic cancer is exocrine or endocrine cancer, or adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, or gelatinous carcinoma. [Brief explanation of the drawing]

[0039] [Figure 1] A schematic diagram of a non-limiting trial design to evaluate the efficacy of topotecan or irinotecan + VCN-01 in lung cancer or ovarian cancer models is shown. [Figure 2]This paper presents the cytotoxic effects of SN-38, irinotecan (IRI), and topotecan (Topo) on a panel of pancreatic cancer cell lines, specifically NP-18(A), NP-9(B), PANC-1(C), BxPC3(D), and HP-2(E). Cell viability curves after exposure to serial dilutions of the drugs are shown. Cell viability was measured on day 5 by BCA assay (mean + SEM). Cell viability curves were adjusted using nonlinear regression analysis (log(inhibitor) vs. normalized response - variable slope). N=3 experiments were conducted for each cell line. The graph shows one representative experiment. [Figure 3] The mean IC50 values ​​(n=3) for a panel of pancreatic cancer cell lines, specifically NP-18(A), NP-9(B), PANC-1(C), BxPC3(D), and HP-1(E), are shown. IC50 values ​​for SN-38, IR, and Topo are also shown for each cell line. IC50 values ​​were estimated by applying nonlinear regression fitting to the cell viability curve. Three replicate trials were performed. [Figure 4] This shows the relative levels of E1A and E2F1 mRNA in NP-18 and NP-9 cells. mRNA levels are shown compared to VCN-01-infected cells (E1A) or untreated cells (E2F1). [Figure 5] A schematic diagram of a non-limiting example of a mouse NP-18 tumor study is shown. [Figure 6] This shows the percentage of weight gain / loss in NP-18 tumor-bearing animals in the tests described in Example 4. [Figure 7] This shows the tumor growth (%) in NP-18 tumor-carrying animals in the tests described in Example 4. [Figure 8] This shows the quantification of VCN-01 viral genome (vg) and E1A mRNA in tumors from the study described in Example 4. Tumors were collected on day 37, at the end of the study. Tumor DNA was isolated and subjected to qPCR to measure vgs, and tumor RNA was isolated and subjected to qPCR to measure the level of E1A mRNA. [Figure 9] A schematic diagram of a non-limiting example of a mouse NP-9 tumor study is shown. [Modes for carrying out the invention]

[0040] Treatment for ovarian cancer In some embodiments, the present disclosure provides a method for treating or preventing ovarian cancer in a patient in need of treatment or prevention, the method comprising administering to the patient (i) an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and optionally a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, and (ii) a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended).

[0041] In some embodiments, the present disclosure provides a method for preventing, eliminating, or mitigating metastatic, secondary malignant, or recurrent disease associated with ovarian cancer in patients in need thereof, the method comprising administering to the patient (i) an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and optionally a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, and (ii) a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended).

[0042] In some embodiments, the Disclosure provides a method for treating or preventing ovarian cancer, cervical cancer, lung cancer, colon cancer, or colorectal cancer and / or pancreatic cancer in a patient in need of treatment or prevention, the method comprising administering to the patient an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is or has been treated with a topoisomerase I inhibitor or a prodrug thereof.

[0043] In some embodiments, the Disclosure provides a method for preventing, eliminating, or mitigating metastatic, secondary malignant, or recurrent disease associated with ovarian cancer in a patient in need thereof, the method comprising administering to the patient an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is or has been treated with a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended).

[0044] In the embodiments, the method reduces or eliminates ovarian tumors or ovarian cancers, or their cells. In the embodiments, the method slows the growth and / or progression and / or metastasis of ovarian tumors or ovarian cancers, or their cells.

[0045] In embodiments, ovarian cancer is either a primary tumor or a metastatic tumor. A primary tumor or cancer that moves from its original location and the major organ where metastases are present can ultimately lead to the death of the subject due to the decline of function of the affected organ. Metastasis is the spread of cancer cells from a primary tumor to other parts of the body, resulting in the occurrence of cancer cells or populations of cancer cells in locations different from the primary tumor. Metastasis can ultimately lead to the death of the subject. For example, in embodiments, ovarian cancer includes benign and malignant cancers, polyps, hyperplasias, and dormant tumors and micrometastases.

[0046] In this embodiment, ovarian cancer is epithelial ovarian cancer, germ cell tumor, or stromal cell tumor.

[0047] In this embodiment, ovarian cancer cells are serous, papillary serous, metastatic, borderline, mucinous, or clear.

[0048] In this embodiment, the ovarian cancer is a grade 1 ovarian cancer characterized by, for example, (i) well-differentiated tissue or (ii) poorly differentiated nuclei with rare mitotic figures.

[0049] In this embodiment, ovarian cancer is a stage I ovarian cancer characterized by (i) the tumor being confined to one ovary, with a complete capsule, no tumor on the ovarian surface, and negative peritoneal lavage fluid (stage IA); (ii) the tumor extending to both ovaries, but with a complete capsule, no tumor on the ovarian surface, and negative peritoneal lavage fluid (stage IB); (iii) intraoperative dissipation (stage IC1); (iv) preoperative capsule rupture or presence of tumor on the ovarian surface (stage IC2); or (v) malignant cells being found in ascites or peritoneal lavage fluid (stage IC3).

[0050] In this embodiment, ovarian cancer is a stage II ovarian cancer characterized, for example, by: (i) extension and / or transplantation of the tumor over the uterus and / or fallopian tubes (stage IIA); or (ii) extension of the tumor into other pelvic and abdominal tissues (stage IIB).

[0051] In the embodiment, ovarian cancer is a stage III ovarian cancer characterized by, for example, spread from the outside of the pelvis to the peritoneum and / or lymph nodes, or to the posterior abdomen. In the embodiment, ovarian cancer is a stage III ovarian cancer characterized by, for example, spread to lymph nodes in the posterior abdominal cavity (stage 3A1); detection of cancer cells in tissue samples from the inner peritoneal layer and / or lymph nodes (stage 3A2); detection of cancer growth of 2 cm or less in the inner peritoneal layer and / or lymph nodes (stage 3B); or detection of cancer growth exceeding 2 cm in the inner peritoneal layer (stage 3C).

[0052] In this embodiment, the ovarian cancer is, for example, a stage IV ovarian cancer characterized by being progressive (metastatic) (e.g., spreading to the liver or lungs).

[0053] In the embodiment, the method delays or prevents progression from Stage I (e.g., IA, IB, or IC) to one of Stage II (e.g., IIA or IIB), Stage III (e.g., IIIA, IIIB, or IIIIC), and Stage IV.

[0054] In the embodiment, the method delays or prevents progression from Stage II (e.g., IIA or IIB) to Stage III (e.g., IIIA, IIIB, or IIIIC) and one of Stage IV.

[0055] In one embodiment, the method delays or prevents progression from Stage III (e.g., IIIA, IIIB, or IIIIC) to Stage IV.

[0056] In embodiments, the treatment of ovarian cancer is further combined with one or more of the following: surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. In embodiments, chemotherapy is one or more of the following: platinum-based drugs (e.g., cisplatin or carboplatin, but not limited) and taxanes (e.g., paclitaxel or docetaxel, but not limited). In embodiments, chemotherapy is one or more of the following: cisplatin, carboplatin, paclitaxel, docetaxel, nab-paclitaxel, altretamine, capecitabine, cyclophosphamide, etoposide, gemcitabine, ifosfamide, doxorubicin, melphalan, pemetrexed, and vinorelbine. In embodiments, targeted therapy is anti-angiogenic therapy (e.g., bevacizumab or ramucirumab). In this embodiment, the immunotherapy is one or more of the following: a drug that targets the PD-1 pathway, such as atezolizumab, durvalumab, semiprimab, nivolumab, and pembrolizumab, and / or a drug that targets the CTLA-4 pathway, such as ipilimumab.

[0057] In this embodiment, the ovarian cancer is metastatic ovarian cancer. In this embodiment, the ovarian cancer is metastatic ovarian cancer that has progressed during or after the initial chemotherapy.

[0058] Treatment for cervical cancer In some embodiments, the present disclosure provides a method for treating or preventing cervical cancer in a patient in need of treatment or prevention, the method comprising administering to the patient (i) an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and optionally a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, and (ii) a topoisomerase I inhibitor or its prodrug, such as topotecan, SN-38, and / or irinotecan.

[0059] In some embodiments, the present disclosure provides a method for treating or preventing cervical cancer in a patient in need of treatment or prevention, the method comprising administering to the patient (i) an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and optionally a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended).

[0060] In some embodiments, the Disclosure provides a method for treating or preventing cervical cancer in a patient in need of treatment or prevention, the method comprising administering to the patient (i) an oncolytic adenovirus comprising a replication mechanism specific to tumor cells and optionally a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is or has been treated with a topoisomerase I inhibitor or its prodrug, such as topotecan, SN-38, and / or irinotecan.

[0061] In some embodiments, the Disclosure provides a method for treating or preventing cervical cancer in a patient in need of treatment or prevention, the method comprising administering to the patient an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and optionally a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is or has been treated with a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended).

[0062] In embodiments, the method reduces or eliminates cervical tumors or cervical cancers, or their cells. In embodiments, the method slows the growth and / or progression and / or metastasis of cervical tumors or cervical cancers, or their cells.

[0063] In embodiments, cervical cancer is either a primary tumor or a metastatic tumor. A primary tumor or cancer that moves from its original location and the major organ where metastases are present can ultimately lead to the death of the subject due to the dysfunction of the affected organ. Metastasis is the spread of cancer cells from a primary tumor to other parts of the body, resulting in the occurrence of cancer cells or populations of cancer cells in locations different from the primary tumor. Metastasis can ultimately lead to the death of the subject. For example, current cervical cancers in embodiments include benign and malignant cancers, polyps, hyperplasia, and dormant tumors and micrometastases.

[0064] In this embodiment, cervical cancer is squamous cell carcinoma, adenocarcinoma, and / or adenosquamous carcinoma (mixed type).

[0065] In this embodiment, cervical cancer is one of the following stages:

[0066] For example, Stage I is characterized by cancer cells growing from the surface of the cervix into deeper tissues of the cervix, but the cancer has not spread to nearby lymph nodes or distant sites. Stage I can be further subdivided as follows: IA: Cancer can only be visualized under a microscope; IA1: The cancer is only visible under a microscope and is less than 3 mm deep. IA2: Cancer is only visible under a microscope and is 3mm to 5mm deep; IB: Stage I cancer that has spread deeper than 5mm but is still confined to the cervix; IB1: The cancer is deeper than 5 mm but less than 2 cm; IB2: The cancer is at least 2 cm in size but less than 4 cm; IB3: The cancer is at least 4 cm in size and is confined to the cervix;

[0067] For example, Stage II is characterized by cancer growing beyond the cervix and uterus, but not spreading to the pelvic wall or lower vagina, nor spreading to nearby lymph nodes or distant sites. Stage II can be further subdivided as follows: IIA: The cancer has grown beyond the cervix and uterus, but has not spread to the parauterine tissue adjacent to the cervix; IIA1: The cancer is 4 cm or smaller; IIA2: The cancer is 4 cm or larger; IIB: The cancer has grown beyond the cervix and uterus and has spread to the parauterine tissue adjacent to the cervix;

[0068] For example, Stage III is characterized by cancer spreading to the lower part of the vagina or the pelvic wall. The cancer may obstruct the ureters. The cancer may have spread to nearby lymph nodes but not to distant sites. Stage III can be further subdivided as follows: IIIA: The cancer has spread to the lower part of the vagina but not to the pelvic wall. IIIB: The cancer has grown within the pelvic wall and / or is blocking one or both ureters, causing hydronephrosis. IIIC: Cancer can be of any size, and imaging or biopsy indicates that the cancer has spread to nearby pelvic lymph nodes (IIIC1) or para-aortic lymph nodes (IIIC2); and

[0069] For example, Stage IV is characterized by the cancer growing in the bladder or rectum, or in more distant organs such as the lungs or bones. Stage IV can be further subdivided as follows: IVA: The cancer has spread to the bladder or rectum, or has grown outside the pelvis. IVB: The cancer has spread to distant organs other than the pelvis, such as distant lymph nodes, lungs, or bones.

[0070] In embodiments, the method delays or prevents progression from Stage I (e.g., IA or IB) to one of Stage II (e.g., IIA or IIB), Stage III (e.g., IIIA, IIIB, or IIIIC), and Stage IV (e.g., IVA or IVB).

[0071] In the embodiment, the method delays or prevents progression from Stage II (e.g., IIA or IIB) to Stage III (e.g., IIIA, IIIB, or IIIIC) and one of Stage IV (e.g., IVA or IVB).

