Oncolytic adenoviruses and topoisomerase I inhibitors used to treat cancer

Abstract

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

Description

Technical Field

[0001] This disclosure especially This involves combination therapy of oncolytic adenoviruses for specific tumors with topotecan, irinotecan, or SN-38.

[0002] Cross-reference to related applications

[0003] This application claims the benefits of U.S. Provisional Application No. 63 / 584,003, filed September 20, 2023, and U.S. Provisional Application No. 63 / 624,896, filed January 25, 2024, the entire contents of which are hereby incorporated by reference.

[0004] Description of XML files submitted electronically

[0005] This application contains a sequence list, which is submitted electronically in XML file format and is hereby incorporated in its entirety by reference. The XML file, created on September 19, 2024, is named "VCN-007_Sequence_Listing.xml" and has a size of 53,098 bytes. Background Technology

[0006] Combination therapy is a core principle of cancer treatment. Compared to monotherapy, combining anticancer drugs can enhance efficacy because it can synergistically target key pathways. This approach has the potential to reduce resistance while providing therapeutic benefits such as reducing tumor growth and limiting tumor metastasis potential.

[0007] There is a need for new treatment strategies that deliver efficient and effective results at an affordable cost, especially those that combine existing treatments. Summary of the Invention

[0008] Therefore, in various aspects, this disclosure provides methods for treating or preventing pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, and / or colonic or colorectal pancreatic cancer in patients in need, the methods comprising co-administering to the patient (i) an oncolytic adenovirus comprising a replication mechanism specific to tumor cells and, optionally, a polynucleotide sequence encoding hyaluronidase inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug of a topoisomerase inhibitor.

[0009] In the implementation scheme, the oncolytic adenovirus is administered intravenously, intra-arterially, intratumorally, and / or intraperitoneally, for example, by injection. In the implementation scheme, the topoisomerase I inhibitor or its prodrug is administered intraperitoneally, intravenously, intra-arterially, or intratumorally, for example, by injection and / or oral administration.

[0010] In one embodiment, the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug are formulated into a single composition. In another embodiment, the single composition is administered simultaneously or concurrently.

[0011] In one implementation scheme, oncolytic adenovirus is administered first, followed by an oncolytic adenovirus inhibitor or its prodrug within approximately 60 minutes of administration. In another implementation scheme, the oncolytic adenovirus inhibitor or its prodrug is administered within approximately 30 minutes, 20 minutes, 10 minutes, 5 minutes, or 1 minute of administration. In yet another implementation scheme, the oncolytic adenovirus is administered first, followed by oncolytic adenovirus administration within approximately 60 minutes of administration. In yet another implementation scheme, the oncolytic adenovirus is administered within approximately 30 minutes, 20 minutes, 10 minutes, 5 minutes, or 1 minute of administration.

[0012] In one implementation scheme, oncolytic adenovirus is administered first, followed by an oncolytic adenovirus inhibitor or its prodrug approximately one day after administration. In another implementation scheme, the oncolytic adenovirus inhibitor or its prodrug is administered approximately 14 days, 10 days, 7 days, 5 days, or 1 day after administration. In yet another implementation scheme, the oncolytic adenovirus is administered first, followed by an oncolytic adenovirus approximately one day after administration. In yet another implementation scheme, the oncolytic adenovirus is administered approximately 14 days, 10 days, 7 days, 5 days, or 1 day after administration.

[0013] In the implementation plan, the topoisomerase I inhibitor is topotecan or SN-38, or the SN-38 prodrug irinotecan, which is converted to SN-38 in vivo.

[0014] In one embodiment, the oncolytic adenovirus optionally includes a polynucleotide sequence encoding hyaluronidase inserted into its genome. In another embodiment, the hyaluronidase is human hyaluronidase PH20. In yet another embodiment, the sequence encoding hyaluronidase is SEQ ID NO: 9 or a sequence having at least about 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 with it.

[0015] In the implementation plan, the oncolytic adenovirus is generated from human adenovirus serotype 5.

[0016] In this implementation, oncolytic adenovirus replication occurs in tumor cells (e.g., solid tumors) with an abnormal Rb-E2F pathway, rather than in healthy, non-tumor, or normal cells. In this implementation, the oncolytic adenovirus is engineered to replicate in tumor cells, rather than in healthy, non-tumor, or normal cells, by deleting the Rb-binding domain from the sequence encoding the E1a protein and inserting four E2F-1 binding sites and one Sp1 binding site into the endogenous promoter of E1a to control E1a expression.

[0017] In this implementation, oncolytic adenovirus replication occurs in tumor cells (e.g., solid tumors) with an abnormal Rb-E2F pathway, rather than in healthy, non-tumor, or normal cells. In this implementation, the oncolytic adenovirus is engineered to replicate in tumor cells, rather than in healthy, non-tumor, or normal cells, by deleting Δ24 from the sequence encoding the E1a protein and inserting four E2F-1 binding sites and one Sp1 binding site into the endogenous promoter of E1a to control E1a expression.

[0018] In the implementation scheme, the capsid of the oncolytic adenovirus is modified to include the heparin sulfate-binding domain present in the adenovirus fibers. 91 KKTK 94 (SEQ ID NO: 6) was replaced.

[0019] In the implementation scheme, the capsid of the oncolytic adenovirus is modified to include the heparin sulfate-binding domain present in the adenovirus fibers. 91 KKTK 94 (SEQ ID NO: 6) is the domain 91 RGDK 94 (SEQ ID NO: 7) Replacement.

[0020] In the implementation scheme, the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3) or a functional variant thereof.

[0021] In the implementation plan, the patient is a human patient. In the implementation plan, the human patient is a female human patient.

[0022] In the implementation plan, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, and / or colon or colorectal cancer are resistant to conventional chemotherapy and / or radiotherapy.

[0023] In the implementation scheme, compared with treatment using a topoisomerase I inhibitor or its prodrug without oncolytic adenovirus, the method improves and / or increases and / or enhances antitumor efficacy. In the implementation scheme, compared with treatment using a topoisomerase I inhibitor or its prodrug without oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with ovarian cancer. In the implementation scheme, compared with treatment using a topoisomerase I inhibitor or its prodrug without oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with cervical cancer. In the implementation scheme, compared with treatment using a topoisomerase I inhibitor or its prodrug without oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with lung cancer. In the implementation scheme, compared with treatment using a topoisomerase I inhibitor or its prodrug without oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with colon or colorectal cancer. In the implementation scheme, compared with treatment using a topoisomerase I inhibitor or its prodrug without oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with pancreatic cancer.

[0024] In various aspects, this disclosure provides methods for treating or preventing pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, and / or colon or colorectal cancer in patients of need, the methods comprising administering to a patient an oncolytic adenovirus comprising a replication mechanism specific to tumor cells and, optionally, an intercalation of a polynucleotide sequence encoding hyaluronidase into the patient's genome, wherein the patient is receiving or has received treatment with a topoisomerase I inhibitor or a prodrug thereof.

[0025] In the implementation scheme, the oncolytic adenovirus is administered intravenously, intra-arterially, intratumorally, and / or intraperitoneally, for example, by injection. In the implementation scheme, the topoisomerase I inhibitor or its prodrug is administered intraperitoneally, intravenously, intra-arterially, intratumorally, and / or orally.

[0026] In the implementation scheme, the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug are formulated into a separate composition.

[0027] In the implementation scheme, oncolytic adenovirus and topoisomerase I inhibitor or its prodrug are formulated into a single composition.

[0028] In the implementation plan, oncolytic adenovirus is administered first, followed by the administration of a topoisomerase I inhibitor or its prodrug within approximately 12 weeks after the administration of the oncolytic adenovirus. In the implementation plan, the topoisomerase I inhibitor or its prodrug 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, or 11 weeks after the administration of the oncolytic adenovirus.

[0029] In the implementation plan, a topoisomerase I inhibitor or its prodrug is first administered, followed by oncolytic adenovirus administration within approximately 12 weeks of administration of the topoisomerase I inhibitor or its prodrug. In the implementation plan, oncolytic adenovirus is administered 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 after administration of the topoisomerase I inhibitor or its prodrug.

[0030] In the implementation plan, the topoisomerase I inhibitor or its prodrug is topotecan, SN-38 and / or irinotecan.

[0031] In the embodiment, the hyaluronidase is human hyaluronidase PH20. In the embodiment, the sequence encoding the hyaluronidase is SEQ ID NO: 9 or a sequence having at least about 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 with it.

[0032] In the implementation plan, the oncolytic adenovirus is generated from human adenovirus serotype 5.

[0033] In this implementation, oncolytic adenovirus replication occurs in tumor cells with an abnormal Rb-E2F pathway, rather than in healthy, non-tumor, or normal cells. In this implementation, oncolytic adenoviruses are engineered to replicate in tumor cells, rather than in healthy, non-tumor, or normal cells, by deleting the Rb-binding domain or deleting Δ24 from the sequence encoding the E1a protein and inserting four E2F-1 binding sites and one Sp1 binding site into the endogenous promoter of E1a to control E1a expression.

[0034] In the implementation scheme, the capsid of the oncolytic adenovirus is modified to include the heparin sulfate-binding domain present in the adenovirus fibers. 91 KKTK 94 (SEQ ID NO: 6) is replaced. In the embodiment, the capsid of the oncolytic adenovirus is modified to allow the heparin sulfate binding domain present in the adenovirus fibers to be replaced. 91 KKTK 94 (SEQ ID NO: 6) is the domain 91 RGDK 94 (SEQ ID NO: 7) Replacement.

[0035] In the implementation scheme, the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3) or a functional variant thereof.

[0036] In the implementation plan, the patient is a human patient. In the implementation plan, the human patient is a female human patient.

[0037] In the implementation plan, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, and / or colon or colorectal cancer are resistant to conventional chemotherapy and / or radiotherapy.