[0072] In one embodiment, the method delays or prevents progression from Stage III (e.g., IIIA, IIIB, or IIIIC) to Stage IV (e.g., IVA or IVB).

[0073] In this embodiment, the cervical cancer is stage IV-B, recurrent, or persistent cervical cancer.

[0074] In this embodiment, the cervical cancer is metastatic cervical cancer. In this embodiment, the cervical cancer is further treated in combination with cisplatin.

[0075] In embodiments, the treatment method for cervical cancer is further combined with one or more of the following: surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. In embodiments, chemotherapy includes one or more of the following: cisplatin, carboplatin, paclitaxel, docetaxel, ifosfamide, 5-fluorouracil (5-FU), gemcitabine, and mitomycin. In embodiments, targeted therapy is anti-angiogenic therapy (e.g., bevacizumab or ramucirumab). In embodiments, immunotherapy includes one or more of the following: agents targeting the PD-1 pathway, such as atezolizumab, durvalumab, semiprimab, nivolumab, and pembrolizumab, and / or agents targeting the CTLA-4 pathway, such as ipilimumab.

[0076] Treatment of lung cancer In some embodiments, the present disclosure provides a method for treating or preventing lung cancer in a patient in need of treatment or prevention, the method comprising administering to the patient (i) an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and optionally a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, and (ii) a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended).

[0077] In some embodiments, the present disclosure provides a method for treating or preventing lung cancer in a patient in need of treatment or prevention, the method comprising administering to the patient (i) an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, and (ii) a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended).

[0078] In some embodiments, the Disclosure provides a method for treating or preventing lung cancer in a patient in need of treatment or prevention, the method comprising administering to the patient an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and optionally a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is or has been treated with a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended).

[0079] In some embodiments, the Disclosure provides a method for treating or preventing lung cancer in a patient in need of treatment or prevention, the method comprising administering to the patient an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is or has been treated with a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended).

[0080] In the embodiments, the method reduces or eliminates lung tumors or lung cancer, or their cells. In the embodiments, the method slows the growth and / or progression and / or metastasis of lung tumors or lung cancer, or their cells.

[0081] In embodiments, lung cancer is either a primary tumor or a metastatic tumor. A primary tumor or cancer that moves from its original location and the major organ where metastases are present can ultimately lead to the death of the subject due to the decline of function of the affected organ. Metastasis is the spread of cancer cells from a primary tumor to other parts of the body, resulting in the occurrence of cancer cells or populations of cancer cells in locations different from the primary tumor. Metastasis can ultimately lead to the death of the subject. For example, in embodiments, lung cancer includes benign and malignant cancers, polyps, hyperplasia, and dormant tumors and micrometastases.

[0082] In this embodiment, lung cancer is small cell lung cancer. In this embodiment, lung cancer is non-small cell lung cancer. In this embodiment, lung cancer is adenocarcinoma of the lung. In this embodiment, lung cancer is squamous cell carcinoma of the lung. In this embodiment, lung cancer is large cell carcinoma of the lung. In this embodiment, lung cancer is mesothelioma. In this embodiment, lung cancer is a carcinoid tumor of the lung.

[0083] In this embodiment, lung cancer is one of the following stages of lung cancer, which are optionally evaluated by the TNM system. For example, an occult stage characterized by the presence of cancer cells in the mucus of the person being coughed; For example, Stage 0 is characterized by a small tumor and a lack of diffusion to deeper lung tissue or outside the lung; For example, Stage I (e.g., IA, e.g., IA1, IA2, IA3, and IB) is characterized by the presence of cancer in lung tissue rather than lymph nodes; Stage II (e.g., IIA or IIB) is characterized by the presence of cancer in lung tissue and lymph nodes near the lungs. For example, Stage III (e.g., IIIA, IIIB, or IIIC) characterized by the presence of cancer in lung tissue and further spread to the lymph nodes or the middle of the chest; and / or For example, Stage IV is characterized by the presence of cancer in lung tissue and widespread spread throughout the body (e.g., brain, bones, and / or liver).

[0084] In the embodiment, the method delays or prevents progression from the occult stage to one of the stages 0, stage I (e.g., IA, IA1, IA2, IA3, IB), stage II (e.g., IIA or IIB), stage III (e.g., IIIA, IIIB, or IIIC), and stage IV.

[0085] In the embodiment, the method delays or prevents progression from Stage 0 to one of Stage I (e.g., IA, IA1, IA2, IA3, IB), Stage II (e.g., IIA or IIB), Stage III (e.g., IIIA, IIIB, or IIIC), and Stage IV.

[0086] In the embodiment, the method delays or prevents progression from Stage I (e.g., IA, IA1, IA2, IA3, IB) to Stage II (e.g., IIA or IIB), Stage III (e.g., IIIA, IIIB, or IIIC), and one of Stage IV.

[0087] In embodiments, the method delays or prevents progression from Stage II (e.g., IIA or IIB) to Stage III (e.g., IIIA, IIIB, or IIIIC) and one of Stage IV.

[0088] In one embodiment, the method delays or prevents progression from Stage III (e.g., IIIA, IIIB, or IIIIC) to Stage IV.

[0089] In this embodiment, the lung cancer is either small cell lung cancer or non-small cell lung cancer.

[0090] In this embodiment, the lung cancer is small cell lung cancer with platinum-sensitive disease. In this embodiment, the lung cancer is small cell lung cancer with platinum-sensitive disease in a subject that has progressed at least approximately 60 days after the initiation of first-line chemotherapy.

[0091] In the embodiment, the lung cancer is non-small cell lung cancer with platinum-sensitive disease. In the embodiment, the lung cancer is small cell lung cancer with platinum-sensitive disease in subjects that have progressed at least approximately 60 days after the initiation of first-line chemotherapy.

[0092] In the embodiment, the treatment method for lung cancer is further combined with one or more of the following: surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. In the embodiment, the chemotherapy is one or more of the following: carboplatin, cisplatin, docetaxel, etoposide, gemcitabine, nab-paclitaxel, paclitaxel, pemetrexed, and vinorelbine. In embodiments, targeted therapies include epidermal growth factor receptor (EGFR) inhibitors (e.g., afatinib, dacomitinib, entrectinib, erlotinib, gefitinib, and osimertinib), agents targeting EGFR exon 20 insertions (e.g., amivantamab or mobocertinib), agents targeting HER2 mutations (e.g., Fam-trastuzumab deruxtecan-nxki), anaplastic lymphoma kinase (ALK) inhibitors (e.g., alectinib, brigatinib, ceritinib, crizotinib, and loratinib), agents targeting ROS1 fusions (e.g., ceritinib, crizotinib, and entrectinib), agents targeting KRAS G12C mutations (e.g., sotrasib), agents targeting NTRK fusions (e.g., lalotrectinib), and BRAF. The immunotherapy is one or more of the following: a drug that targets the V600E mutation (e.g., dabrafenib or trametinib), a drug that targets the MET exon 14 skip (e.g., captinib or tepotinib), a drug that targets the RET fusion (e.g., pralcetinib or serpercatinib), and an anti-angiogenic therapy (e.g., bevacizumab or ramucirumab). In embodiments, the immunotherapy is one or more of the following: a drug that targets the PD-1 pathway (e.g., atezolizumab, durvalumab, semiprimab, nivolumab, and pembrolizumab), and / or a drug that targets the CTLA-4 pathway (e.g., ipilimumab).

[0093] Treatment of colon cancer or colorectal cancer In some embodiments, the present disclosure provides a method for treating or preventing colon cancer or colorectal cancer in a patient in need of treatment or prevention, the method comprising administering to the patient (i) an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and optionally a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, and (ii) a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended).

[0094] In some embodiments, the present disclosure provides a method for treating or preventing colon cancer or colorectal cancer in a patient in need of treatment or prevention, the method comprising administering to the patient (i) an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, and (ii) a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended).

[0095] In some embodiments, the Disclosure provides a method for treating or preventing colon cancer or colorectal cancer in a patient in need of treatment or prevention, the method comprising administering to the patient an oncolytic adenovirus comprising a replication mechanism specific to tumor cells and optionally a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is or has been treated with a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended).

[0096] In some embodiments, the Disclosure provides a method for treating or preventing colon cancer or colorectal cancer in a patient in need of treatment or prevention, the method comprising administering to the patient an oncolytic adenovirus comprising a replication mechanism specific to tumor cells and a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is or has been treated with a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended).

[0097] In embodiments, the method reduces or eliminates colon tumors or colorectal tumors or colon cancer or colorectal cancer, or their cells. In embodiments, the method slows down the proliferation and / or progression and / or metastasis of colon tumors or colorectal tumors or colon cancer or colorectal cancer, or their cells.

[0098] In embodiments, colon cancer or colorectal cancer is either a primary or metastatic tumor. A primary tumor or cancer that moves from its original location and the major organ where metastatic lesions are located can ultimately lead to the death of the subject due to the decline of function of the affected organ. Metastasis is the spread of cancer cells from a primary tumor to other parts of the body, resulting in the occurrence of cancer cells or populations of cancer cells in locations different from the primary tumor. Metastasis can ultimately lead to the death of the subject. For example, current colon cancer or colorectal cancer in embodiments includes benign and malignant cancers, polyps, hyperplasia, and dormant tumors and micrometastases.

[0099] In this embodiment, colon cancer or colorectal cancer is adenocarcinoma, gastrointestinal stromal tumor (GIST), squamous cell carcinoma, carcinoid tumor, lymphoma, or colon sarcoma.

[0100] In this embodiment, colon cancer or colorectal cancer is one of the following stages of colon cancer or colorectal cancer, which are optionally evaluated by the TNM system. For example, Stage 0 cancer, characterized by early-stage cancer in the innermost layer of the intestine; For example, Stage I cancer is characterized by the presence of cancer in the inner lining of the colon; For example, Stage II cancer characterized by cancer spreading to the muscular wall of the colon; For example, stage III cancer characterized by spread to the lymph nodes; and / or For example, Stage IV cancer is characterized by the spread of cancer to other organs outside the colon.

[0101] In one embodiment, the method delays or prevents progression from Stage 0 to one of Stages I, II, III, and IV.

[0102] In one embodiment, the method delays or prevents progression from Stage I to one of Stages II, III, and IV.

[0103] In one embodiment, the method delays or prevents progression from Stage II to one of Stage III and Stage IV.

[0104] In one embodiment, the method delays or prevents progression from Stage III to Stage IV.

[0105] In embodiments, the treatment method for colon cancer or colorectal cancer is further combined with one or more of the following: surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. In embodiments, chemotherapy is one or more of the following: 5-FU, leucovorin, oxaliplatin, capecitabine, regorafenib, trifluridine, and tipiracil. In embodiments, chemotherapy is one or more of the following regimens: FOLFOX (5-FU, leucovorin, and oxaliplatin), CapeOx (capecitabine and oxaliplatin), or 5-FU with leucovorin or capecitabine. In embodiments, targeted therapy is one or more of the following: VEGF-targeting agents (e.g., bevacizumab [Avastin], ziu-aflibercept [Zaltrap], or ramucirumab [Cyramza]) or EGFR-targeting agents (e.g., cetuximab [Erbitux] or panitumumab [Vectibix]). In this embodiment, the immunotherapy is one or more of the following: a drug that targets the PD-1 pathway, such as atezolizumab, durvalumab, semiprimab, nivolumab, and pembrolizumab, and / or a drug that targets the CTLA-4 pathway, such as ipilimumab.

[0106] In one embodiment, a method for treating colon cancer or colorectal cancer is a further combination with an anti-telangiectatic and Rad3-related (ATR) inhibitor, such as belzocertib. In another embodiment, a method for treating colon cancer or colorectal cancer includes the administration of topotecan and belzocertib.

[0107] Treatment of pancreatic cancer In some embodiments, the present disclosure provides a method for treating or preventing pancreatic cancer in a patient in need of treatment or prevention, the method comprising administering to the patient (i) an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and optionally a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, and (ii) a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended).

[0108] In some embodiments, the present disclosure provides a method for treating or preventing pancreatic cancer in a patient in need of treatment or prevention, the method comprising administering to the patient (i) an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, and (ii) a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended).

[0109] In some embodiments, the Disclosure provides a method for treating or preventing pancreatic cancer in a patient in need of treatment or prevention, the method comprising administering to the patient an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and optionally a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is or has been treated with a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended).

[0110] In some embodiments, the Disclosure provides a method for treating or preventing pancreatic cancer in a patient in need of treatment or prevention, the method comprising administering to the patient an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is or has been treated with a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended).

[0111] In the embodiments, the method reduces or eliminates pancreatic tumors or pancreatic cancer, or their cells. In the embodiments, the method slows the growth and / or progression and / or metastasis of pancreatic tumors or pancreatic cancer, or their cells.

[0112] In embodiments, pancreatic cancer is either a primary tumor or a metastatic tumor. A primary tumor or cancer that moves from its original location and the major organ where metastases are present can ultimately lead to the death of the subject due to the decline of function of the affected organ. Metastasis is the spread of cancer cells from a primary tumor to other parts of the body, resulting in the occurrence of cancer cells or populations of cancer cells in locations different from the primary tumor. Metastasis can ultimately lead to the death of the subject. For example, current pancreatic cancers in embodiments include benign and malignant cancers, polyps, hyperplasia, and dormant tumors and micrometastases.

[0113] In this embodiment, the pancreatic cancer is an exocrine carcinoma or an endocrine carcinoma, or an adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, or gelatinous carcinoma.

[0114] In this embodiment, pancreatic cancer is one of the following stages of pancreatic cancer, as optionally evaluated by the TNM system. • Stage 0: Characterized by, for example, "carcinoma in situ," where the cancer has not yet grown outside the lumen from which it originated (Tis, N0, M0); • Stage IA: Characterized, for example, by a tumor less than 2 cm in size in the pancreas. It has not spread to lymph nodes or other parts of the body (T1, N0, M0); Stage IB: Characterized, for example, by a tumor larger than 2 cm in the pancreas. It has not spread to lymph nodes or other parts of the body (T2, N0, M0); Stage IIA: Characterized, for example, by a tumor larger than 4 cm and extending beyond the pancreas. It has not spread to nearby arteries, veins, lymph nodes, or other parts of the body (T3, N0, M0); Stage IIB: Characterized, for example, by a tumor of any size that has not spread to nearby arteries or veins. It has spread to 1-3 regional lymph nodes but not to other parts of the body (T1, T2, or T3; N1; M0); Stage III: Characterized by a tumor of any size that has spread to four or more regional lymph nodes but not to nearby arteries, veins, or other parts of the body (T1, T2, or T3, N2, M0). Or, characterized by a tumor that has spread to nearby arteries and veins and may also have spread to regional lymph nodes. Not spread to other parts of the body (T4, any N, M0); and Stage IV: Characterized, for example, any tumor (any T, any N, M1) that has spread to other parts of the body.

[0115] In one embodiment, the method delays or prevents progression from Stage 0 to one of Stages IA or IB, Stage IIA or IIB, Stage III, and Stage IV.

[0116] In the embodiment, the method delays or prevents progression from stage IA or IB to one of stages IIA or IIB, stage III, and stage IV.

[0117] In one embodiment, the method delays or prevents progression from stage IIA or IIB to one of stage III and stage IV.

[0118] In one embodiment, the method delays or prevents progression from Stage III to Stage IV.

[0119] In embodiments, the treatment method for pancreatic cancer is further combined with one or more of the following: surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. In embodiments, chemotherapy is one or more of gemcitabine, 5-FU, oxaliplatin, paclitaxel, capecitabine, and cisplatin. In embodiments, targeted therapy is one or more of EGFR inhibitors (e.g., erlotinib), PARP inhibitors (e.g., olaparib), and NTRK inhibitors (e.g., larotrectinib or entrectinib). In embodiments, immunotherapy is one or more of the following: agents targeting the PD-1 pathway, e.g., atezolizumab, durvalumab, semiprimab, nivolumab, and pembrolizumab, and / or agents targeting the CTLA-4 pathway, e.g., ipilimumab.

[0120] In the embodiment, the method for treating pancreatic cancer includes the FOLFIRINOX (leucovorin, fluorouracil / 5FU, irinotecan, and oxaliplatin) regimen or the NALIRIFOX (irinotecan, 5-FU, leucovorin, and oxaliplatin) regimen.

[0121] Simultaneous administration of oncolytic adenovirus and topoisomerase inhibitors In some embodiments, the present disclosure provides a method for improving and / or enhancing and / or enhancing an antitumor effect in a patient in need thereof, the method comprising: (i) administering to the patient an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and optionally a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome; (ii) administering a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended); and (iii) observing an improved antitumor effect compared to treatment with a topoisomerase I inhibitor or its prodrug in the absence of the oncolytic adenovirus.

[0122] In one embodiment, the present disclosure provides a method for improving and / or enhancing and / or enhancing an antitumor effect in a patient in need thereof, the method comprising: (i) administering to the patient an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome; (ii) a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended); and (iii) observing an improved antitumor effect compared to treatment with a topoisomerase I inhibitor or its prodrug in the absence of the oncolytic adenovirus.

[0123] In one embodiment, the present disclosure provides a method for improving and / or enhancing and / or enhancing an antitumor effect in a patient in need thereof, the method comprising administering to the patient an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and optionally a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is or has been treated with a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended), and the method comprises observing an improved antitumor effect compared to treatment with a topoisomerase I inhibitor or its prodrug in the absence of the oncolytic adenovirus.

[0124] In one embodiment, the Disclosure provides a method for improving and / or enhancing and / or enhancing an antitumor effect in a patient in need thereof, the method comprising administering to the patient an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is or has been treated with a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended), and the method comprises observing an improved antitumor effect compared to treatment with a topoisomerase I inhibitor or its prodrug in the absence of the oncolytic adenovirus.

[0125] In some embodiments, the oncolytic adenovirus, topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended) of the present disclosure are administered co-administered. In some embodiments, co-administration may occur simultaneously or sequentially.

[0126] For example, an oncolytic adenovirus and a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited) are administered to the subject simultaneously. As used herein, the term “simultaneously” means that the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited) are administered with a time interval of no more than about 60 minutes, e.g., no more than about 30 minutes, no more than about 20 minutes, no more than about 10 minutes, no more than about 5 minutes, or no more than about 1 minute. Administration of oncolytic adenovirus and a topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited)) may be by co-administration of a single formulation (e.g., a formulation comprising oncolytic adenovirus and a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited)) or separate formulations (e.g., a first formulation comprising oncolytic adenovirus and a second formulation comprising a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited)).

[0127] In the embodiment, the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited) are administered to the subject simultaneously, but the release of the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited) may occur sequentially from their respective dosage forms (or a single unit dosage form if co-formulated).

[0128] Co-administration is, for example, when the timing of their administrations overlaps in time with the pharmacological activity of oncolytic adenovirus and topoisomerase I inhibitors or their prodrugs (e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited)). It is not necessary for liposomal irinotecan (e.g., ONIVYDE, not to limit) to be administered simultaneously without limitation. For example, oncolytic adenovirus and topoisomerase I inhibitors or their prodrugs, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to limit) may be administered sequentially or simultaneously. The terms “sequentially” and “simultaneously” as used herein refer to oncolytic adenovirus This means that the oncolytic adenovirus and a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., non-limiting ONIVYDE) are administered at time intervals of more than approximately 60 minutes. For example, an oncolytic adenovirus and a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., non-limiting ONIVYDE) are administered at time intervals of more than approximately 60 minutes. The time between consecutive administrations of IVYDE may be more than approximately 60 minutes, more than approximately 2 hours, more than approximately 5 hours, more than approximately 10 hours, more than approximately 1 day, more than approximately 2 days, more than approximately 3 days, or more than approximately 1 week. The administration time will depend on the metabolic rate, excretion rate, and / or pharmacodynamic activity of the oncolytic adenovirus being administered and the topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited).Either an oncolytic adenovirus or a topoisomerase I inhibitor or its prodrug (e.g., topotecan or SN-38) may be administered first.

[0129] In this embodiment, an oncolytic adenovirus is administered first, and a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited) is administered about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks later.

[0130] In this embodiment, a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the effect of limitation) is administered first, and the oncolytic adenovirus is administered approximately 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks later.

[0131] In embodiments, co-administration also does not require that the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited) be administered to the subject via the same route of administration. Rather, in embodiments, each therapeutic agent can be administered via any suitable route, e.g., intravenously, intraventricularly, intratumorally, intraperitoneally, or orally, non-intraventricularly, non-tumorally, non-peritoneally, or parenterally.

[0132] In embodiments, administration of compositions and formulations of the present invention comprising an oncolytic adenovirus and / or a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited) may be used in combination with additional agents such as one or more chemotherapeutic agents and / or one or more additional therapies conventionally used to treat or prevent ovarian cancer, cervical cancer, lung cancer, colon cancer, or colorectal cancer, and / or pancreatic cancer, including but not limited to, one or more additional antitumor agents. In embodiments, the co-administration of the additional agents and the compositions / formulations of the present invention may be simultaneous or sequential. In embodiments, the additional agents may be included in compositions and formulations of the present invention comprising an oncolytic adenovirus and / or a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited). In embodiments, the additional agent may be administered in a composition separate from the compositions and formulations of the present invention, which contain an oncolytic adenovirus and / or a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited). In embodiments, the additional agent may be administered simultaneously with the compositions and formulations of the present invention, which contain an oncolytic adenovirus and / or a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited). In embodiments, additional agents are administered prior to the compositions and formulations of the present invention comprising an oncolytic adenovirus and / or a topoisomerase I inhibitor or its prodrug, such as topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended).In embodiments, additional agents are administered after compositions and formulations of the present invention comprising an oncolytic adenovirus and / or a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited). In embodiments, compositions and formulations of the present invention comprising an oncolytic adenovirus and / or a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited) can be used alone in therapeutic regimens for treating or preventing ovarian cancer, cervical cancer, lung cancer, colon cancer, or colorectal cancer, and / or pancreatic cancer, without the use of other antitumor agents and / or other conventional therapies.

[0133] Furthermore, the compositions / formulations of the present invention may contain additional agents (e.g., via co-formulation). For example, additional agents and oncolytic adenoviruses and / or topoisomerase I inhibitors or their prodrugs, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited) may be combined into a single formulation. Furthermore, additional agents and oncolytic adenoviruses and / or topoisomerase I inhibitors or their prodrugs, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited) may be formulated separately.

[0134] Oncolytic adenovirus This disclosure partially covers pharmaceutical compositions, formulations, and uses of one or more oncolytic adenoviruses.

[0135] When used in this document, "oncolytic adenovirus" and its plural form refer to adenoviruses that can replicate themselves or have the ability to replicate within tumor cells. Oncolytic adenoviruses are distinguished from non-replicating adenoviruses because non-replicating adenoviruses cannot replicate themselves within target cells.

[0136] Adenovirus In embodiments, the oncolytic adenovirus used in this disclosure is an oncolytic adenovirus having a replication mechanism and a capsid that enables infection and replication in human cancer cells. In embodiments, the oncolytic adenovirus is produced from an adenovirus that infects humans. Examples of adenoviruses that infect humans include, but are not limited to, human adenoviruses of serotypes 1-51, such as adenoviruses of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, and 51, or combinations thereof, such as hybrid recombinants of two or more different serotypes of human adenovirus.

[0137] The 51 human adenovirus serotypes identified to date are classified into six groups, A through F. Human adenovirus serotype 5 (Ad5), belonging to group C, is a virus formed by an icosahedral protein capsid containing 36 kilobases of linear deoxyribonucleic acid (DNA). In adults, Ad5 infection is usually asymptomatic, while in children it causes colds and conjunctivitis. Ad5 generally infects epithelial cells, which are cells of the bronchial epithelium, during spontaneous infection. It enters cells through the interaction of fibrils, which are viral proteins extending like antennae from the 12 vertices of the capsid, with cellular proteins involved in cell-cell adhesion called coxsackie adenovirus receptors (CARs). Once the viral DNA reaches the interior of the nucleus, systematic transcription of the virus's initial genes (E1-E4) begins. The first viral gene to be expressed corresponds to the initial region gene 1A (E1A). E1A binds to retinoblastoma cell proteins and releases E2F, thus activating the transcription of other viral genes such as E2, E3, and E4, as well as cellular genes that activate the cell cycle. Part of E1B binds to the p53 protein, activating the cell cycle and inhibiting apoptosis in infected cells. E2 codes for viral replication proteins, E3 codes for proteins that inhibit the antiviral immune response, and E4 codes for proteins that transport viral RNA. Early gene expression leads to viral DNA replication, which, once replicated, activates the major late promoter, leading to the expression of messenger ribonucleic acid (RNA) transcripts, which, through cleavage and differential splicing, produce all the RNAs that code for the structural proteins that form the capsid.