[0038] In the implementation scheme, compared with treatment using a topoisomerase I inhibitor or its prodrug without oncolytic adenovirus, the method improves and / or increases and / or enhances antitumor efficacy. In the implementation scheme, compared with treatment using a topoisomerase I inhibitor or its prodrug without oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with ovarian cancer. In the implementation scheme, compared with treatment using a topoisomerase I inhibitor or its prodrug without oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with cervical cancer. In the implementation scheme, compared with treatment using a topoisomerase I inhibitor or its prodrug without oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with lung cancer. In the implementation scheme, compared with treatment using a topoisomerase I inhibitor or its prodrug without oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with colon or colorectal cancer. In the implementation scheme, compared with treatment using a topoisomerase I inhibitor or its prodrug without oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with pancreatic cancer.

[0039] In the implementation scheme, compared with treatment using a topoisomerase I inhibitor or its prodrug without oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with colon or colorectal cancer. In the implementation scheme, compared with treatment using a topoisomerase I inhibitor or its prodrug without oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies, or recurrent diseases associated with pancreatic cancer.

[0040] In the implementation scheme, ovarian cancer is epithelial ovarian cancer, germ cell tumor, and / or stromal cell tumor. In the implementation scheme, cervical cancer is squamous cell carcinoma, adenocarcinoma, and / or adenosquamous carcinoma (mixed type). In the implementation scheme, lung cancer is small cell lung cancer (also known as oat cell carcinoma), non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, large cell lung carcinoma, mesothelioma, and / or lung carcinoid. In the implementation scheme, colon or colorectal cancer is colonic adenocarcinoma, gastrointestinal stromal tumor (GIST), squamous cell carcinoma, carcinoid, lymphoma, or sarcoma. In the implementation scheme, pancreatic cancer is exocrine or endocrine carcinoma, or adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, or colloid carcinoma. Attached Figure Description

[0041] Figure 1 A schematic diagram of a non-limiting study design for evaluating the effects of topotecan or irinotecan + VCN-01 in lung cancer or ovarian cancer models is shown.

[0042] Figures 2A to 2E The results of cytotoxicity of SN-38, irinotecan (IRI), and topotecan (Topo) in a group of pancreatic cancer cell lines are presented, specifically NP-18. Figure 2A ), NP-9 ( Figure 2B ), PANC-1 ( Figure 2C ) and BxPC3 ( Figure 2D ) and HP-2 ( Figure 2E Cell viability curves after exposure to a series of drug dilutions are presented. Cell viability was measured by BCA assay on day 5 (expressed as mean + SEM). Cell viability curves were adjusted for nonlinear regression fitting: log(inhibitor) versus normalized response - variable slope. N=3 experiments were performed for each cell line. The figure shows a representative experiment.

[0043] Figures 3A to 3E The mean IC50 values ​​(n=3) for a group of pancreatic cancer cell lines are shown, specifically NP-18 ( Figure 3A ), NP-9 ( Figure 3B ), PANC-1 ( Figure 3CBxPC3 Figure 3D ) and HP-1 ( Figure 3E IC50 values ​​for SN-38, IR, and Topo in each cell line. IC50 values ​​were estimated by applying nonlinear regression fitting to the cell viability curves. Three replicates were performed.

[0044] Figure 4 The relative levels of E1A and E2F1 mRNA in NP-18 and NP-9 cells are shown. mRNA levels are shown relative to cells infected with VCN-01 alone (E1A) or untreated cells (E2F1).

[0045] Figure 5 A schematic diagram of a non-limiting example of a mouse NP-18 tumor study is shown.

[0046] Figure 6 The percentage increase / decrease in body weight of the NP-18 tumor-bearing animals in the study described in Example 4 is shown.

[0047] Figure 7 Tumor growth (%) in NP-18 tumor-bearing animals of the study described in Example 4 is shown.

[0048] Figure 8 The quantification of VCN-01 viral genome (vgs) and E1A mRNA in the tumors of the study described in Example 4 is shown. Tumors were collected at the end of the study (day 37). Tumor DNA was isolated and qPCR was performed to measure vgs, and tumor RNA was isolated and qPCR was performed to measure E1A mRNA levels.

[0049] Figure 9 A schematic diagram illustrating a non-limiting example of NP-9 tumor research is shown. Detailed Implementation

[0050] Treatment of ovarian cancer

[0051] In various respects, this disclosure provides methods for treating or preventing ovarian cancer in patients in need, the methods comprising administering to the patient (i) an oncolytic adenovirus comprising a replication mechanism specific to tumor cells and, optionally, a polynucleotide sequence encoding hyaluronidase inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as, but not limited to, ONIVYDE).

[0052] In various respects, this disclosure provides methods for preventing, removing, or reducing metastatic, secondary malignant, or recurrent disease associated with ovarian cancer in patients of need, the methods comprising administering to the patient (i) an oncolytic adenovirus comprising a replication mechanism specific to tumor cells and, optionally, an intercalation of a polynucleotide sequence encoding hyaluronidase into its genome, and (ii) a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as, but not limited to, ONIVYDE).

[0053] In various respects, this disclosure provides methods for treating or preventing ovarian, cervical, lung, colon or colorectal, and / or pancreatic cancer in patients of need, the methods comprising administering to a patient an oncolytic adenovirus comprising a replication mechanism specific to tumor cells and, optionally, an intercalation of a polynucleotide sequence encoding hyaluronidase into the patient’s genome, wherein the patient is receiving or has received treatment with a topoisomerase I inhibitor or a prodrug thereof.

[0054] In various respects, this disclosure provides methods for preventing, removing, or reducing metastatic, secondary malignant, or recurrent disease associated with ovarian cancer in patients of need, the methods comprising administering to a patient an oncolytic adenovirus containing a replication mechanism specific to tumor cells and, optionally, an intercalation of a polynucleotide sequence encoding hyaluronidase into its genome, wherein the patient is receiving or has received treatment with a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as, but not limited to, ONIVYDE).

[0055] In one implementation, the method reduces or eliminates ovarian tumors or cancer, or their cells. In another implementation, the method slows the growth and / or progression and / or metastasis of ovarian tumors or cancer, or their cells.

[0056] In the implementation scheme, ovarian cancer is a primary or metastatic tumor. Primary tumors or cancers that migrate from their initial location and inoculate vital organs can ultimately lead to the subject's death through the deterioration of function in the affected organs. Metastasis refers to the spread of cancer cells or clusters of cancer cells from the primary tumor to other parts of the body, locations different from the primary tumor. Metastasis can ultimately lead to the subject's death. For example, in the implementation scheme, the ovarian cancer of the present invention includes benign and malignant cancers, polyps, hyperplasia, and dormant tumors or micrometastases.

[0057] In the implementation plan, ovarian cancer is defined as epithelial ovarian cancer, germ cell tumor, or stromal cell tumor.

[0058] In the implementation plan, ovarian cancer is defined as serous carcinoma, papillary serous carcinoma, metastatic carcinoma, borderline carcinoma, mucinous carcinoma, or clear cell carcinoma.

[0059] In the implementation scheme, the ovarian cancer is grade 1 ovarian cancer, characterized for example: (i) well-differentiated tissue; or (ii) low-grade nuclei with uncommon mitotic figures.

[0060] In the implementation scheme, the ovarian cancer is stage I ovarian cancer, characterized for example: (i) the tumor is confined to one ovary, with an intact capsule, no tumor on the surface of the ovary, and negative lavage fluid (stage IA); (ii) the tumor involves both ovaries, with an intact capsule, no tumor on the surface of the ovaries, and negative lavage fluid (stage IB); (iii) intraoperative tumor rupture (stage IC1); (iv) preoperative capsule rupture or presence of tumor on the surface of the ovary (stage IC2); or (v) presence of malignant cells in ascites or peritoneal lavage fluid (stage IC3).

[0061] In the implementation scheme, the ovarian cancer is stage II ovarian cancer, characterized for example: (i) the tumor has spread and / or implanted in the uterus and / or fallopian tubes (stage IIA); or (ii) the tumor has spread to other pelvic peritoneal tissues (stage IIB).

[0062] In the implementation scheme, the ovarian cancer is stage III ovarian cancer, characterized, for example, by spread to the peritoneum and / or lymph nodes outside the pelvic cavity, such as lymph nodes in the posterior abdomen. In the implementation scheme, the ovarian cancer is stage III ovarian cancer, characterized, for example, by spread to lymph nodes in the posterior abdomen (stage 3A1); the presence of cancer cells in tissue samples from the peritoneal lining and / or lymph nodes (stage 3A2); cancer growth of 2 cm or smaller on the peritoneal lining and / or lymph nodes (stage 3B); or cancer growth larger than 2 cm on the peritoneal lining (stage 3C).

[0063] In the implementation scheme, the ovarian cancer is stage IV ovarian cancer, characterized, for example, as advanced (metastatic) cancer (e.g., spread to the liver or lungs).

[0064] In an implementation, the method slows down or prevents progression from Phase I (e.g., IA, IB, or IC) to one of Phase II (e.g., IIA or IIB), Phase III (e.g., IIIA, IIIB, or IIIC), or Phase IV.

[0065] In an implementation, the method slows down or prevents progression from stage II (e.g., IIA or IIB) to one of stage III (e.g., IIIA, IIIB, or IIIC) and stage IV.

[0066] In the implementation, the method slows down or prevents progression from stage III (e.g., IIIA, IIIB, or IIIIC) to stage IV.

[0067] In the implementation scheme, the method for treating ovarian cancer is further combined with one or more of surgery, radiation therapy, chemotherapy, targeted therapy, and immunotherapy. In the implementation scheme, chemotherapy is one or more of platinum-based agents (e.g., but not limited to cisplatin or carboplatin) and taxanes (e.g., but not limited to paclitaxel or docetaxel). In the implementation scheme, chemotherapy is one or more of cisplatin, carboplatin, paclitaxel, docetaxel, albumin-bound paclitaxel, altretamine, capecitabine, cyclophosphamide, etoposide, gemcitabine, ifosfamide, doxorubicin, melphalan, pemetrexed, and vinorelbine. In the implementation scheme, targeted therapy is an anti-angiogenic therapy (e.g., bevacizumab or ramucirumab). In the implementation plan, the immunotherapy is one or more of the following: agents targeting the PD-1 pathway (e.g., atezolizumab, durvalumab, cemiplimab, nivolumab, and pembrolizumab) and / or agents targeting the CTLA-4 pathway (e.g., ipilimumab).