[0138] In the embodiments, the oncolytic adenovirus used in the present disclosure is generated from human adenovirus serotype 5.

[0139] Hyaluronidase In embodiments, the oncolytic adenovirus used in this disclosure includes a sequence encoding a hyaluronidase enzyme inserted into its genome. In embodiments, the oncolytic adenovirus used in this disclosure does not include a sequence encoding a hyaluronidase enzyme inserted into its genome.

[0140] Hyaluronidase enzymes are a family of enzymes responsible for the breakdown of hyaluronic acid. In the human species, six genes encoding hyaluronidase enzymes with different characteristics and arrangements have been identified to date. Isoforms Hyal1 and Hyal2 are found in most tissues, with Hyal1 being the dominant form in human plasma. Hyal3 is located in the bone marrow and testes, but its function has not been fully characterized. Hyaluronidase enzyme PH20 is highly expressed in the testes and is involved in the process of oocyte fertilization by sperm. Hyaluronidase enzyme PH20 is fixed to the cell membrane and inner lining of the sperm head, enabling sperm to penetrate the extracellular matrix of the cumulus cell (which is rich in hyaluronic acid) and reach the zona pellucida of the oocyte. During the acrosome reaction, a portion of the hyaluronidase enzyme fixed to the sperm membrane is enzymatically processed to produce a soluble protein, which is then released from the acrosome membrane. Membrane protein PH20 is the only enzyme in the mammalian hyaluronidase family that is active at neutral pH.

[0141] While we do not wish to be bound by theory, it is thought that the expression of hyaluronidase enzyme by oncolytic adenovirus can help degrade the dense physical and immunosuppressive stromal barrier surrounding solid tumors, thereby ensuring tumor penetration by oncolytic adenovirus and combination therapies. While we do not wish to be bound by theory, this would ensure that the adenovirus can reach and infect more tumor cells. By degrading the tumor stroma, tumor neoantigens can be exposed, thereby enabling an anti-tumor immune response by the patient's immune system.

[0142] In this embodiment, the hyaluronidase enzyme is a mammalian testicular hyaluronidase enzyme, optionally a human one (e.g., GenBank gene ID: 6677), also known as SPAM1, sperm adhesion molecule 1, or PH20. In this embodiment, the sequence of the hyaluronidase enzyme has a sequence corresponding to an enzyme with a deleted membrane-bound carboxyl-terminal domain, and as a result, the enzyme is soluble. When this carboxyl-terminal domain is deleted, the resulting enzyme is secreted into the extracellular environment.

[0143] In this embodiment, the sequence encoding the hyaluronidase enzyme inserted into the oncolytic adenovirus genome is Sequence ID No. 1, from which nucleotides 1471-1527 corresponding to the carboxyl terminal domain are deleted.

[0144] In the embodiments, the hyaluronidase enzyme is a hyaluronidase mutant. The hyaluronidase mutant has at least one amino acid modification, generally an amino acid substitution, compared to the parental wild-type sequence. In the embodiments, the hyaluronidase of the Disclosure has at least about 60% (for example, about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%) of any of the sequences disclosed herein. Or they contain amino sequences having sequence identity of approximately 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Furthermore, in embodiments, the hyaluronidase mutants retain most or all of their biochemical activity, as measured by any suitable method known in the art.

[0145] In embodiments, the hyaluronidase of the present disclosure contains at least about 60% (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%) of the amino acid encoded by SEQ ID NO: 1. It contains an amino sequence having sequence identity of approximately 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0146] In embodiments, the hyaluronidase of the present disclosure contains at least about 60% (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%) of the amino acid encoded by SEQ ID NO: 1. It contains an amino sequence with sequence identity of approximately 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and sequence number 1 is missing nucleotides 1471-1527 corresponding to the carboxy terminal domain.

[0147] In embodiments, the hyaluronidase of the present disclosure contains at least about 60% (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%) of the amino acid encoded by SEQ ID NO: 9. It contains an amino sequence having sequence identity of approximately 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0148] In embodiments, the hyaluronidase of the present disclosure contains at least about 60% (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%) of SEQ ID NO: 8. It contains an amino sequence having sequence identity of %, or approximately 79%, or approximately 80%, or approximately 81%, or approximately 82%, or approximately 83%, or approximately 84%, or approximately 85%, or approximately 86%, or approximately 87%, or approximately 88%, or approximately 89%, or approximately 90%, or approximately 91%, or approximately 92%, or approximately 93%, or approximately 94%, or approximately 95%, or approximately 96%, or approximately 97%, or approximately 98%, or approximately 99%.

[0149] In embodiments, the hyaluronidase of the present disclosure contains at least about 60% (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%) of SEQ ID NO: 10. It contains an amino sequence having sequence identity of %, or approximately 79%, or approximately 80%, or approximately 81%, or approximately 82%, or approximately 83%, or approximately 84%, or approximately 85%, or approximately 86%, or approximately 87%, or approximately 88%, or approximately 89%, or approximately 90%, or approximately 91%, or approximately 92%, or approximately 93%, or approximately 94%, or approximately 95%, or approximately 96%, or approximately 97%, or approximately 98%, or approximately 99%.

[0150] In embodiments, the oncolytic adenovirus used in this disclosure comprises one or more regulatory elements, modifications, or mutations that result in the expression of a tumor-specific enzyme or viral replication.

[0151] In the embodiments, the expression of the hyaluronidase enzyme is controlled by a promoter that can be manipulated in tumor cells. In the embodiments, the expression of the hyaluronidase enzyme is controlled by a promoter that can be manipulated in the ovarian cancer cells, cervical cancer cells, lung cancer cells, colon cancer or colorectal cancer cells, and / or pancreatic cancer cells being treated. Optionally, in the embodiments, the expression of the enzyme is controlled by a promoter that can be manipulated in animal cells. Optionally, in the embodiments, the promoter is selected from the cytomegalovirus promoter, the adenovirus major late promoter, the SV40 promoter, the herpes simplex virus thymidine kinase promoter, the RSV promoter, the EF1-α promoter, the beta-actin promoter, the human IL-2 promoter, the human IL-4 promoter, the IFN promoter, the E2F promoter, the human GM-CSF promoter, or a combination thereof.

[0152] In embodiments, the promoter that regulates enzyme expression can be naturally present in the adenovirus, as in the case of the adenovirus major late promoter. In embodiments, the promoter can also be inserted together with the enzyme-coding sequence. In embodiments, the promoter is the adenovirus major late promoter, which is already located in the oncolytic adenovirus genome. In embodiments, it is not necessary to introduce the promoter together with the hyaluronidase enzyme sequence; rather, the latter is introduced into the oncolytic adenovirus genome so that it remains under the control of the promoter.

[0153] In embodiments, the oncolytic adenovirus used in the disclosure includes additional sequences that enable the promotion or optimization of protein translation of a sequence encoding a hyaluronidase enzyme. In embodiments, the sequences are located inside or outside the hyaluronidase enzyme gene. For example, in embodiments, the additional sequences are selected from cleavage and splicing sequences that enable RNA to be processed, IRES (intrasequence ribosome entry site) sequences, picornavirus sequence 2A, or a combination thereof.

[0154] Specific replication mechanisms of tumor cells In embodiments, the tumor cell-specific replication mechanism contained in the oncolytic virus used in this disclosure is a mechanism that causes the oncolytic adenovirus to replicate itself in a specific form in tumor cells and not in healthy, non-tumor, or normal cells. In embodiments, the mechanism may take different forms if, as a result, it provides an oncolytic adenovirus that is capable of replicating only in tumor cells, e.g., ovarian cancer, cervical cancer, lung cancer, colon cancer, or colorectal cancer, and / or pancreatic cancer cells, or primarily. In other words, in embodiments, the replication mechanism is an adenovirus with selective replication, requiring the virus to enter cancer cells or tumor cells in order for the virus to replicate itself. In embodiments, the oncolytic adenovirus used may have modifications in its genomic sequence that result in selective replication within tumor cells.

[0155] In embodiments, this is achieved by incorporating a specific tissue promoter or a specific tumor promoter, where the promoter controls the expression of one or more genes in groups E1a, E1b, E2, and E4. In embodiments, the promoter is selected from artificial promoters formed by various transcription factor binding sites, such as the E2F promoter, telomerase hTERT promoter, tyrosinase promoter, prostate-specific antigen (PSA) promoter, alpha-fetoprotein promoter, COX-2 promoter, as well as the binding sites for hypoxia-inducible factor (HIF-1), ETS transcription factor, tumor cytotoxic factor (TCF), E2F transcription factor, or Sp1 transcription factor. In embodiments, the promoter controls the expression of E1a.

[0156] In embodiments, the treatment is for tumors with an abnormal Rb-E2F pathway. In healthy, non-tumor, or normal cells, the E1A protein expressed by adenovirus dissociates the Rb-E2F complex in host cells, allowing the free E2F protein to drive both cell division and viral replication. In tumor cells with an abnormal Rb-E2F mechanism, free E2F allows cells to divide continuously (and the virus to replicate) without being initiated by viral E1A. Genetic engineering that suppresses normal viral E1A expression prevents viral replication in healthy, non-tumor, or normal cells while allowing replication in tumor cells. Almost all solid tumors, including but not limited to ovarian cancer, cervical cancer, lung cancer, colon cancer, or colorectal cancer, and / or pancreatic cancer, have an abnormal Rb-E2F pathway. In embodiments, the oncolytic adenovirus used in this disclosure is characterized by a deletion of the Rb-binding domain or a deletion of Δ24, which affects the interaction between E1a and retinoblastoma protein, as well as the insertion of four sites that bind to E2F-1 and one site that binds to Sp1 into the endogenous E1a promoter, thereby controlling E1a expression. The DNA sequence corresponds to Sequence ID No. 2.

[0157] Furthermore, the oncolytic adenoviruses used in this disclosure are, in embodiments, expected to include one or more modifications to the capsid, enabling improved in vivo distribution and reduced clearance of the oncolytic adenovirus from the body. In embodiments, the oncolytic adenoviruses used in this disclosure have a capsid modified to reduce their isolation and destruction in the liver, so that the heparan sulfate binding domain KKTK present in the adenovirus fibrils is replaced, for example, with the domain RGDK. The modification relates to positions 91-94 of the adenovirus fibrils with reference to the standard sequence of adenovirus serotype 5 fibrils. Sequence ID 4 shows the complete sequence of the adenovirus type 5 fibril protein with a modified version (modified RGDK) in the heparan sulfate binding domain.

[0158] Therefore, in embodiments, the oncolytic adenovirus used in this disclosure is generated from human adenovirus serotype 5 and comprises the following: • A hyaluronidase enzyme inserted into the genome; optionally, a sequence encoding a human testicular hyaluronidase enzyme sequence (PH20, SEQ ID NO: 1) with a deletion corresponding to the membrane-bound carboxyl terminal domain for enzyme solubilization; optionally, a sequence encoding a hyaluronidase enzyme having the amino acid sequence of SEQ ID NO: 10; • Replication of oncolytic adenoviruses occurs in tumor cells with abnormalities in the Rb-E2F pathway, and not in healthy cells, non-tumor cells, or normal cells; optionally, oncolytic adenoviruses have a deletion of the Rb-binding domain or Δ24 deletion, which affects the interaction between E1a and the Rb protein, and the insertion of four binding sites to E2F-1 and one binding site to Sp1 into the endogenous E1a promoter to control E1a expression; Modification of the adenovirus capsid to improve biological distribution and reduce clearance of oncolytic adenoviruses from the body; optionally, the heparan sulfate binding domain KKTK present in the adenovirus fibrils is replaced with, for example, the domain RGDK.

[0159] In embodiments, the oncolytic adenovirus of the present disclosure contains at least about 60% (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%) of the sequence of SEQ ID NO: 3. It contains a nucleotide sequence having sequence identity of 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%. In the embodiment, the oncolytic adenovirus has the nucleotide sequence of SEQ ID NO: 3.

[0160] Method for producing oncolytic adenovirus according to the present disclosure In embodiments, any method for constructing genetically engineered adenoviruses known in the fields of gene therapy and virology using adenoviruses is used to construct the oncolytic adenoviruses used in this disclosure. The most commonly used method is based on first constructing the desired genetic engineering in a plasmid containing the adenovirus region to be modified, and then performing homologous recombination in bacteria with a plasmid containing the remainder of the viral genome. As will be understood by those skilled in the art, due to the degeneracy of the genetic code, the protein sequences described herein may be encoded by any number of possible nucleic acid sequences.