[0068] In the implementation plan, ovarian cancer refers to metastatic ovarian cancer that occurs after disease progression during or following initial or subsequent chemotherapy.

[0069] Treatment of cervical cancer

[0070] In various respects, this disclosure provides methods for treating or preventing cervical cancer in patients in need, the methods comprising administering to the patient (i) an oncolytic adenovirus comprising a polynucleotide sequence encoding hyaluronidase and optionally inserted into its genome, and having a specific replication mechanism for tumor cells, and (ii) a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38 and / or irinotecan.

[0071] In various respects, this disclosure provides methods for treating or preventing cervical cancer in patients of need, the methods comprising administering to the patient (i) an oncolytic adenovirus containing a polynucleotide sequence encoding hyaluronidase and an insertion into its genome that is specific to tumor cells and a specific replication mechanism for tumor cells, and (ii) a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as but not limited to ONIVYDE).

[0072] In various respects, this disclosure provides methods for treating or preventing cervical cancer in patients of need, the methods comprising administering to a patient an oncolytic adenovirus containing a polynucleotide sequence encoding hyaluronidase and optionally inserted into its genome, which has a specific replication mechanism against tumor cells and a specific replication mechanism against tumor cells, wherein the patient is receiving or has received treatment with a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38 and / or irinotecan.

[0073] In various respects, this disclosure provides methods for treating or preventing cervical cancer in patients of need, the methods comprising administering to a patient an oncolytic adenovirus containing a polynucleotide sequence encoding hyaluronidase and an insertion into the patient’s genome that has a specific replication mechanism against tumor cells, wherein the patient is receiving or has received treatment with a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as but not limited to ONIVYDE).

[0074] In the implementation scheme, the method reduces or eliminates cervical tumors or cancer, or their cells. In the implementation scheme, the method slows the growth and / or progression and / or metastasis of cervical tumors or cancer, or their cells.

[0075] In the implementation scheme, cervical cancer is a primary or metastatic tumor. Primary tumors or cancers that migrate from their initial location and inoculate vital organs can ultimately lead to the subject's death through the deterioration of the function of the affected organs. Metastasis refers to the spread of cancer cells or clusters of cancer cells from the primary tumor to other parts of the body, which are located in a different location than the primary tumor. Metastasis can ultimately lead to the subject's death. For example, in the implementation scheme, the cervical cancer of the present invention includes benign and malignant cancers, polyps, hyperplasia, and dormant tumors or micrometastases.

[0076] In the implementation plan, cervical cancer is squamous cell carcinoma, adenocarcinoma, and / or adenosquamous carcinoma (mixed type).

[0077] In the implementation plan, cervical cancer is defined as falling into any of the following stages: Stage I is characterized, for example, by cancer cells growing from the surface of the cervix into the deeper tissues of the cervix, but without spreading to nearby lymph nodes or distant sites. Stage I can be further subdivided into: IA: Cancers that can only be observed under a microscope; IA1: Cancer observed only under a microscope and less than 3 mm deep; IA2: Cancer observed only under a microscope, with a depth between 3 mm and 5 mm; IB: Stage I cancer that has spread to less than 5 mm in the cervix but is still confined to the cervix; IB1: Cancer depth greater than 5 mm, but size not exceeding 2 cm; IB2: The size of the cancer is at least 2 cm but no more than 4 cm; IB3: The cancer is at least 4 cm in size and is confined to the cervix; Stage II is characterized, for example, by cancer that has grown beyond the cervix and uterus, but has not yet spread to the pelvic wall or lower vagina, nor to nearby lymph nodes or distant sites. Stage II can be further subdivided into: IIA: The cancer has grown beyond the cervix and uterus, but has not yet spread to the tissues surrounding the cervix (paracervical tissues); IIA1: Cancer no larger than 4 cm; IIA2: Cancer is 4 cm or larger; IIB: The cancer has grown beyond the cervix and uterus and has spread to the tissues surrounding the cervix (paracervical tissue). Stage III is characterized, for example, by the cancer spreading to the lower vagina or pelvic wall. The cancer may be obstructing the ureter. The cancer may have spread to nearby lymph nodes, but not yet to distant sites. Stage III can be further subdivided into: IIIA: The cancer has spread to the lower part of the vagina, but has not yet spread to the pelvic wall.

[0078] Stage IIIB: The cancer has grown into the pelvic wall and / or obstructs one or both ureters, leading to hydronephrosis.

[0079] Stage IIIC: The cancer may be of any size, and imaging or biopsy shows that the cancer has spread to nearby pelvic lymph nodes (IIIC1) or para-aortic lymph nodes (IIIC2); and

[0080] Stage IV is characterized, for example, by cancer cells having grown into the bladder or rectum, or into more distant organs such as the lungs or bones. Stage IV can be further subdivided into: IVA: The cancer has spread to the bladder or rectum, or has grown out of the pelvic cavity.

[0081] IVB: The cancer has spread to distant organs outside the pelvic region, such as distant lymph nodes, lungs, or bones.

[0082] In an implementation, the method slows down or prevents progression from Phase I (e.g., IA or IB) to one of Phase II (e.g., IIA or IIB), Phase III (e.g., IIIA, IIIB, or IIIC), or Phase IV (e.g., IVA or IVB).

[0083] In an implementation, the method slows down or prevents progression from stage II (e.g., IIA or IIB) to one of stage III (e.g., IIIA, IIIB, or IIIC) and stage IV (e.g., IVA or IVB).

[0084] In an implementation, the method slows down or prevents progression from stage III (e.g., IIIA, IIIB, or IIIIC) to stage IV (e.g., IVA or IVB).

[0085] In the implementation plan, cervical cancer is defined as stage IV-B, recurrent, or persistent cervical cancer.

[0086] In the implementation plan, the cervical cancer is metastatic cervical cancer. In the implementation plan, the cervical cancer is further treated with cisplatin.

[0087] In the implementation scheme, the method for treating cervical cancer is further combined with one or more of surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. In the implementation scheme, chemotherapy is one or more of cisplatin, carboplatin, paclitaxel, docetaxel, ifosfamide, 5-fluorouracil (5-FU), gemcitabine, and mitomycin. In the implementation scheme, targeted therapy is an anti-angiogenic therapy (e.g., bevacizumab or ramucirumab). In the implementation scheme, immunotherapy is one or more of agents targeting the PD-1 pathway (e.g., atezolizumab, durvalumab, cimiprimab, nivolumab, and pembrolizumab) and / or agents targeting the CTLA-4 pathway (e.g., ipilimumab).

[0088] Treatment of lung cancer

[0089] In various respects, this disclosure provides methods for treating or preventing lung cancer in patients in need, the methods comprising administering to the patient (i) an oncolytic adenovirus comprising a polynucleotide sequence encoding hyaluronidase and optionally inserted into its genome, which has a specific replication mechanism against tumor cells and a specific replication mechanism against tumor cells, and (ii) a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as but not limited to ONIVYDE).

[0090] In various respects, this disclosure provides methods for treating or preventing lung cancer in patients in need, the methods comprising administering to the patient (i) an oncolytic adenovirus comprising a polynucleotide sequence encoding hyaluronidase and an insertion into its genome that is specific to tumor cells and a specific replication mechanism for tumor cells, and (ii) a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as but not limited to ONIVYDE).

[0091] In various respects, this disclosure provides methods for treating or preventing lung cancer in patients of need, the methods comprising administering to a patient an oncolytic adenovirus containing a polynucleotide sequence encoding hyaluronidase and optionally, inserted into its genome, having a specific replication mechanism against tumor cells, wherein the patient is receiving or has received treatment with a topoisomerase I inhibitor or a prodrug thereof, for example ， Topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE).

[0092] In various respects, this disclosure provides methods for treating or preventing lung cancer in patients of need, the methods comprising administering to a patient an oncolytic adenovirus containing a polynucleotide sequence encoding hyaluronidase and an insertion into the tumor cell genome that has a specific replication mechanism against tumor cells, wherein the patient is receiving or has received treatment with a topoisomerase I inhibitor or a prodrug thereof, for example ， Topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE).

[0093] In one implementation, the method reduces or eliminates lung tumors or cancer, or their cells. In another implementation, the method slows the growth and / or progression and / or metastasis of lung tumors or cancer, or their cells.

[0094] In the implementation scheme, lung cancer is a primary or metastatic tumor. A primary tumor or cancer that migrates from its initial location and infects vital organs can ultimately lead to the subject's death through the deterioration of function in the affected organs. Metastasis refers to the spread of cancer cells or clusters of cancer cells from the primary tumor to other parts of the body, locations different from the primary tumor. Metastasis can ultimately lead to the subject's death. For example, in the implementation scheme, the lung cancer of the present invention includes benign and malignant cancers, polyps, hyperplasia, and dormant tumors or micrometastases.

[0095] In the implementation plan, the lung cancer is small cell lung cancer. In the implementation plan, the lung cancer is non-small cell lung cancer. In the implementation plan, the lung cancer is lung adenocarcinoma. In the implementation plan, the lung cancer is lung cell carcinoma. In the implementation plan, the lung cancer is large cell lung carcinoma. In the implementation plan, the lung cancer is mesothelioma. In the implementation plan, the lung cancer is carcinoid tumor of the lung.

[0096] In the implementation plan, lung cancer is defined as one of the following lung cancer stages, optionally assessed using the TNM system: The latent period is characterized, for example, by the presence of cancer cells in the mucus coughed up by the subject; Stage 0 is characterized, for example, by a small tumor that has not spread to deeper lung tissue or outside the lungs; Stage I (e.g., IA, such as IA1, IA2, IA3 and IB) is characterized, for example, by the presence of cancer in the lung tissue but not in the lymph nodes; Stage II (e.g., IIA or IIB) is characterized, for example, by the presence of cancer in lung tissue and lymph nodes near the lungs; Stage III (e.g., IIIA, IIIB, or IIIC), characterized, for example, by the presence of cancer in the lung tissue and its further spread to the lymph nodes and the central thoracic cavity; and / or Stage IV is characterized, for example, by the presence of cancer cells in lung tissue and their widespread spread to other parts of the body (e.g., to the brain, bones, and / or liver).