[0161] In embodiments, nucleic acids encoding the components of the present disclosure can be incorporated into an oncolytic adenovirus as known in the art and, depending on the host cell, can be used to produce the hyaluronidase of the present disclosure. Generally, nucleic acids are operably linked to any number of regulatory elements (promoters, origins of replication, selection markers, ribosome binding sites, inducers, etc.).

[0162] In embodiments, the oncolytic adenoviruses used in this disclosure are grown and amplified in cell lines commonly used in the fields of gene therapy and viral therapy, such as HEK-293 (registration number: ATCC CRL-1573) and A549 (registration number: ATCC CCL185). In embodiments, the method of adenovirus growth is by infection of a cell line that enables adenovirus replication. The lung adenocarcinoma cell line A549 is an example of a cell line with such characteristics. In embodiments, growth is carried out, for example, as follows: A549 cells are grown on a plastic cell culture plate and infected using 100 virus particles per cell. After 2 days, a cytopathic effect reflecting virus production can be observed as cell clustering. In embodiments, the cells are harvested and stored in tubes. In embodiments, the cell pellet is frozen after centrifugation at approximately 1,000 g for approximately 5 minutes, and then thawed three times to lyse the cells. In the embodiment, the obtained cell extract is centrifuged at approximately 1,000 g for approximately 5 minutes, and the supernatant containing the virus is loaded onto a cesium chloride gradient and centrifuged at approximately 35,000 g for approximately 1 hour. In the embodiment, the virus band obtained from the gradient is loaded again onto another cesium chloride gradient and centrifuged at approximately 35,000 g for approximately 16 hours. In the embodiment, the virus band is collected and dialyzed against PBS-10% glycerol. In the embodiment, the viral dialyze is dispensed and stored at approximately -80°C. The number of plaque-forming units and particles may be quantified according to standard protocols known in the prior art. Phosphate-buffered saline (PBS) containing 5% glycerol is a standard preparation for preserving adenovirus and is used in the embodiment. However, newer preparations that improve viral stability have been described and are used in the embodiment.

[0163] Topoisomerase I inhibitor Topoisomerase inhibitors can inhibit cell proliferation, for example, by preventing DNA replication, stimulating DNA damage, and inducing cell cycle arrest.

[0164] Topotecan is a semi-synthetic derivative of camptothecin, a cytotoxic alkaloid. SN-38 is a metabolite of the prodrug irinotecan that is inhibited by active topoisomerase I. Generally, topoisomerase I inhibitors induce cell cycle arrest in S phase by stabilizing the complex between topoisomerase I and DNA, thereby inhibiting the religation of topoisomerase I-mediated single-strand DNA breaks and generating potentially lethal double-strand DNA breaks.

[0165] In embodiments, topoisomerase I inhibitors or their prodrugs, such as topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended) are administered in various ways, including systemic, intra-arterial, intratumoral, intraperitoneal, and / or oral administration, to treat ovarian cancer, cervical cancer, lung cancer, colon cancer, or colorectal cancer, and / or pancreatic cancer.

[0166] In the embodiment, for systemic administration, a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited) is administered intravenously or orally. The topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited) then enters the bloodstream and is distributed throughout the body. Irinotecan is converted to the active topoisomerase I inhibitor SN-38 by carboxylesterase enzymes in the liver and tumors.

[0167] In the embodiments, topoisomerase I inhibitors or their prodrugs, such as topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended) are administered intravenously, intra-arterially, intraperitoneally, or orally.

[0168] In the embodiments, liposomal irinotecan (e.g., by ONIVYDE, to the extent not limited thereto) is administered by intravenous infusion.

[0169] In embodiments, the method of the present disclosure improves and / or enhances and / or enhances the antitumor effect compared to treatment with a topoisomerase I inhibitor or its prodrug without oncolytic adenovirus. In embodiments, the method makes the patient more suitable for treatment with combination therapy of one or more cancer therapies.

[0170] formulation This disclosure provides oncolytic adenoviruses and / or topoisomerase I inhibitors or their prodrugs described in various formulations, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not limited to), compositions. Any oncolytic adenoviruses and / or topoisomerase I inhibitors or their prodrugs described herein, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not limited to), may take the form of a solution, emulsion, suspension, delayed-release formulation, sustained-release formulation, controlled-release formulation, liposomal formulation, or any other form suitable for parenteral or oral use.

[0171] It will be understood that the compositions used in this disclosure are for use in pharmaceutically acceptable presentations. This means that any oncolytic adenovirus and / or topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent intended) described herein may be administered to a subject as a component of a composition comprising a pharmaceutically acceptable carrier or medium. Such compositions may optionally contain pharmaceutically acceptable excipients in suitable amounts to obtain a form for appropriate administration.

[0172] Pharmaceutical excipients may be liquids such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, and sesame oil. Examples of pharmaceutical excipients include saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, and analogues. In addition, adjuvants, stabilizers, thickeners, lubricants, and colorants may be used. In embodiments, pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any of the drugs described herein are administered intravenously. Saline and aqueous solutions of dextrose and glycerol can also be used as liquid excipients, particularly for injections. Suitable pharmaceutically acceptable excipients include starch, glucose, cellulose, hypromellose, lactose, sucrose, trehalose, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, povidone, crospovidone, water, and ethanol. Any of the agents described herein may also contain small amounts of wetting agents or emulsifiers or pH buffers, if desired. Other examples of suitable pharmaceutically acceptable excipients are described in Remington's Pharmaceutica lSciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), which is incorporated herein by reference.

[0173] Dosage and Administration It will be understood that the actual amount of oncolytic adenovirus and / or topoisomerase I inhibitors or their prodrugs administered pursuant to this disclosure, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the effect of limitation), will vary according to different parameters, e.g., size and stage of ovarian cancer, cervical cancer, lung cancer, colon cancer, or colorectal cancer, and / or pancreatic cancer, the tumor being treated, the age and weight of the patient being treated, the specific dosage form, and the mode of administration. It will be understood that the doses of the oncolytic adenovirus and the co-administered topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE's method, not to the extent intended) must be sufficient to ensure that the combination of methods produces a positive therapeutic effect against ovarian cancer, cervical cancer, lung cancer, colon cancer, or colorectal cancer, and / or pancreatic cancer. In embodiments, a positive therapeutic effect against ovarian cancer, cervical cancer, lung cancer, colon cancer, or colorectal cancer, and / or pancreatic cancer refers to cessation of tumor growth, reduction of tumor volume, prevention of tumor invasion into surrounding tissues, or prevention of metastasis.

[0174] In this embodiment, for example, in the case of ovarian cancer, the dose of topotecan is approximately 1.5 mg / m². 2 The treatment is administered intravenously once daily over approximately 30 minutes, starting on day 1 of a 21-day cycle, for approximately 5 consecutive days until disease progression or unacceptable toxicity occurs.

[0175] In this embodiment, for example, in the case of cervical cancer, the dose of topotecan is approximately 0.75 mg / m². 2 Therefore, the cisplatin dose on day 1 of the 21-day cycle is approximately 50 mg / m². 2 In combination with the above, administer intravenously by drip infusion over approximately 30 minutes once daily on days 1, 2, and 3, until disease progression or unacceptable toxicity is observed.

[0176] In this embodiment, for example, in the case of lung cancer, the dose of topotecan is approximately 1.5 mg / m².2 The treatment is administered intravenously once daily over approximately 30 minutes, starting on day 1 of a 21-day cycle, for approximately 5 consecutive days until disease progression or unacceptable toxicity occurs.

[0177] In some embodiments, topotecan may be administered orally for the treatment of lung cancer, for example, an oral capsule dose of topotecan of 2.3 mg / m2 (rounded to the nearest 0.25 mg) is administered once daily for approximately 5 consecutive days, starting on day 1 of a 21-day cycle, until disease progression or the appearance of unacceptable toxicity.

[0178] In this embodiment, for example, in the case of colon cancer or colorectal cancer, the dose of topotecan is approximately 1.25 mg / m². 2 Approximately 1.5mg / m 2 It is administered intravenously by infusion, for example, in cycles of approximately 5 days or approximately 21 days.

[0179] In this embodiment, for example, in the case of pancreatic cancer, the dose of topotecan is approximately 1.25 mg / m². 2 Approximately 1.5mg / m 2 For example, it is administered intravenously by infusion over a period of about 5 days, or in a cycle of about 21 days. In one embodiment, for example, for pancreatic cancer, topotecan is administered at a dose of about 350 mg / m² over about 30 minutes, approximately once every 3 weeks. 2 It is injected as follows. In one embodiment, for example, in pancreatic cancer, topotecan is administered at approximately 100 mg / m². 2 Once a week or 150 mg / m² 2 It is injected every two weeks.

[0180] Many factors (e.g., body weight, sex, diet, timing of administration, route of administration, excretion rate, subject condition, drug combinations, genetic predisposition, and response susceptibility) that may modify the action of oncolytic adenoviruses and / or topoisomerase I inhibitors or their prodrugs, such as topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., the ONIVYDE method, not to the extent of the intent) can be considered by those skilled in the art. Administration can be carried out continuously up to the maximum tolerated dose or in one or more divided doses. The optimal administration rate under a given set of conditions can be determined by those skilled in the art using conventional assessments of dose and tolerability.

[0181] The compositions of this disclosure, for example, compositions comprising oncolytic adenovirus disclosed herein, are administered by any route that ensures the required amount of oncolytic adenovirus reaches the tumor site. In the case of ovarian cancer, cervical cancer, lung cancer, colon cancer or colorectal cancer, and / or pancreatic cancer, the compositions of this disclosure, for example, compositions comprising oncolytic adenovirus disclosed herein, are injected by any route that ensures the required amount of oncolytic adenovirus reaches the tumor site. Any route of administration includes, but is not limited to, intravenous injection, intra-arterial injection, intratumoral injection, and / or intraperitoneal injection. Accordingly, in embodiments, preferred compositions comprising oncolytic adenovirus disclosed herein take appropriate forms for intravenous, intra-arterial, intratumoral, and / or intraperitoneal administration or injection.

[0182] In embodiments, oncolytic adenovirus and / or topoisomerase I inhibitors, or their prodrugs, such as topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not intended to be limited) may be administered, for example, more than once a day (e.g., about twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times per day), about once a day, about every other day, about once every three days, about once a week, about once every two weeks, about once a month, about once every two months, about once every three months, about once every six months, or about once a year.

[0183] definition As used herein, "a," "an," or "the" may mean one or more.

[0184] Furthermore, when used in relation to a mentioned numerical value, the term "approximately" means adding or subtracting up to 10% of that value. For example, the phrase "approximately 50%" encompasses a range from 45% to 55%.

[0185] When used in connection with medical applications, an "effective dose" is the amount effective in providing a measurable treatment, prevention, or reduction in the incidence of the disorder in question.

[0186] As used herein, if, in the presence of a drug or irritant, the readings of activity and / or effect are reduced by a substantial amount, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more (including at least about 100%) compared to the absence of such modification, then some are "decreased". As will be understood by those skilled in the art, in embodiments, activity may decrease and some downstream readings may decrease, while others may increase.

[0187] Conversely, if, in the presence of a drug or stimuli, the readings of activity and / or effect increase by a substantial amount, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, at least about 100%, or more, then the activity is "increased".

[0188] Where used herein, all compositional percentages refer to the total weight of the composition unless otherwise specified. The word “include” and its variations as used herein are intended to be non-limiting so as not to exclude other similar items that may also be useful in the compositions and methods of the present art. Similarly, the terms “may” and “may” and their variations are intended to be non-limiting so as not to exclude other embodiments of the art of the present invention that do not include those elements or features, even if an embodiment may include or may include a particular element or feature.

[0189] For the purposes of describing and claiming this disclosure, the open-ended term “including” is used herein as a synonym for terms such as “including,” “containing,” or “having,” but this disclosure or its embodiments may be described using alternative terms such as “consisting of” or “essentially consisting of.”

[0190] As used herein, the words “preferred” and “preferred” refer to embodiments of the technology that provide a particular benefit under certain circumstances. However, other embodiments may be preferred under the same or other circumstances. Furthermore, the enumeration of one or more preferred embodiments does not mean that other embodiments are unhelpful and is not intended to exclude other embodiments from the scope of this technology.

[0191] In embodiments, the terms “patient” and “subject” are used interchangeably. In embodiments, the subject and / or animal is a mammal, such as a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or a non-human primate such as a monkey, chimpanzee, or baboon. In embodiments, the subject and / or animal is a non-mammal, such as a zebrafish.

[0192] In embodiments, the method of the present invention is useful for treating human subjects. In embodiments, the human is an adult human. In embodiments, the human is an elderly human. In embodiments, the human may be called a patient. In embodiments, the human is female. In embodiments, the human is male.

[0193] In the embodiment, the human being has an age in the range of approximately 1 to 18 months, approximately 18 to 36 months, approximately 1 to 5 years, approximately 5 to 10 years, approximately 10 to 15 years, approximately 15 to 20 years, approximately 20 to 25 years, approximately 25 to 30 years, approximately 30 to 35 years, approximately 35 to 40 years, approximately 40 to 45 years, approximately 45 to 50 years, approximately 50 to 55 years, approximately 55 to 60 years, approximately 60 to 65 years, approximately 65 to 70 years, approximately 70 to 75 years, approximately 75 to 80 years, approximately 80 to 85 years, approximately 85 to 90 years, approximately 90 to 95 years, or approximately 95 to 100 years.

[0194] The amount of each component in the compositions described herein required to achieve a therapeutic effect may be determined empirically according to conventional procedures for a particular purpose. Generally, for the purpose of administering a therapeutic agent (e.g., oncolytic adenovirus and / or topoisomerase I inhibitors or their prodrugs, e.g., topotecan, SN-38, and / or irinotecan (e.g., liposomal irinotecan, e.g., ONIVYDE, not to the extent of being limited), the compositions described herein) for therapeutic purposes, the therapeutic agent is given in a pharmacologically effective amount. "Pharmacologically effective amount," "pharmacologically effective dose," "therapeutic effective amount," or "effective amount" refers to an amount sufficient to produce a desired physiological effect or to achieve a desired outcome, particularly for treating or preventing a disorder or disease. When used herein, an effective amount would be, for example, a sufficient amount to delay the onset of symptoms of a disorder or disease, to alter the course of symptoms of a disorder or disease (for example, to slow the progression of symptoms of a disease), to reduce or eliminate one or more symptoms or signs of a disorder or disease, and to reverse the symptoms of a disorder or disease. Therapeutic effects also include stopping or slowing the progression of an underlying disease or disorder, regardless of whether improvement is achieved.

[0195] The effective dose, toxicity, and therapeutic efficacy can be determined, for example, by standard pharmaceutical procedures in cell cultures, tissue samples, tissue homogenates, or experimental animals to determine the LD50 (lethal dose for approximately 50% of the population) and ED50 (therapeutably effective dose for approximately 50% of the population) or maximum tolerated dose.

[0196] The dosage may vary depending on the form of drug administration used and the route of administration utilized. The dose ratio between toxic effect and therapeutic effect is the therapeutic index and can be expressed as the LD50 / ED50 ratio. In embodiments, compositions and methods exhibiting a large therapeutic index are preferred. The therapeutically effective dose can be estimated in the initial stages from in vitro assays, including, for example, cell culture assays and measurement of methane production in fecal samples. Doses can also be formulated in animal models to achieve a circulating plasma concentration range including IC50 determined in cell cultures or appropriate animal models. Plasma levels of the compositions described herein can be measured, for example, by high-performance liquid chromatography. The effect of any particular dosage can be observed by appropriate bioassays. The dosage is determined by a physician and may be adjusted, if necessary, to suit the observed therapeutic effect.

[0197] In embodiments, the effect results in a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In embodiments, the effect results in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even more than about 90%. Therapeutic effects also include stopping or slowing the progression of the underlying disease or disability, regardless of whether improvement is achieved.

[0198] As used herein, “method of treatment” is equally applicable to the use of a composition for treating a disease or disorder described herein, and / or the use of a composition for the manufacture of a drug for treating a disease or disorder described herein. [Examples]

[0199] Example 1: Effect of topotecan on VCN-01 replication in cell lines derived from lung cancer and ovarian cancer. VCN-01 replication + / - topotecan was evaluated in A549 adherent and suspension cells, as well as two ovarian cancer cell lines, PA-1 and SW-626. Readings included viral particles (vp) by qPCR and infectivity titers (transduction units, TU) by hexon staining at three time points: 4 hours (input), 48 hours, and 72 hours post-infection. See Figure 1.

[0200] The study design will include evaluation of topoisomerase I inhibitors, topotecan, irinotecan, irinotecan metabolite SN-38, cell cycle inhibitors, hydroxyurea, and other agents including carboplatin and melphalan. Topotecan and SN-38 are expected to enhance VCN-01 replication in these cell lines. Gemcitabine will be used as a negative control, as it has been shown to inhibit adenovirus replication in vitro.

[0201] Example 2: In vitro sensitivity testing of pancreatic tumor cell lines to topoisomerase I (topoI) inhibitor, irinotecan (IRI), its active metabolite, SN-38, and topotecan (Topo). A panel of various human pancreatic tumor cell lines was examined to evaluate the cytotoxicity of IRI and Topo as monotherapies. Human pancreatic cell lines such as BxPC3 (ATCC-CRL1687), Panc-I (ATCC CRL 1469), NP-9, or NP-18 are susceptible to human adenovirus infection, and VCN-01 virus replication has been demonstrated in these cell lines. The HP-1 hamster pancreatic tumor cell line was also examined to assess its sensitivity to topoI inhibitors.

[0202] The concentrations of SN-38, IRI, or Topo required to halve the viability of cell cultures were determined for each cell line (IC50 value). For this purpose, tumor cells (15,000 cells / well) were seeded in 96-well plates and maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 5% fetal bovine serum at 37°C and 5% CO2. After 24 hours, serial 1 / 10 dilutions of each chemotherapeutic agent, IRI, SN-38, or Topo in DMEM + 5% FBS were prepared, and the chemotherapeutic agent-containing medium was added to the wells in sets of four. After incubating the cells for a further 5 days under standard culture conditions, the cells were washed once with PBS and stained for total protein content using a commercially available bicinchoninic acid assay (BCA; Pierce Biotechnology) (4 μl reagent A + 196 μl reagent B / well). After incubation at 37°C for 30 minutes, absorbance (A 540 nm) was quantified. The concentration (IC50 value) of each chemotherapeutic agent required to reduce the protein level by half compared to the untreated well was estimated using standard nonlinear regression with Prism software, and then plotted on a dose-response curve. At least three replicate trials were performed for each cell line. The results are shown in Figures 2A-2E, 3A-3E, and Table 1. [Table 1]

[0203] Example 3: Evaluation of viral (E1A) and cellular (E2F1) gene expression in NP-18 and NP-9 human pancreatic cancer cell lines exposed to VCN-01 + topoI inhibitor, SN-38, IRI, and Topo. The cells are divided into 2 × 10 6Cells were seeded in 6-well culture plates at a cell / well density. Cells were infected with VCN-01 at an MOI of 1. 24 hours post-infection, cells were treated with SN-38, IRI, or Topo at 100 times the previously determined IC50. For NP-18 cells, the topoI concentrations used were 0.1 μM SN-38, 200 μM IRI, and 2 μM Topo. For NP-9 cells, these concentrations were 2 μM SN-38, 1500 μM IRI, and 0.5 μM Topo. Cell pellets were harvested 24 hours after drug exposure, and RNA was isolated using the Qiagen RNAeasy Mini Kit and treated with DNAse. RNA was quantified and subjected to reverse transcription, followed by qPCR specific to E1A or E2F1. RNA levels for E1A and E2F1 were normalized to human β-actin control levels.

[0204] As shown in Figure 4, in NP-18 cells, viral E1A mRNA expression increased approximately 4 to 6 times with the topoI inhibitor compared to cells infected with tVCN-01 alone. In NP-9 cells, E1A expression increased more than 10 times with SN-38 and IRI, and approximately 2 times with Topo, compared to cells infected with VCN-01. Similarly, E2F1 mRNA levels increased 2 to 3 times in NP-18 cells containing the topoI inhibitor and 1 to 3 times in NP-9 cells containing the topoI inhibitor compared to untreated cells.

[0205] While we do not wish to be bound by theory, these data are consistent with the idea that topoI inhibitors increase the expression of the cellular transcription factor E2F1, thereby activating a modified VCN-01 E1A initial viral promoter and increasing the expression of viral genes.

[0206] Example 4: Evaluation of the antitumor effect of VCN-01 + liposomal irinotecan in an immunodeficient mouse model of human pancreatic cancer NP-18. The objective of this study was to evaluate the antitumor effect of the combination of VCN-01 and liposomal irinotecan (nal-IRI; ONIVYDE) in immunodeficient nude mice carrying human pancreatic NP-18 subcutaneous tumors. Liposomal irinotecan is a component of the NALIRIFOX chemotherapy cocktail, which consists of liposomal irinotecan + 5-fluorouracil (5-FU) + leucovorin + oxaliplatin.

[0207] Athymic nude mice (n=54, 6 cohorts @ 8 male mice + 6 additional animals per cohort) were subjected to 4 × 10⁻¹⁰ 6 NP-18 cells / flank (0.2 ml of cell suspension in PBS) were subcutaneously inoculated into two flanks of animals using a 25G needle. After transplantation, tumor growth was monitored twice a week by palpation, and the length and width of tumor nodules were measured with calipers. The tumor was 150 mm. 3 Upon reaching this stage, the animals were treated as shown in Figure 5. On day 1, six groups of eight mice each (16 tumors in total) were treated with VCN-01 or PBS IV via the tail vein. On days 3, 10, and 17, the animals were administered PBS or Na-IRI via intravenous injection at the indicated doses. The animals' body weight and tumor growth were measured weekly. At the end of the study, on day 37, the animals were sacrificed, blood was collected by cardiac puncture, and serum was analyzed for the indicated hematological and biochemical parameters. Tumors were collected on day 37 and subjected to qPCR analysis of vial genomes (DNA) and E1A mRNA.

[0208] Animal body weight was measured throughout the study (Figure 6). Animals treated with VCN-01 and VCN-01+naI-IRI showed initial weight loss. One animal in the VCN-01+naI-IRI (10 mg / kg) cohort was sacrificed due to extreme weight loss (>30%). All other animals recovered by the end of the study.

[0209] Tumor growth was tracked (Figure 7). Animals treated with VCN-01 alone or naI-IRI alone at doses of 5 mg / kg and 10 mg / kg showed a significant reduction in tumor growth compared to the PBS cohort. Animals treated with combination therapy of VCN-01 + naI-IRI at both doses of 5 mg / kg and 10 mg / kg showed a significant reduction in tumor growth compared to each monotherapy. There was no significant difference in tumor growth between the two combination therapy cohorts, VCN-01 + naI-IRI (5 mg / kg) and VCN-01 + naI-IRI (10 mg / kg).

[0210] Hematological and biochemical results (data not shown). At the end of the study, on day 37, blood was collected from each animal and subjected to hematological and biochemical analysis. Hematological parameters evaluated included leukocytes, erythrocytes, platelets, hemoglobin, lymphocytes, monocytes, neutrophils, eosinophils, and basophils. Platelets showed a significant decrease with high-dose naI-IRI treatment (10 mg / kg) alone or in combination with VCN-01. Thrombocytopenia is a known side effect of irinotecan. Other hematological parameters did not show significant differences between PBS or treatment cohorts. Biochemical parameters examined included ALT, AST, alkaline phosphatase, albumin, amylase, GGT, lipase, total bilirubin, total protein, creatinine, and urea. Alkaline phosphatase levels were significantly elevated in combination treatment (VCN-01 + naI-IRI) with both high-dose and low-dose naI-IRI. Other biochemical parameters evaluated did not show significant differences between treatment cohorts.

[0211] Tumors were collected on day 37, at the end of the study. Isolated tumor DNA was evaluated for the presence of VCN-01 viral genome (vg) by qPCR, and tumor RNA was analyzed for the presence of viral E1A mRNA by reverse transcription and qPCR (Figure 8). Vg was detected in the tumors of animals treated with VCN-01, demonstrating that VCN-01 was retained in the tumors throughout the study and suggesting ongoing viral replication. Compared to VCN-01 alone, tumor vg was significantly reduced in animals treated with VCN-01 + naI-IRI (5 mg / kg), while there was no significant difference in tumor vg between animals treated with VCN-01 alone and those treated with VCN-01 + naI-IRI 10 mg / kg. E1A mRNA analysis revealed the presence of E1A mRNA in all animals treated with VCN-01, indicating ongoing viral transcription. No differences in E1A mRNA levels were observed among the VCN-01 treated cohorts.

[0212] While we do not wish to be bound by theory, these data are consistent with the idea that the topoI inhibitor irinotecan (NAI-IRI) increases viral gene expression and enhances antitumor effects by increasing the expression of the cellular transcription factor E2F1 and activating a modified VCN-01 E1A early viral promoter.