[0097] In an implementation, the method slows down or prevents progression from the occult period to one of the following: stage 0, stage I (e.g., IA, such as IA1, IA2, IA3, and IB), stage II (e.g., IIA or IIB), stage III (e.g., stage IIIA, IIIB, or IIIC), and stage IV.

[0098] In an implementation, the method mitigates or prevents progression from stage 0 to one of stage I (e.g., IA, such as IA1, IA2, IA3, and IB), stage II (e.g., IIA or IIB), stage III (e.g., stage IIIA, IIIB, or IIIC), and stage IV.

[0099] In an implementation, the method slows down or prevents progression from stage I (e.g., IA, such as IA1, IA2, IA3, and IB) to one of stage II (e.g., IIA or IIB), stage III (e.g., stage IIIA, IIIB, or IIIC), and stage IV.

[0100] In an implementation, the method slows down or prevents progression from stage II (e.g., IIA or IIB) to one of stage III (e.g., IIIA, IIIB, or IIIC) and stage IV.

[0101] In the implementation, the method slows down or prevents progression from stage III (e.g., IIIA, IIIB, or IIIC) to stage IV.

[0102] In the implementation plan, lung cancer is either small cell lung cancer or non-small cell lung cancer.

[0103] In the implementation plan, the lung cancer is small cell lung cancer with platinum sensitivity. Specifically, the lung cancer is small cell lung cancer in the subject who has platinum sensitivity and whose disease progresses at least approximately 60 days after initiation of first-line chemotherapy.

[0104] In the implementation plan, lung cancer is non-small cell lung cancer with platinum sensitivity. In the implementation plan, lung cancer is small cell lung cancer with platinum sensitivity in the subject, and the subject has progressed at least approximately 60 days after initiating first-line chemotherapy.

[0105] In the implementation plan, the method for treating lung cancer is further combined with one or more of surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. In the implementation plan, chemotherapy is one or more of carboplatin, cisplatin, docetaxel, etoposide, gemcitabine, albumin-bound paclitaxel, paclitaxel, pemetrexed, and vinorelbine. In the implementation plan, targeted therapy is one or more of the following: 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), and agents targeting HER2 mutations (e.g., Fam-Dexi). Trastuzumab (nxki), anaplastic lymphoma kinase (ALK) inhibitors (e.g., alectinib, brigatinib, ceritinib, crizotinib, and lorlatinib), agents targeting ROS1 fusions (e.g., ceritinib, crizotinib, and entrectinib), agents targeting KRASG12C mutations (e.g., sotorasib), agents targeting NTRK fusions (e.g., larotrectinib), and agents targeting BRAF... Agents targeting V600E mutations (e.g., dabrafenib or trametinib), agents targeting MET exon 14 skipping (e.g., capmatinib or tepotinib), agents targeting RET fusions (e.g., pralsetinib or selpercatinib), and anti-angiogenic therapies (e.g., bevacizumab or ramucirumab). In the implementation scheme, immunotherapy is one or more of agents targeting the PD-1 pathway (e.g., atezolizumab, durvalumab, cimiprimab, nivolumab, and pembrolizumab) and / or agents targeting the CTLA-4 pathway (e.g., ipilimumab).

[0106] Treatment of colon or colorectal cancer

[0107] In various aspects, this disclosure provides methods for treating or preventing colorectal cancer in patients of need, the methods comprising administering to the patient (i) an oncolytic adenovirus comprising a polynucleotide sequence encoding hyaluronidase and optionally inserted into its genome, and having a specific replication mechanism for tumor cells, and (ii) a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as, but not limited to, ONIVYDE).

[0108] In various respects, this disclosure provides methods for treating or preventing colorectal cancer in patients of need, the methods comprising administering to the patient (i) an oncolytic adenovirus comprising a polynucleotide sequence encoding hyaluronidase and an insertion into its genome that is specific to tumor cells and a specific mechanism for tumor cell replication, and (ii) a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as, but not limited to, ONIVYDE).

[0109] In various aspects, this disclosure provides methods for treating or preventing colorectal cancer in patients of need, methods comprising administering to a patient an oncolytic adenovirus containing a polynucleotide sequence encoding hyaluronidase and optionally, inserted into its genome, which has a specific replication mechanism against tumor cells and a specific replication mechanism against tumor cells, wherein the patient is receiving or has received treatment with a topoisomerase I inhibitor or a prodrug thereof, for example ， Topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE).

[0110] In various aspects, this disclosure provides methods for treating or preventing colorectal cancer in patients of need, methods comprising administering to a patient an oncolytic adenovirus containing a polynucleotide sequence encoding hyaluronidase and an insertion into its genome that has a specific replication mechanism against tumor cells, wherein the patient is receiving or has received treatment with a topoisomerase I inhibitor or a prodrug thereof, for example ， Topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE).

[0111] In the implementation scheme, the method reduces or eliminates colonic or colorectal tumors or cancers, or their cells. In the implementation scheme, the method slows the growth and / or progression and / or metastasis of colonic or colorectal tumors or cancers, or their cells.

[0112] In the implementation scheme, colon or colorectal cancer is a primary or metastatic tumor. A primary tumor or cancer that migrates from its initial location and infects vital organs can ultimately lead to the subject's death through the deterioration of function in the affected organs. Metastasis refers to the spread of cancer cells or clusters of cancer cells from the primary tumor to other parts of the body, locations different from the primary tumor. Metastasis can ultimately lead to the subject's death. For example, in the implementation scheme, the colon or colorectal cancer of the present invention includes benign and malignant cancers, polyps, hyperplasia, and dormant tumors or micrometastases.

[0113] In the implementation plan, colon or colorectal cancer is colonic adenocarcinoma, gastrointestinal stromal tumor (GIST), squamous cell carcinoma, carcinoid, lymphoma, or sarcoma.

[0114] In the implementation plan, colon or colorectal cancer is defined as one of the following colon or colorectal cancer stages, if optionally assessed using the TNM system: Stage 0, characterized, for example, early-stage cancer of the innermost layer of the intestine; Stage I, characterized, for example, by the presence of cancer in the lining of the colon; Stage II is characterized, for example, by the cancer spreading to the muscular wall of the colon; Stage III, characterized, for example, by the cancer having spread to the lymph nodes; and / or Stage IV is characterized, for example, by the cancer spreading to other organs besides the colon.

[0115] In the implementation scheme, the method slows down or prevents progression from stage 0 to one of stages I, II, III, and IV.

[0116] In the implementation scheme, the method slows down or prevents progression from Phase I to one of Phase II, Phase III, or Phase IV.

[0117] In the implementation scheme, the method slows down or prevents progression from Phase II to either Phase III or Phase IV.

[0118] In the implementation scheme, the method slows down or prevents progression from stage III to stage IV.

[0119] In the implementation scheme, the method for treating colon or colorectal cancer is further combined with one or more of surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. In the implementation scheme, chemotherapy is one or more of 5-FU, leucovorin, oxaliplatin, capecitabine, regorafenib, trifluorouridine, and tipiracil. In the implementation scheme, chemotherapy is one or more of FOLFOX (5-FU, leucovorin, and oxaliplatin), CapeOx (capecitabine and oxaliplatin), or a regimen of 5-FU with leucovorin or capecitabine. In the implementation scheme, targeted therapy is one or more of an agent targeting VEGF (e.g., bevacizumab [Avastin], ziv-aflibercept [Zaltrap], or ramucirumab [Cyramza]) or an agent targeting EGFR (e.g., cetuximab [Erbitux] or panitumumab [Vectibix]). In the implementation plan, the immunotherapy is one or more of an agent that targets the PD-1 pathway (e.g., atezolizumab, durvalumab, cimiprimab, nivolumab, and pembrolizumab) and / or an agent that targets the CTLA-4 pathway (e.g., ipilimumab).

[0120] In the implementation scheme, the method for treating colon or colorectal cancer is further combined with ataxia-telangiectasia and Rad3-related (ATR) inhibitors (such as berzosertib). In the implementation scheme, the method for treating colon or colorectal cancer includes administration of topotecan and berzosertib.

[0121] Treatment of pancreatic cancer

[0122] In various respects, this disclosure provides methods for treating or preventing pancreatic cancer in patients of need, the methods comprising administering to the patient (i) an oncolytic adenovirus comprising a polynucleotide sequence encoding hyaluronidase and optionally inserted into its genome, which has a specific replication mechanism against tumor cells and a specific replication mechanism against tumor cells, and (ii) a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as but not limited to ONIVYDE).

[0123] In various respects, this disclosure provides methods for treating or preventing pancreatic cancer in patients of need, the methods comprising administering to the patient (i) an oncolytic adenovirus containing a polynucleotide sequence encoding hyaluronidase and an insertion into its genome that is specific to tumor cells and a specific replication mechanism for tumor cells, and (ii) a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as, but not limited to, ONIVYDE).

[0124] In various respects, this disclosure provides methods for treating or preventing pancreatic cancer in patients of need, methods comprising administering to a patient an oncolytic adenovirus containing a polynucleotide sequence encoding hyaluronidase and optionally, inserted into its genome, which has a specific replication mechanism against tumor cells and a specific replication mechanism against tumor cells, wherein the patient is receiving or has received treatment with a topoisomerase I inhibitor or a prodrug thereof, for example ， Topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE).

[0125] In various respects, this disclosure provides methods for treating or preventing pancreatic cancer in patients of need, methods comprising administering to a patient an oncolytic adenovirus containing a polynucleotide sequence encoding hyaluronidase and an insertion into its genome that has a specific replication mechanism against tumor cells, wherein the patient is receiving or has received treatment with a topoisomerase I inhibitor or a prodrug thereof, for example ， Topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE).

[0126] In one implementation, the method reduces or eliminates pancreatic tumors or cancer, or their cells. In another implementation, the method slows the growth and / or progression and / or metastasis of pancreatic tumors or cancer, or their cells.

[0127] In the implementation scheme, pancreatic cancer is a primary or metastatic tumor. Primary tumors or cancers that migrate from their initial location and inoculate vital organs can ultimately lead to the subject's death through functional deterioration of the affected organs. Metastasis refers to the spread of cancer cells or clusters of cancer cells from the primary tumor to other parts of the body, locations different from the primary tumor. Metastasis can ultimately lead to the subject's death. For example, in the implementation scheme, the pancreatic cancer of the present invention includes benign and malignant cancers, polyps, hyperplasia, and dormant tumors or micrometastases.

[0128] In the implementation plan, pancreatic cancer is an exocrine or endocrine carcinoma, or an adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, or colloid carcinoma.

[0129] In the implementation plan, pancreatic cancer is defined as one of the following pancreatic cancer stages, optionally assessed using the TNM system: Stage 0: characterized, for example, carcinoma in situ, wherein the cancer has not yet grown beyond the duct from which it originated (Tis, N0, M0); Stage IA: Characterized, for example, by a tumor in the pancreas that is 2 cm or smaller. It has not yet 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 yet 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 yet spread to nearby arteries, veins, lymph nodes, or other parts of the body (T3, N0, M0). Stage IIB: Characterized by, for example, that the tumor, regardless of size, has not yet spread to nearby arteries or veins. It has spread to 1 to 3 regional lymph nodes, but has not spread to other parts of the body (T1, T2, or T3; N1; M0). Stage III: Characterized by, for example, a tumor of any size that has spread to four or more regional lymph nodes, but has not spread to nearby arteries, veins, or other parts of the body (T1, T2, or T3, N2, M0), or a tumor that has spread to nearby arteries and veins and may have spread to regional lymph nodes, but has not yet spread to other parts of the body (T4, any N, M0); and Stage IV: characterized, for example, by any tumor that has spread to other parts of the body (any T, any N, M1).

[0130] In the implementation scheme, the method slows down or prevents progression from stage 0 to one of stage IA or IB, stage IIA or IIB, stage III, and stage IV.

[0131] In the implementation scheme, the method slows down or prevents progression from stage IA or IB to one of stage IIA or IIB, stage III, or stage IV.

[0132] In the implementation scheme, the method slows down or prevents progression from stage IIA or IIB to one of stage III or IV.

[0133] In the implementation scheme, the method slows down or prevents progression from stage III to stage IV.

[0134] In the implementation scheme, the method for treating pancreatic cancer is further combined with one or more of surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. In the implementation scheme, chemotherapy is one or more of gemcitabine, 5-FU, oxaliplatin, paclitaxel, capecitabine, and cisplatin. In the implementation scheme, 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 the implementation scheme, immunotherapy is one or more of agents targeting the PD-1 pathway (e.g., atezolizumab, durvalumab, cimiprimab, nivolumab, and pembrolizumab) and / or agents targeting the CTLA-4 pathway (e.g., ipilimumab).

[0135] In the implementation plan, methods for treating pancreatic cancer include the FOLFIRINOX (leucovorin, fluorouracil / 5FU, irinotecan and oxaliplatin) or NALIRIFOX (irinotecan, 5-FU, leucovorin, oxaliplatin) regimen.

[0136] Co-administration of oncolytic adenovirus and topoisomerase inhibitor

[0137] In one aspect, this disclosure provides a method for improving and / or increasing and / or enhancing antitumor efficacy in patients in need, the method comprising administering to a patient (i) an oncolytic adenovirus comprising a multinucleotide sequence encoding hyaluronidase that has a replication mechanism specific to tumor cells and optionally is inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as but not limited to ONIVYDE), and (iii) observing improved antitumor efficacy compared to treatment with a topoisomerase I inhibitor or a prodrug thereof without the use of an oncolytic adenovirus.

[0138] In one aspect, this disclosure provides a method for improving and / or increasing and / or enhancing antitumor efficacy in a patient in need, the method comprising administering to the patient (i) an oncolytic adenovirus comprising a replication mechanism specific to tumor cells and a polynucleotide sequence encoding hyaluronidase inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as but not limited to ONIVYDE), and (iii) observing the improved antitumor efficacy compared to treatment with a topoisomerase I inhibitor or a prodrug thereof without the use of an oncolytic adenovirus.

[0139] In one aspect, this disclosure provides a method for improving and / or increasing and / or enhancing antitumor efficacy in patients in need, the method comprising administering to a patient an oncolytic adenovirus comprising a replication mechanism specific to tumor cells and, optionally, an intercalation of a polynucleotide sequence encoding hyaluronidase into the patient's genome, wherein the patient is receiving or has received treatment with a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as, but not limited to, ONIVYDE), and observing improved antitumor efficacy compared to treatment with a topoisomerase I inhibitor or a prodrug thereof without the use of an oncolytic adenovirus.

[0140] In one aspect, this disclosure provides a method for improving and / or increasing and / or enhancing antitumor efficacy in patients in need, the method comprising administering to a patient an oncolytic adenovirus comprising a replication mechanism specific to tumor cells and a polynucleotide sequence encoding hyaluronidase inserted into the patient's genome, wherein the patient is receiving or has received treatment with a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as, but not limited to, ONIVYDE), and observing improved antitumor efficacy compared to treatment with a topoisomerase I inhibitor or a prodrug thereof without the use of an oncolytic adenovirus.

[0141] In the embodiments, the oncolytic adenovirus, topoisomerase I inhibitor, or prodrug thereof disclosed herein, such as topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as but not limited to ONIVYDE), are co-administered. In the embodiments, co-administration may occur simultaneously or sequentially.

[0142] In the implementation plan, the oncolytic adenovirus and a topoisomerase I inhibitor or a prodrug thereof, such as topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE), are administered to the subject simultaneously. As used herein, the term “simultaneously” means that the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof (e.g., topotecan, SN-38, or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE) are administered at time intervals not exceeding about 60 minutes, such as not exceeding about 30 minutes, not exceeding about 20 minutes, not exceeding about 10 minutes, not exceeding about 5 minutes, or not exceeding about 1 minute. The administration of oncolytic adenovirus and topoisomerase I inhibitors or their prodrugs (e.g., topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE)) can be performed by concurrent administration of a single formulation (e.g., a formulation containing oncolytic adenovirus and topoisomerase I inhibitors or their prodrugs, e.g., topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE)) or a single formulation (e.g., a first formulation containing oncolytic adenovirus and a second formulation containing a topoisomerase I inhibitor or its prodrug, e.g., topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE)).

[0143] In the implementation scheme, the oncolytic adenovirus and a topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE)) 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., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE) from their respective dosage forms (or a single unit dosage form if co-formulated) may occur sequentially.

[0144] If, for example, the timing of administration of an oncolytic adenovirus and a topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as but not limited to ONIVYDE) results in temporal overlap of the pharmacological activities of the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as but not limited to ONIVYDE)), co-administration does not require simultaneous administration. For example, an oncolytic adenovirus and a topoisomerase I inhibitor or its prodrug, such as topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as but not limited to ONIVYDE), may be administered sequentially or concurrently. As used herein, the terms “sequentially” and “simultaneously” mean that oncolytic adenovirus and a topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE)) are administered at intervals exceeding approximately 60 minutes. For example, the time interval between sequential administrations of oncolytic adenovirus and a topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE) may exceed approximately 60 minutes, approximately 2 hours, approximately 5 hours, approximately 10 hours, approximately 1 day, approximately 2 days, approximately 3 days, or approximately 1 week. The timing of administration will depend on the metabolic rate, excretion rate, and / or pharmacokinetic activity of the oncolytic adenovirus and topoisomerase I inhibitor or their prodrug (e.g., topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as but not limited to ONIVYDE)). The oncolytic adenovirus or topoisomerase I inhibitor or its prodrug, such as topotecan or SN-38, may be administered first.

[0145] In the implementation plan, oncolytic adenovirus is first administered, followed by administration of a topoisomerase I inhibitor or its prodrug, such as topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as but not limited to ONIVYDE), after 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 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks, approximately 8 weeks, approximately 9 weeks, approximately 10 weeks, approximately 11 weeks, or approximately 12 weeks.

[0146] In the implementation plan, a topoisomerase I inhibitor or its prodrug, such as topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE), is first administered, followed by oncolytic adenovirus at 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, approximately 11 weeks, or approximately 12 weeks.

[0147] In the implementation plan, co-administration does not require administering the oncolytic adenovirus and topoisomerase I inhibitor or their prodrug, such as topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE), to the subject via the same route of administration. Instead, in the implementation plan, each therapeutic agent may be administered via any appropriate route, such as intravenous, intraventricular, intratumoral, intraperitoneal, oral, non-intraventricular, non-tumoral, non-peritoneal, or non-oral.

[0148] In embodiments, the administration of the compositions and formulations of the present invention comprising oncolytic adenovirus and / or a topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as, but not limited to, ONIVYDE)) may be combined with additional agents, such as one or more additional antitumor agents, including but not limited to one or more chemotherapeutic agents and / or one or more additional therapies conventionally used for the treatment or prevention of ovarian cancer, cervical cancer, lung cancer, colon or colorectal cancer, and / or pancreatic cancer. 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 the compositions and formulations of the present invention comprising oncolytic adenovirus and / or a topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as, but not limited to, ONIVYDE)). In embodiments, the additional agent may be administered separately from the compositions and formulations of the present invention, which comprise oncolytic adenovirus and / or topoisomerase I inhibitors or prodrugs thereof, such as topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as, but not limited to, ONIVYDE). In embodiments, the additional agent is administered concurrently with the compositions and formulations of the present invention comprising oncolytic adenovirus and / or topoisomerase I inhibitors or prodrugs thereof (e.g., topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as, but not limited to, ONIVYDE)). In embodiments, the additional agent is administered prior to the administration of the compositions and formulations of the present invention comprising oncolytic adenovirus and / or topoisomerase I inhibitors or prodrugs thereof (e.g., topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as, but not limited to, ONIVYDE)). In embodiments, an additional agent is administered after the application of the compositions and formulations of the present invention comprising oncolytic adenovirus and / or a topoisomerase I inhibitor or a prodrug thereof (e.g., topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as, but not limited to, ONIVYDE)). In embodiments, the compositions and formulations of the present invention comprising oncolytic adenovirus and / or a topoisomerase I inhibitor or a prodrug thereof (e.g., topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as, but not limited to, ONIVYDE)) may be used alone in treatment regimens for the treatment or prevention of ovarian cancer, cervical cancer, lung cancer, colon or colorectal cancer, and / or pancreatic cancer. Right now No other antitumor agents and / or other conventional therapies are required.