[0213] Example 5: Evaluation of the antitumor effect of VCN-01 + liposomal irinotecan in an immunodeficient mouse model of human pancreatic cancer NP-9. The purpose of this study is to evaluate the antitumor effect of the combination of VCN-01 and liposomal irinotecan (nal-IRI;ONIVYDE) in immunodeficient nude mice carrying human pancreatic NP-9 subcutaneous tumors.

[0214] Athymic nude mice (n=54, 6 cohorts @ 8 female mice + 6 additional animals per cohort) received 7.5 × 10⁻⁶ doses. 6NP-9 cells / flank (0.2 ml of cell suspension in PBS) were subcutaneously injected into two flanks of animals using a 25G needle. After transplantation, tumor growth is monitored twice a week by palpation. The tumor is 150 mm. 3 Once the target is reached, the animals are treated as shown in Figure 9. On day 1, six groups of eight mice each (16 tumors in total) are treated with VCN-01 or PBS IV via the tail vein. On days 3, 10, and 17, the animals are administered PBS or Na-IRI via IP at the dose indicated. The animals' body weight and tumor growth are measured weekly. At the end of the study, the animals are sacrificed, blood is collected by cardiac puncture, and serum is analyzed for hematological and biochemical parameters. The tumors are collected at the end of the study and subjected to vial genome (DNA) and E1A mRNA qPCR analysis.

[0215] Compared to PBS alone, antitumor effects are expected with each treatment alone, VCN-01, naI-IRI at 5 mg / kg, and naI-IRI at 2.5 mg / kg. Improved antitumor effects are expected with combination therapy of VCN-01 and naI-IRI. Efficacy is expected to be observed with both high-dose (5 mg / kg) and low-dose (2.5 mg / kg) naI-IRI combination therapies.

[0216] Built-in by reference All patents and publications referenced herein are incorporated herein by reference in their entirety.

[0217] The publications discussed herein merely present disclosures made prior to the filing date of this application. Nothing in this specification shall be construed as acknowledging that this disclosure does not have prior rights to such publications for reasons of prior disclosure.

[0218] Where used herein, all headings are for structural purposes only and are not intended to restrict disclosure in any way. The content of any individual section may be equally applicable to all sections.

[0219] Embodiment Various additional embodiments of the present disclosure are provided by the following enumerated embodiments, which can be combined in any number and any combination.

[0220] Embodiment 1. A method for treating or preventing ovarian cancer, cervical cancer, lung cancer, colon cancer, or colorectal cancer and / or pancreatic cancer in a patient in need of treatment or prevention, comprising co-administering to the patient (i) an oncolytic adenovirus comprising a tumor cell-specific replication mechanism and optionally a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug thereof.

[0221] Embodiment 2. The method according to Embodiment 1, wherein the oncolytic adenovirus is administered, for example, by intravenous, intra-arterial, intratumoral, and / or intraperitoneal administration by injection.

[0222] Embodiment 3. The method according to Embodiment 1 or 2, wherein the topoisomerase I inhibitor or its prodrug is administered by intraperitoneal, intravenous, intra-arterial, intratumoral, and / or oral administration.

[0223] Embodiment 4. The method according to any one of Embodiments 1 to 3, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug are formulated in separate compositions.

[0224] Embodiment 5. The method according to any one of Embodiments 1 to 3, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug are formulated in a single composition.

[0225] Embodiment 6. The method according to any one of Embodiments 1 to 5, wherein the separate compositions are administered simultaneously or at the same time.

[0226] Embodiment 7. The method according to any one of Embodiments 1 to 6, wherein the oncolytic adenovirus is administered first, and the topoisomerase I inhibitor or its prodrug is administered within approximately 14 days, approximately 10 days, approximately 7 days, approximately 5 days, approximately 1 day, or within approximately 60 minutes of the administration of the oncolytic adenovirus.

[0227] Embodiment 8. The method according to Embodiment 7, wherein the topoisomerase I inhibitor or its prodrug is administered within approximately 30 minutes, approximately 20 minutes, approximately 10 minutes, approximately 5 minutes, or approximately 1 minute after administration of the oncolytic adenovirus.

[0228] Embodiment 9. The method according to any one of Embodiments 1 to 6, wherein the topoisomerase I inhibitor or its prodrug is administered first, the oncolytic adenovirus is administered within approximately 14 days, approximately 10 days, approximately 7 days, approximately 5 days, approximately 1 day, or approximately 60 minutes from the administration of the topoisomerase I inhibitor or its prodrug, and / or the oncolytic adenovirus is administered within approximately 30 minutes, approximately 20 minutes, approximately 10 minutes, approximately 5 minutes, or approximately 1 minute from the administration of the topoisomerase I inhibitor or its prodrug.

[0229] Embodiment 10. The method according to any one of Embodiments 1 to 9, wherein (a) the ovarian cancer is epithelial ovarian cancer, germ cell tumor, or stromal cell tumor; (b) the cervical cancer is squamous cell carcinoma, adenocarcinoma, and / or adenosquamous carcinoma (mixed) cancer; (c) the lung cancer is small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, lung large cell carcinoma, mesothelioma, and / or lung carcinoid tumor; (d) the colon cancer or colorectal cancer is adenocarcinoma, gastrointestinal stromal tumor (GIST), squamous cell carcinoma, carcinoid tumor, lymphoma, or colon sarcoma. In the embodiment, and / or (e) the pancreatic cancer is exocrine or endocrine carcinoma, or adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, or gelatinous carcinoma.

[0230] Embodiment 11. The method according to any one of Embodiments 1 to 10, wherein the topoisomerase I inhibitor is topotecan or SN-38, and / or the topoisomerase I inhibitor prodrug is irinotecan, optionally liposomal irinotecan, or optionally ONIVYDE.

[0231] Embodiment 12. The method according to any one of Embodiments 1 to 11, wherein the hyaluronidase enzyme is human hyaluronidase enzyme PH20.

[0232] Embodiment 13. The method according to Embodiment 12, wherein the sequence encoding the hyaluronidase enzyme is sequence number 9 or a sequence having at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto.

[0233] Embodiment 14. The method according to any one of Embodiments 1 to 13, wherein the oncolytic adenovirus is produced from human adenovirus serotype 5.

[0234] Embodiment 15. The method according to any one of Embodiments 1 to 14, wherein replication of the oncolytic adenovirus occurs in tumor cells having an abnormal Rb-E2F pathway, but not in healthy, non-tumorous, or normal cells.

[0235] Embodiment 16. The method according to Embodiment 15, wherein the oncolytic adenovirus is modified to control E1a expression by deleting the Rb-binding domain or Δ24 from the sequence encoding the E1a protein, and by inserting four E2F-1 binding sites and one Sp1 binding site into the endogenous promoter of E1a, thereby replicating only in tumor cells and not in healthy cells, non-tumor cells, or normal cells.

[0236] Embodiment 17. The oncolytic adenovirus has a heparan sulfate binding domain present in the adenovirus fibrils. 91 KKTK 94(Sequence ID 6) is the domain 91 RGDK 94 The method according to any one of Embodiments 1 to 16, having a capsid modified to be optionally substituted by (SEQ ID NO: 7).

[0237] Embodiment 18. The method according to any one of Embodiments 1 to 17, wherein the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3).

[0238] Embodiment 19. The method according to any one of claims 1 to 18, wherein the patient is a human patient.

[0239] Embodiment 20. The method according to Embodiment 19, wherein the human patient is a female human patient.

[0240] Embodiment 21. The method according to any one of Embodiments 1 to 20, wherein the ovarian cancer, cervical cancer, lung cancer, colon cancer, or colorectal cancer, and / or pancreatic cancer are resistant to conventional chemotherapy and / or radiotherapy.

[0241] Embodiment 22. The method according to any one of Embodiments 1 to 21, wherein the method improves and / or enhances and / or enhances the antitumor effect compared to treatment with the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

[0242] Embodiment 23. The method according to any one of Embodiments 1 to 22, wherein the method results in a reduction or maintenance of tumor size, and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with ovarian cancer, compared to treatment with the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

[0243] Embodiment 24. The method according to any one of Embodiments 1 to 22, wherein the method results in a reduction or maintenance of tumor size and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with cervical cancer, compared to treatment with the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

[0244] Embodiment 25. The method according to any one of Embodiments 1 to 22, wherein the method results in a reduction or maintenance of tumor size and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with lung cancer, compared to treatment with the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

[0245] Embodiment 26. The method according to any one of Embodiments 1 to 22, wherein the method results in a reduction or maintenance of tumor size and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with colon cancer or colorectal cancer, compared to treatment with the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

[0246] Embodiment 27. The method according to any one of Embodiments 1 to 22, wherein the method results in a reduction or maintenance of tumor size, and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with pancreatic cancer, compared to treatment with the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

[0247] Embodiment 28. A method for treating or preventing ovarian cancer, cervical cancer, lung cancer, colon cancer, or colorectal cancer and / or pancreatic cancer in a patient in need of treatment or prevention, comprising administering to the patient an oncolytic adenovirus comprising a replication mechanism specific to tumor cells and optionally a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is or has been treated with a topoisomerase I inhibitor or a prodrug thereof.

[0248] Embodiment 29. The method according to Embodiment 28, wherein the oncolytic adenovirus is administered by, for example, intravenous, intraarterial, intratumoral, and / or intraperitoneal administration via injection.

[0249] Embodiment 30. The method according to Embodiment 28 or 29, wherein the topoisomerase I inhibitor or a prodrug thereof is administered by intraperitoneal, intravenous, intraarterial, intratumoral, and / or oral administration.

[0250] Embodiment 31. The method according to any one of Embodiments 28 to 30, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are formulated in separate compositions.

[0251] Embodiment 32. The method according to any one of Embodiments 28 to 30, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are formulated in a single composition.

[0252] Embodiment 33. The method according to any one of Embodiments 28 to 32, wherein the oncolytic adenovirus is administered first, and the topoisomerase I inhibitor or a prodrug thereof is administered within about 12 weeks from the administration of the oncolytic adenovirus, or the topoisomerase I inhibitor or a prodrug thereof is administered first, and the oncolytic adenovirus is administered within about 12 weeks from the administration of the topoisomerase I inhibitor or a prodrug thereof.

[0253] Embodiment 34. The method of Embodiment 33, wherein the topoisomerase I inhibitor or a prodrug thereof is administered within about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or about 11 weeks of administration of the oncolytic adenovirus.

[0254] Embodiment 35. The method of any one of Embodiments 28 to 34, wherein the topoisomerase I inhibitor is topotecan or SN-38, and / or the prodrug of the topoisomerase I inhibitor is irinotecan, optionally liposomal irinotecan, optionally ONIVYDE.

[0255] Embodiment 36. The method of any one of Embodiments 28 to 35, wherein the hyaluronidase enzyme is human hyaluronidase enzyme PH20.

[0256] Embodiment 37. The method of Embodiment 36, wherein the sequence encoding the hyaluronidase enzyme is SEQ ID NO:9 or a sequence having at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto.

[0257] Embodiment 38. The method of any one of Embodiments 28 to 37, wherein the oncolytic adenovirus is produced from human adenovirus serotype 5.

[0258] Embodiment 39. The method of any one of Embodiments 28 to 38, wherein replication of the oncolytic adenovirus occurs in tumor cells having an aberrant Rb-E2F pathway and does not occur in healthy, non-neoplastic, or normal cells.

[0259] Embodiment 40. The method according to Embodiment 39, wherein the oncolytic adenovirus is modified by deleting the Rb-binding domain or Δ24 from the sequence encoding the E1a protein, and by inserting four binding sites to E2F-1 and one binding site to Sp1 into the endogenous promoter of E1a, thereby controlling the expression of E1a and causing it to replicate only in tumor cells and not in healthy cells, non-tumor cells, or normal cells.

[0260] Embodiment 41. The oncolytic adenovirus has a heparan sulfate binding domain present in the adenovirus fibrils. 91 KKTK 94 (Sequence ID 6) is the domain 91 RGDK 94 The method according to any one of embodiments 28 to 40, having a capsid modified to be optionally substituted by (SEQ ID NO: 7).

[0261] Embodiment 42. The method according to any one of Embodiments 28 to 41, wherein the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3).

[0262] Embodiment 43. The method according to any one of Embodiments 28 to 42, wherein the patient is a human patient.

[0263] Embodiment 44. The method according to Embodiment 43, wherein the human patient is a female human patient.

[0264] Embodiment 45. The method according to any one of Embodiments 28 to 44, wherein the ovarian cancer, cervical cancer, lung cancer, colon cancer, or colorectal cancer, and / or pancreatic cancer are resistant to conventional chemotherapy and / or radiotherapy.