[0149] Furthermore, the compositions / formulations of the present invention may contain additional agents (e.g., via co-formulation). For example, additional agents may be combined with oncolytic adenovirus and / or topoisomerase I inhibitors or their prodrugs (e.g., topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE)) into a single formulation. Alternatively, additional agents and oncolytic adenovirus and / or topoisomerase I inhibitors or their prodrugs, such as topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE), may be formulated separately.

[0150] Oncolytic adenovirus

[0151] This disclosure relates in part to pharmaceutical compositions, formulations, and uses of one or more oncolytic adenoviruses.

[0152] As used in this document, "oncolytic adenovirus" and its plural form refer to adenoviruses that are capable of self-replication or replication in tumor cells. Oncolytic adenoviruses are different from non-replicating adenoviruses because the latter cannot self-replicate in target cells.

[0153] adenovirus

[0154] In the embodiments, the oncolytic adenovirus used in this disclosure is an oncolytic adenovirus having a replication mechanism and a capsid that allows infection and replication in human cancer cells. In the embodiments, the oncolytic adenovirus is generated from an adenovirus that infects humans. Examples of adenoviruses that infect humans include, but are not limited to, human adenovirus serotypes 1 to 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 adenoviruses.

[0155] The 51 identified human adenovirus serotypes were divided into six groups, from A to F. Human adenovirus serotype 5 (Ad5), belonging to group C, is a virus formed from an icosahedral protein capsid containing 36 kcal baseline deoxyribonucleic acid (DNA). In adults, Ad5 infection is usually asymptomatic, while in children it causes the common cold and conjunctivitis. Ad5 typically infects epithelial cells, specifically bronchial epithelial cells during natural infection. It enters the cell via fibrils (viral proteins that extend like antennas from the twelve vertices of the capsid) interacting with cellular proteins involved in intercellular adhesion, called the Coxsackie-adenovirus receptor (CAR). Once the viral DNA reaches the cell nucleus, it begins systematic transcription of the early viral genes (E1 to E4). The first viral gene to be expressed corresponds to the early region gene 1A (E1A). E1A binds to retinoblastoma cellular proteins to release E2F, thereby activating the transcription of other viral genes, such as E2, E3, E4, and cellular genes that activate the cell cycle. In itself, E1B binds to the p53 protein to activate the cell cycle and prevent apoptosis in infected cells. E2 encodes viral replication proteins; E3 encodes proteins that suppress antiviral immune responses; and E4 encodes proteins that transport viral RNA. Expression of early genes leads to viral DNA replication, and once this is replicated, the major late promoter is activated, resulting in the expression of messenger RNA (RNA) transcripts, which, through cleavage and differential splicing, generate all the RNA encoding the structural proteins that form the capsid.

[0156] In the implementation scheme, the oncolytic adenovirus used in this disclosure is generated from human adenovirus serotype 5.

[0157] Hyaluronidase

[0158] In one embodiment, the oncolytic adenovirus used in this disclosure contains a sequence encoding hyaluronidase inserted into its genome. In another embodiment, the oncolytic adenovirus used in this disclosure does not contain a sequence encoding hyaluronidase inserted into its genome.

[0159] Hyaluronidase is a class of enzymes responsible for degrading hyaluronic acid. In the human species, six genes encoding hyaluronidase have been located to date, each with distinct characteristics and locations. Isotypes Hyal1 and Hyal2 are present in most tissues, with Hyal1 being the predominant form in human plasma. Hyal3 is located in the bone marrow and testes, but its function remains unclear. Hyaluronidase PH20 is highly expressed in the testes and is involved in the fertilization process of oocytes with sperm. Hyaluronidase PH20 is anchored to the sperm cell membrane and the inner acrosome membrane, giving sperm the ability to penetrate the extracellular matrix of cumulus cells (which is rich in hyaluronic acid) and reach the zona pellucida of the oocyte. During the acrosome reaction, some hyaluronidases anchored to the sperm membrane are enzymatically processed to produce a soluble form of protein, which is released from the acrosome membrane. The membrane protein PH20 is the only enzyme in the mammalian hyaluronidase family that is active at neutral pH.

[0160] Unwilling to be bound by theory, it is believed that hyaluronidase expression in oncolytic adenoviruses can help degrade the dense physical and immunosuppressive matrix barrier surrounding solid tumors, thereby ensuring better penetration of the tumor by the oncolytic adenovirus and co-administered therapies. This ensures that the adenovirus can reach and infect a large number of tumor cells. Degrading the tumor matrix can also expose tumor neoantigens, thereby stimulating an anti-tumor immune response in the patient's immune system.

[0161] In this embodiment, the hyaluronidase is mammalian testicular hyaluronidase, optionally human hyaluronidase (e.g., GenBank gene ID: 6677), also known as SPAM1 or sperm adhesion molecule 1 or PH20. In this embodiment, the hyaluronidase sequence has a sequence corresponding to that of an enzyme from which a membrane-binding carboxyl-terminal domain is deleted, such that the enzyme is soluble. When the carboxyl-terminal domain is deleted, the resulting enzyme is secreted into the extracellular environment.

[0162] In the implementation scheme, the sequence encoding hyaluronidase inserted into the oncolytic adenovirus genome is SEQ ID NO:1, wherein nucleotides 1471 to 1527 corresponding to the carboxyl-terminal domain are deleted.

[0163] Furthermore, in the implementation scheme, the hyaluronidase is a hyaluronidase variant. Compared to the parental wild-type sequence, the hyaluronidase variant has at least one or more amino acid modifications, typically amino acid substitutions. In embodiments, the hyaluronidase of this disclosure comprises an amino sequence having 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%, 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%) sequence identity with any sequence disclosed herein. Furthermore, in the implementation scheme, the hyaluronidase variant retains most or all of its biochemical activity, as measured by any suitable method known in the art.

[0164] In embodiments, the hyaluronidase of this disclosure comprises an amino sequence having 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%, 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%) sequence identity with the amino acid encoded by SEQ ID NO: 1.

[0165] In the embodiments, the hyaluronidase of this disclosure comprises having at least about 60% of the amino acids encoded by SEQ ID NO: 1 (wherein nucleotides 1471 to 1527 corresponding to the carboxyl-terminal domain are deleted). (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%, or about 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%) amino sequences with sequence identity.

[0166] In embodiments, the hyaluronidase of this disclosure comprises an amino sequence having 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%, 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%) sequence identity with the amino acid encoded by SEQ ID NO: 9.

[0167] In an embodiment, the hyaluronidase of this disclosure comprises an amino sequence having 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%, 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%) sequence identity with SEQ ID NO: 8.

[0168] In embodiments, the hyaluronidase of this disclosure comprises an amino sequence having 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%, 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%) sequence identity with SEQ ID NO: 10.

[0169] In the embodiments, the oncolytic adenovirus used in this disclosure comprises one or more regulatory elements, modifications, or variants that provide tumor-specific enzyme expression or viral replication.

[0170] In this embodiment, hyaluronidase expression is controlled by a promoter operated on in tumor cells. Alternatively, in this embodiment, hyaluronidase expression is controlled by a promoter operated on in ovarian cancer, cervical cancer, lung cancer, colon or colorectal cancer, and / or pancreatic cancer cells. Optionally, in this embodiment, enzyme expression is controlled by a promoter operated on in animal cells. Optionally, in this embodiment, the promoter is selected from cytomegalovirus promoters, adenovirus major late promoters, SV40 promoters, herpes simplex virus thymidine kinase promoters, RSV promoters, EF1-α promoters, β-actin promoters, human IL-2 promoters, human IL-4 promoters, IFN promoters, E2F promoters, human GM-CSF promoters, or combinations thereof.

[0171] In one implementation, the promoter regulating enzyme expression may be naturally present in the adenovirus, as is the case with the major late promoter of adenovirus. In another implementation, the promoter may be inserted along with the sequence encoding the enzyme. In yet another implementation, the promoter is the major late promoter of the adenovirus and is already located in the oncolytic adenovirus genome. In yet another implementation, it is not necessary to introduce the promoter along with the hyaluronidase sequence; instead, the latter is introduced into the oncolytic adenovirus genome so that it remains under the control of the promoter.

[0172] In embodiments, the oncolytic adenovirus used in this disclosure includes additional sequences that allow for the translation of proteins encoding hyaluronidase sequences that facilitate or optimize the translation of such proteins. In embodiments, the sequences are located inside or outside the hyaluronidase gene. For example, in embodiments, the additional sequences are selected from cutting and splicing sequences that allow RNA processing, IRES (internal ribosome entry site) sequences, microRNA virus sequence 2A, or combinations thereof.

[0173] It has a specific replication mechanism for tumor cells.

[0174] In embodiments, the tumor cell-specific replication mechanism of the oncolytic viruses used in this disclosure is a mechanism that causes the oncolytic adenovirus to self-replicate in a specific form in tumor cells rather than in healthy, non-tumor, or normal cells. In embodiments, the mechanism may take different forms if it provides the oncolytic adenovirus with the ability to replicate only or primarily in tumor cells (e.g., ovarian cancer, cervical cancer, lung cancer, colon or colorectal cancer, and / or pancreatic cancer). In other words, in embodiments, the adenoviruses exhibit selective replication, wherein their replication mechanism requires viral entry into cancer or tumor cells for the virus to self-replicate. In embodiments, the oncolytic adenoviruses used may have modifications in their genomic sequence that impart selective replication to them in tumor cells.

[0175] In this implementation, it is achieved by incorporating a tissue-specific promoter or a tumor-specific promoter, wherein the promoter controls the expression of one or more genes in the E1a, E1b, E2, and E4 groups. In this implementation, the promoter is selected from E2F promoters, telomerase hTERT promoters, tyrosinase promoters, prostate-specific antigen (PSA) promoters, alpha-fetoprotein promoters, COX-2 promoters, and artificial promoters formed from various transcription factor binding sites, such as binding sites for hypoxia-inducible factor (HIF-1), ETS transcription factor, tumor cytotoxic factor (TCF), E2F transcription factor, or Sp1 transcription factor. In this implementation, the promoter controls the expression of E1a.

[0176] In the implementation plan, the treatment is the treatment of tumors with an aberrant Rb-E2F pathway. In healthy, non-tumor, or normal cells, the E1A protein expressed by adenovirus dissociates the host cell Rb-E2F complex, allowing free E2F protein to drive both cell division and viral replication. In tumor cells with an aberrant Rb-E2F mechanism, free E2F allows for continuous cell division (and viral replication) without initiation by viral E1A. Therefore, genetic modifications that inhibit normal viral E1A expression prevent viral replication in healthy, non-tumor, or normal cells while allowing viral replication in tumor cells. Almost all solid tumors have an aberrant Rb-E2F pathway, including but not limited to ovarian cancer, cervical cancer, lung cancer, colon or colorectal cancer, and / or pancreatic cancer. In the embodiments, the oncolytic adenovirus used in this disclosure is characterized by the deletion of the Rb-binding domain or the deletion of Δ24, which affects the interaction between E1a and retinoblastoma proteins, and the insertion of four E2F-1 binding sites and one Sp1 binding site into the endogenous E1a promoter to control E1a expression. The DNA sequence corresponds to SEQ ID NO:2.

[0177] Furthermore, in embodiments, it is envisioned that the oncolytic adenovirus used in this disclosure comprises one or more modifications in its capsid, thereby allowing for improved biodistribution of the oncolytic adenovirus and reduced clearance of the oncolytic adenovirus from the body. In embodiments, the capsid of the oncolytic adenovirus used in this disclosure is modified to reduce its isolation and destruction in the liver, such that the heparin-binding domain KKTK present in the adenoviral fibrils is replaced, for example, by the domain RGDK. The modification relates to positions 91 to 94 of the adenoviral fibrils, with reference to the standard sequence of adenoviral serotype 5 fibrils. Sequence SEQ ID NO: 4 shows the complete sequence of adenoviral serotype 5 fibrin, whose heparin-binding domain has a modified version (modified RGDK).

[0178] Therefore, in the embodiments, the oncolytic adenovirus used in this disclosure is generated from human adenovirus serotype 5 and comprises: A sequence encoding hyaluronidase is inserted into its genome, optionally a human testicular hyaluronidase sequence (PH20, SEQ ID NO: 1), wherein the sequence corresponding to the membrane-bound carboxyl-terminal domain has been deleted to make the enzyme soluble, optionally the sequence encoding hyaluronidase having the amino acid sequence of SEQ ID NO: 10. Oncolytic adenovirus replication occurs in tumor cells with an aberrant Rb-E2F pathway, but not in healthy, non-tumor, or normal cells; optionally, the oncolytic adenovirus contains a deletion of the Rb-binding domain or Δ24 deletion, which affects the interaction between E1a and Rb proteins, and the insertion of four E2F-1 binding sites and one Sp1 binding site into the endogenous E1a promoter to control E1a expression; and / or The adenovirus capsid is modified to improve the biodistribution of oncolytic adenovirus and reduce the clearance of oncolytic adenovirus from the body; optionally, the heparin-binding domain KKTK present in the adenovirus fibrils has been replaced, for example, by the domain RGDK.

[0179] In an embodiment, the oncolytic adenovirus of this disclosure comprises a nucleotide sequence having 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%, 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%) sequence identity with the sequence of SEQ ID NO: 3. In the implementation scheme, the oncolytic adenovirus has the nucleotide sequence of SEQ ID NO: 3.

[0180] Method for preparing oncolytic adenovirus of this disclosure

[0181] In the embodiments, in order to construct the oncolytic adenovirus to be used in this disclosure, any method known in the fields of gene therapy and viral therapy using adenoviruses for constructing genetically modified adenoviruses is used. The most commonly used method is based on first constructing the desired genetic modification in a plasmid containing the region of the adenovirus to be modified, and then performing homologous recombination in bacteria with a plasmid containing the remainder of the viral genome. Those skilled in the art will understand that, due to the degeneracy of the genetic code, the protein sequences described herein can be encoded by any number of possible nucleic acid sequences.

[0182] In embodiments, nucleic acids encoding components of this disclosure may be incorporated into oncolytic adenoviruses as known in the art, and, depending on the host cell, for the production of hyaluronidases of this disclosure. Typically, the nucleic acid is operatively linked to any number of regulatory elements (promoters, origins of replication, selectable markers, ribosome binding sites, inducers, etc.).

[0183] In the embodiments, the oncolytic adenovirus used in this disclosure is propagated and amplified in cell lines commonly used in the fields of gene therapy and viral therapy, such as cell lines HEK-293 (reference number: ATCC CRL-1573) and A549 (reference number: ATCC CCL185). In the embodiments, the adenovirus is propagated by infecting cell lines that allow adenovirus replication. The lung adenocarcinoma cell line A549 is an example of a cell line with such characteristics. In the embodiments, propagation is performed, for example, as follows: A549 cells are grown on plastic cell culture plates and infected with 100 virus particles / cell. Two days later, a cytopathic effect reflecting viral production as cell aggregation can be observed. In the embodiments, the cells are collected and stored in tubes. In the embodiments, after centrifugation at approximately 1,000 g for approximately 5 minutes, the cell pellet is frozen and thawed three times to lyse the cells. In one 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 another embodiment, the viral bands obtained from the gradient are reloaded onto another cesium chloride gradient and centrifuged at approximately 35,000 g for approximately 16 hours. In yet another embodiment, the viral bands are collected and dialyzed in PBS-10% glycerol. In yet another embodiment, the viral dialysate is aliquoted and stored at approximately -80°C. The number of plaque-forming units and particles can be quantified according to standard protocols known in the art. Phosphate-buffered saline (PBS) containing 5% glycerol is a standard preparation for storing adenovirus and is used in this embodiment. However, new preparations for improving viral stability have been described and are used in this embodiment.

[0184] Topoisomerase I inhibitors

[0185] Topoisomerase inhibitors can inhibit cell proliferation by, for example, preventing DNA replication, stimulating DNA damage, and inducing cell cycle arrest.

[0186] Topotecan is a semi-synthetic derivative of the cytotoxic alkaloid camptothecin. SN-38 is the active topoisomerase I-inhibiting metabolite of the prodrug irinotecan. Typically, topoisomerase I inhibitors cause cell cycle arrest in the S phase by stabilizing the complex between topoisomerase I and DNA, thereby inhibiting the rejoining of topoisomerase I-mediated single-strand DNA breaks and generating potentially lethal double-strand DNA breaks.

[0187] In the implementation plan, a topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE)) is administered in various ways to treat ovarian cancer, cervical cancer, lung cancer, colon or colorectal cancer and / or pancreatic cancer, including systemic, intra-arterial, intratumoral, intraperitoneal and / oral administration.

[0188] In the implementation scheme, for systemic administration, a topoisomerase I inhibitor or its prodrug, such as topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as but not limited to ONIVYDE), is administered intravenously or orally. The topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as but not limited to ONIVYDE)) then enters the bloodstream and is distributed throughout the body. Irinotecan is converted to the active topoisomerase I inhibitor SN-38 by the liver and tumor carboxylesterases.

[0189] In the implementation scheme, the topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE)) is administered intravenously, intra-arterially, intraperitoneally or orally.

[0190] In the implementation scheme, liposomal irinotecan (e.g., but not limited to ONIVYDE) is administered via intravenous infusion.

[0191] In the embodiments, compared with treatment using a topoisomerase I inhibitor or its prodrug without oncolytic adenovirus, the methods of this disclosure improve and / or increase and / or enhance antitumor efficacy. In the embodiments, the methods make patients suitable for combination therapy with more than one cancer treatment.

[0192] preparation

[0193] This disclosure provides compositions of oncolytic adenovirus and / or topoisomerase I inhibitors or their prodrugs (e.g., topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as, but not limited to, ONIVYDE)) in various formulations. Any oncolytic adenovirus and / or topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, such as, but not limited to, ONIVYDE)) described herein may be in the form of solutions, emulsions, suspensions, delayed-release formulations, sustained-release formulations, controlled-release formulations, liposomal formulations, or any other form suitable for parenteral or oral administration.

[0194] It should be understood that the compositions used in this disclosure are used in a pharmaceutically acceptable form. This means that any oncolytic adenovirus and / or topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE)) described herein may be administered to a subject as a component of a composition comprising a pharmaceutically acceptable carrier or mediator. Such compositions may optionally contain a suitable amount of a pharmaceutically acceptable excipient to provide a form suitable for appropriate administration.

[0195] Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Examples of pharmaceutical excipients include saline, gum arabic, gelatin, starch paste, talc, keratin, silica gel, urea, etc. Additionally, adjuvants, stabilizers, thickeners, lubricants, and colorants may be used. In the implementation plan, pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any of the agents described herein is administered intravenously. Saline solutions, dextran solutions, and glycerol solutions can also be used as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, cellulose, hydroxypropyl methylcellulose, lactose, sucrose, trehalose, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, povidone, crospovidone, water, ethanol, etc. If desired, any agent described herein may also contain small amounts of wetting agents, emulsifiers, or pH buffers. Other examples of suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences 1447-1676 (edited by Alfonso R. Gennaro, 19th edition, 1995), which is incorporated herein by reference.