[0265] Embodiment 46. The method according to any one of Embodiments 28 to 45, wherein the method improves and / or enhances and / or enhances the antitumor effect compared to treatment with the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

[0266] Embodiment 47. The method according to any one of Embodiments 28 to 46, wherein the method results in a reduction or maintenance of tumor size, and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with ovarian cancer, compared to treatment with the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

[0267] Embodiment 48. The method according to any one of Embodiments 28 to 46, wherein the method results in a reduction or maintenance of tumor size and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with cervical cancer, compared to treatment with the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

[0268] Embodiment 49. The method according to any one of Embodiments 28 to 46, wherein the method results in a reduction or maintenance of tumor size and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with lung cancer, compared to treatment with the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

[0269] Embodiment 50. The method according to any one of Embodiments 28 to 46, wherein the method results in a reduction or maintenance of tumor size and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with colon cancer or colorectal cancer, compared to treatment with the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

[0270] Embodiment 51. The method according to any one of Embodiments 28 to 46, wherein the method results in a reduction or maintenance of tumor size, and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with pancreatic cancer, compared to treatment with the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

[0271] Embodiment 52. The method of any one of Embodiments 1 to 22, 27, 28 to 46, or 51, wherein the prodrug of the topoisomerase I inhibitor is irinotecan, and the irinotecan is administered as part of the NALIRIFOX (irinotecan, 5-FU, leucovorin, oxaliplatin) regimen.

[0272] Embodiment 53. The method of any one of Embodiments 1 to 22, 27, 28 to 46, or 51, wherein the prodrug of the topoisomerase I inhibitor is liposomal irinotecan, optionally ONIVYDE, and the liposomal irinotecan is administered as part of the NALIRIFOX (irinotecan, 5-FU, leucovorin, oxaliplatin) regimen.

[0273] Embodiment 54. The method of any one of Embodiments 1 to 22, 27, 28 to 46, 51 or 53, wherein the prodrug of the topoisomerase I inhibitor is liposomal irinotecan, optionally ONIVYDE, and is administered by intravenous infusion.

[0274] Embodiment 55. The method of any one of Embodiments 1 to 54, wherein the method results in increased expression of the cellular transcription factor E2F1 compared to treatment with the oncolytic virus without the topoisomerase I inhibitor or prodrug thereof.

[0275] Embodiment 56. The method of any one of Embodiments 1 to 55, wherein the method results in increased expression of one or more viral genes compared to treatment with the oncolytic virus without the topoisomerase I inhibitor or prodrug thereof.

[0276] Sequence SEQ ID NO: 1 The complete PH20 cDNA sequence including the carboxy-terminal domain. (Both from ATG to the stop codon are included, and nt 1471-1527 were underlined and bolded) ##STR## Sequence ID 2 Sequence of the modified endogenous promoter E1a + VCN-01 E1a-Delta24 coding region (including four binding sites to E2F-1, a binding site to Sp1, and the E1a-Delta24 coding region) Sequence ID 3 Complete sequence of VCN-01 Sequence ID 4 Amino acid sequence of modified version of adenovirus serotype fibers with RGDK modification MKRARPSEDTFNPVYPYDTETGPPTVPFLTPPFVSPNGFQESPPGVLSLRLSEPLVTSNGMLALKMGNGLSLDEAGNLTSQNVTTVSPPLRGDKSNINLEISAPLTVTSEALTVAAAAPLMVAGNTLTMGSQAPLTVHDSKLSI ATQGPLTVSEGKLALQTSGPLTTTDSSTLTITASPPLTTATGSLGIDLKEPIYTQNGKLGLKYGAPLHVTDDLNTLTVATGPGVTINNTSLGTKVTGALGFDSQGNMQLNVAGGLRIDSQNRRLILDVSYPFDAQNQLNLRLGQ GPLFINSAHNLDINYNKGLYLFTASNNSKKLEVNLSTAKGLMFDATAIAINAGDGLEFGSPNAPNTNPLKTKIIGHGLEFDSNKAMVPKLGTGLSFDSTGAITVGNKNNDKLTLWTTPAPSPNCDLNAEKDAKLTLVLTKCGSQ ILATVSVLAVKGSLAPISGTVQSAHLIIRFDENGVLLNNSFLDPEYWNFRNGDLTEGTAYTNAVGFMPNLSAYPKSHGKTAKSNIVSQVYLNGDKTKPVTLTITLNGTQETGDTTPSAYSMSFSWDWSGHNYINEIFATSSYTF Sequence ID 5 RGD-C peptide CDCRGDCFC Sequence ID 6 Joint domain KKTK Sequence ID 7 Joint domain RGDK Sequence ID 8 Translation of Sequence ID No. 1 (PH20 with a carboxyl-terminal domain) MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKA KKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNE STALYPSIYLNTQQSPVAATLYVRNRVREAAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQ EQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATMFIVSILFLIISSVASL- Sequence ID 9 cDNA encoding PH20 with a deleted carboxyl-terminal domain. Sequence ID 10 Translation of Sequence ID No. 9 (PH20 with deleted carboxyl-terminal domain) MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQD HLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR NDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIIN VTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS-

Claims

1. A method for treating or preventing ovarian cancer, cervical cancer, lung cancer, colon cancer, or colorectal cancer in a patient in need of treatment or prevention, comprising co-administering to the patient (i) an oncolytic adenovirus comprising a replication mechanism specific to tumor cells and optionally a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug thereof.

2. The method according to claim 1, wherein the oncolytic adenovirus is administered, for example, by intravenous, intra-arterial, intratumoral, and / or intraperitoneal injection.

3. The method according to claim 1 or 2, wherein the topoisomerase I inhibitor or its prodrug is administered by intraperitoneal, intravenous, intra-arterial, intratumoral, and / or oral administration.

4. The method according to any one of claims 1 to 3, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug are formulated in separate compositions.

5. The method according to any one of claims 1 to 3, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug are formulated in a single composition.

6. The method according to any one of claims 1 to 5, wherein the separate compositions are administered simultaneously or at the same time.

7. The method according to any one of claims 1 to 6, wherein the oncolytic adenovirus is administered first, and the topoisomerase I inhibitor or its prodrug is administered within approximately 14 days, approximately 10 days, approximately 7 days, approximately 5 days, approximately 1 day, or approximately 60 minutes after the administration of the oncolytic adenovirus.

8. The method according to claim 7, wherein the topoisomerase I inhibitor or its prodrug is administered within approximately 30 minutes, approximately 20 minutes, approximately 10 minutes, approximately 5 minutes, or approximately 1 minute after administration of the oncolytic adenovirus.

9. The method according to any one of claims 1 to 6, wherein the topoisomerase I inhibitor or its prodrug is administered first, the oncolytic adenovirus is administered within approximately 14 days, approximately 10 days, approximately 7 days, approximately 5 days, approximately 1 day, or approximately 60 minutes from the administration of the topoisomerase I inhibitor or its prodrug, and / or the oncolytic adenovirus is administered within approximately 30 minutes, approximately 20 minutes, approximately 10 minutes, approximately 5 minutes, or approximately 1 minute from the administration of the topoisomerase I inhibitor or its prodrug.

10. Pancreatic cancer is exocrine or endocrine cancer, or adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, or gelatinous carcinoma; Ovarian cancer can be epithelial ovarian cancer, germ cell tumor, or stromal cell tumor; Cervical cancer is squamous cell carcinoma, adenocarcinoma, and / or adenosquamous carcinoma (mixed type); Lung cancer is small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer, mesothelioma, and / or lung carcinoid tumor, and / or The method according to any one of claims 1 to 9, wherein the colon cancer or colorectal cancer is an adenocarcinoma, gastrointestinal stromal tumor (GIST), squamous cell carcinoma, carcinoid tumor, lymphoma, or colon sarcoma.

11. The method according to any one of claims 1 to 10, wherein the topoisomerase I inhibitor is topotecan or SN-38, and / or the topoisomerase I inhibitor prodrug is irinotecan, optionally liposomal irinotecan, or optionally ONIVYDE.

12. The method according to any one of claims 1 to 11, wherein the hyaluronidase enzyme is human hyaluronidase enzyme PH20.

13. The method according to claim 12, wherein the sequence encoding the hyaluronidase enzyme is sequence number 9 or a sequence having at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto.

14. The method according to any one of claims 1 to 13, wherein the oncolytic adenovirus is produced from human adenovirus serotype 5.

15. The method according to any one of claims 1 to 14, wherein replication of the oncolytic adenovirus occurs in tumor cells having an abnormal Rb-E2F pathway, but not in healthy, non-tumorous, or normal cells.

16. The method according to claim 15, wherein the oncolytic adenovirus is modified by deleting the Rb-binding domain or Δ24 from the sequence encoding the E1a protein, and by inserting four binding sites to E2F-1 and one binding site to Sp1 into the endogenous promoter of E1a, thereby controlling the expression of E1a and causing it to replicate only in tumor cells and not in healthy cells, non-tumor cells, or normal cells.

17. The aforementioned oncolytic adenovirus has a heparan sulfate binding domain present in the adenovirus fibrils. ９１ KKTK ９４ (Sequence ID 6) is the domain ９１ RGDK ９４ The method according to any one of claims 1 to 16, comprising a capsid modified to be optionally substituted by (SEQ ID NO: 7).

18. The method according to any one of claims 1 to 17, wherein the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3).

19. The method according to any one of claims 1 to 18, wherein the patient is a human patient.

20. The method according to claim 19, wherein the human patient is a female human patient.

21. The method according to any one of claims 1 to 20, wherein the pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, and / or colon cancer or colorectal cancer is resistant to conventional chemotherapy and / or radiotherapy.

22. The method according to any one of claims 1 to 21, wherein the method improves and / or enhances and / or enhances the antitumor effect compared to treatment with the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

23. The method according to any one of claims 1 to 22, wherein the method results in a reduction or maintenance of tumor size, and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with pancreatic cancer, compared to treatment with the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

24. The method according to any one of claims 1 to 22, wherein the method results in a reduction or maintenance of tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with ovarian cancer, compared to treatment with the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

25. The method according to any one of claims 1 to 22, wherein the method results in a reduction or maintenance of tumor size, and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with cervical cancer, compared to treatment with the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

26. The method according to any one of claims 1 to 22, wherein the method results in a reduction or maintenance of tumor size, and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with lung cancer, compared to treatment with the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

27. The method according to any one of claims 1 to 22, wherein the method results in a reduction or maintenance of tumor size, and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with colon cancer or colorectal cancer, compared to treatment with the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

28. The method according to any one of claims 1 to 22 or 27, wherein the prodrug of the topoisomerase I inhibitor is irinotecan, and the irinotecan is administered as part of the NALIRIFOX (irinotecan, 5-FU, leucovorin, oxaliplatin) regimen.

29. The method according to any one of claims 1 to 22 or 27, wherein the prodrug of the topoisomerase I inhibitor is liposomal irinotecan, optionally ONIVYDE, which is administered as part of the NALIRIFOX (irinotecan, 5-FU, leucovorin, oxaliplatin) regimen.

30. The method according to any one of claims 1 to 22, 27, or 29, wherein the prodrug of the topoisomerase I inhibitor is liposomal irinotecan, optionally ONIVYDE, and is administered by intravenous infusion.

31. The method according to any one of claims 1 to 30, wherein the method results in an increase in the expression of cellular transcription factor E2F1 compared to treatment with the oncolytic virus without the topoisomerase I inhibitor or its prodrug.

32. The method according to any one of claims 1 to 31, wherein the method results in an increase in the expression of one or more viral genes compared to treatment with the oncolytic virus without the topoisomerase I inhibitor or its prodrug.

33. A method for treating or preventing pancreatic cancer in a patient requiring treatment or prevention of pancreatic cancer, comprising co-administering to the patient (i) an oncolytic adenovirus and (ii) a prodrug of a topoisomerase I inhibitor, wherein the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3), and the prodrug of the topoisomerase I inhibitor is liposomal irinotecan, optionally ONIVYDE, administered as part of the NALIRIFOX (irinotecan, 5-FU, leucovorin, oxaliplatin) regimen.

34. A prodrug of an oncolytic adenovirus and a topoisomerase I inhibitor for use in the treatment or prevention of pancreatic cancer, wherein the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3), and the prodrug of the topoisomerase I inhibitor is liposomal irinotecan, optionally ONIVYDE, administered as part of the NALIRIFOX (irinotecan, 5-FU, leucovorin, oxaliplatin) regimen.

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