[0196] Application and dosage

[0197] It will be understood that the actual dose of the oncolytic adenovirus and / or topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE) to be administered according to this disclosure will vary based on various parameters, such as the size and stage of the tumor to be treated (ovarian, cervical, lung, colon or colorectal, and / or pancreatic cancer), the age and weight of the patient to be treated, the specific dosage form, and the method of administration. It should be understood that the dose of the oncolytic adenovirus and the co-administered topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE)) must be sufficient to ensure a positive therapeutic effect against ovarian, cervical, lung, colon or colorectal, and / or pancreatic cancer. In the implementation plan, a positive therapeutic effect on ovarian cancer, cervical cancer, lung cancer, colon or colorectal cancer and / or pancreatic cancer refers to the cessation of tumor growth, or reduction of tumor volume, or prevention of tumor infiltration into surrounding tissues, or prevention of metastasis.

[0198] In the implementation plan, for example, for ovarian cancer, the dose of topotecan is approximately 1.5 mg / m². 2 For example, by intravenous infusion for approximately 30 minutes per day for approximately 5 consecutive days, starting on day 1 of a 21-day cycle, until disease progression or unacceptable toxicity occurs.

[0199] In the implementation plan, for example for cervical cancer, the dose of topotecan is approximately 0.75 mg / m². 2 For example, by intravenous infusion over approximately 30 minutes daily on days 1, 2, and 3 of a 21-day cycle, such as with cisplatin (approximately 50 mg / m²) on day 1. 2 () combination, until the disease progresses or unacceptable toxicity occurs.

[0200] In the implementation plan, for example, for lung cancer, the dosage of topotecan is approximately 1.5 mg / m². 2 For example, by intravenous infusion for approximately 30 minutes daily for approximately 5 consecutive days, starting on day 1 of a 21-day cycle, until disease progression or unacceptable toxicity occurs.

[0201] In the implementation plan, topotecan can be administered orally for the treatment of lung cancer; for example, the oral topotecan capsule dose is 2.3 mg / m². 2 (Rounded to the nearest 0.25 mg), administered once daily for approximately 5 consecutive days, starting on day 1 of a 21-day cycle, until disease progression or unacceptable toxicity occurs.

[0202] In the implementation plan, for example for colon or colorectal cancer, the dose of topotecan is approximately 1.25 mg / m².2 Approximately 1.5 mg / m 2 For example, it can be administered intravenously over a period of approximately 5 days via an infusion over a 21-day cycle.

[0203] In the implementation plan, for example for pancreatic cancer, the dose of topotecan is approximately 1.25 mg / m². 2 Approximately 1.5 mg / m 2 For example, it can be administered intravenously over a period of approximately 5 days within a 21-day cycle. In one implementation method, for example for pancreatic cancer, it is administered every 3 weeks over approximately 30 minutes at approximately 350 mg / m². 2 Infusion of topotecan. In the administration of the drug, for example for pancreatic cancer, at approximately 100 mg / m² weekly. 2 Or every 2 weeks at 150 mg / m 2 Infusion of topotecan.

[0204] Those skilled in the art can consider numerous factors that can alter the effects of oncolytic adenoviruses and / or topoisomerase I inhibitors or their prodrugs (e.g., topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE)) (e.g., body weight, sex, diet, time of administration, route of administration, excretion rate, subject condition, drug combination, genetic predisposition, and response sensitivity). Administration may be continuous or in one or more discrete doses up to the maximum tolerated dose. Those skilled in the art can use routine assessments of dose administration and dose tolerability to determine the optimal rate of administration for a given set of conditions.

[0205] The compositions disclosed herein (e.g., compositions comprising the oncolytic adenovirus disclosed herein) are administered via any route that ensures the necessary amount of oncolytic adenovirus reaches the tumor site. In the cases of ovarian cancer, cervical cancer, lung cancer, colon or colorectal cancer, and / or pancreatic cancer, the compositions disclosed herein, such as those comprising the oncolytic adenovirus disclosed herein, are injected via any route that ensures the necessary amount of oncolytic adenovirus reaches the tumor site. Optional routes of administration include, but are not limited to, intravenous injection, intra-arterial injection, intratumoral injection, and / or intraperitoneal injection. Therefore, in embodiments, preferred compositions comprising the oncolytic adenovirus disclosed herein are used in suitable forms for intravenous, intra-arterial, intratumoral, and / or intraperitoneal administration or injection.

[0206] In the implementation scheme, oncolytic adenovirus and / or topoisomerase I inhibitors or their prodrugs (e.g., topotecan, SN-38 and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE)) may be administered, for example, more than once daily (e.g., about twice, about three, about four, about five, about six, about seven, about eight, about nine or about ten times daily), about once daily, about every other day, about every three days, about once weekly, about once every two weeks, about once monthly, about once every two months, about once every three months, about once every six months or about once a year.

[0207] definition

[0208] As used herein, “a / an” or “the” may mean one or more species.

[0209] Additionally, when used in conjunction with a reference figure, the term “about” means a reference figure plus or minus up to 10% of that reference figure. For example, the language “about 50%” covers a range of 45% to 55%.

[0210] When used in conjunction with medical purposes, "effective amount" is the amount that is effective in providing measurable treatment, prevention, or reduction of the incidence of a target condition.

[0211] As used herein, in the presence of an agent or stimulant, relative to the absence of such regulation, if the readings of activity and / or effect are reduced by a significant amount, such as 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 (up to and including at least about 100%), something is “reduced.” As those skilled in the art will understand, in embodiments, activity is reduced and some downstream readings will decrease, but others may increase.

[0212] Conversely, in the presence of an agent or stimulant, relative to the absence of such an agent or stimulant, if the readings of activity and / or effect increase by a significant amount, such as 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 most and including at least about 100% or more), at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 50 times, or at least about 100 times, then the activity is “increased”.

[0213] As mentioned herein, unless otherwise specified, all percentages of components are by weight of the total composition. As used herein, the word “comprising” and its variations are intended to be non-limiting, such that a detailed description of items in the list does not exclude other similar items that may also be applicable to compositions and methods of this technology. Similarly, the terms “can” and “may” and their variations are intended to be non-limiting, such that a detailed description of embodiments that may or may include certain elements or features does not exclude other embodiments of the invention that do not include those elements or features.

[0214] Although the open-ended term “comprising”, as a synonym for terms such as including, containing, or having, is used herein to describe and claim protection of this disclosure, alternative terms such as “consisting of” or “substantially consisting of” may be used to describe this disclosure or its embodiments.

[0215] As used herein, the terms "preferred" and "ideally" refer to embodiments of the present technology that provide certain benefits in certain circumstances. However, other embodiments may also be preferred in the same or other circumstances. Furthermore, the detailed description of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the present technology.

[0216] In the implementation plan, the terms "patient" and "subject" are used interchangeably. In the implementation plan, 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 the implementation plan, the subject and / or animal is a non-mammal, such as, for example, a zebrafish.

[0217] In one embodiment, the method of the present invention can be used to treat a human subject. In another embodiment, the person is an adult. In yet another embodiment, the person is an elderly person. In a further embodiment, the person may be referred to as a patient. In yet another embodiment, the person is female. In yet another embodiment, the person is male.

[0218] In the implementation plan, the age of the person is in the range of about 1 to about 18 months, about 18 to about 36 months, about 1 to about 5 years, about 5 to about 10 years, about 10 to about 15 years, about 15 to about 20 years, about 20 to about 25 years, about 25 to about 30 years, about 30 to about 35 years, about 35 to about 40 years, about 40 to about 45 years, about 45 to about 50 years, about 50 to about 55 years, about 55 to about 60 years, about 60 to about 65 years, about 65 to about 70 years, about 70 to about 75 years, about 75 to about 80 years, about 80 to about 85 years, about 85 to about 90 years, about 90 to about 95 years, or about 95 to about 100 years.

[0219] The amount of each component in the composition described herein required to achieve the therapeutic effect may be empirically determined according to routine procedures for a specific purpose. Typically, therapeutic agents (e.g., oncolytic adenoviruses and / or topoisomerase I inhibitors or their prodrugs (e.g., topotecan, SN-38, and / or irinotecan (e.g., but not limited to liposomal irinotecan, e.g., but not limited to ONIVYDE)), compositions) are administered for therapeutic purposes at a pharmacologically effective dose. “Pharmacologically effective dose,” “therapeuticly effective dose,” “effective dose,” or “effective dose” means an amount sufficient to produce the desired physiological effect or to achieve the desired outcome (particularly the treatment or prevention of a condition or disease). As used herein, an effective dose includes amounts sufficient, for example, to delay the development of symptoms of a condition or disease, to alter the course of symptoms of a condition or disease (e.g., to slow the progression of disease symptoms), to reduce or eliminate one or more symptoms or manifestations of a condition or disease, and to reverse the symptoms of a condition or disease. Therapeutic benefits also include stopping or slowing the progression of an underlying disease or condition, whether or not improvement is achieved.

[0220] Effective doses, toxicity, and therapeutic efficacy can be determined in cell cultures, tissue samples, tissue homogenates, or laboratory animals using standard pharmaceutical procedures, such as determining the LD50 (the dose lethal to approximately 50% of the population) and ED50 (the dose therapeutically effective in approximately 50% of the population) or the maximum tolerated dose. Dosage can vary depending on the dosage form used and the route of administration employed. The dose ratio between toxicity and therapeutic effect is the therapeutic index and can be expressed as the ratio LD50 / ED50. In embodiments, compositions and methods exhibiting a large therapeutic index are preferred. The therapeutically effective dose can be initially assessed by in vitro assays (including, for example, cell culture assays or measurements of methane production in fecal samples). Additionally, doses can be formulated in animal models to achieve a range of circulating plasma concentrations, including the IC50 as determined in cell cultures or in suitable animal models. The levels of the composition in plasma can be measured, for example, by high-performance liquid chromatography. The effect of any particular dose can be monitored by appropriate bioassays. The dose can be determined by a physician and adjusted as necessary to suit the observed therapeutic effect.

[0221] In the implementation plan, the effect will result 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 the implementation plan, the effect will result in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or greater. Treatment benefits also include stopping or slowing the progression of the underlying disease or condition, whether or not improvement is achieved.

[0222] As used herein, the term "treatment method" also applies to the use of compositions for treating the diseases or conditions described herein and / or to the manufacture of medicaments for treating the diseases or conditions described herein.

[0223] Example

[0224] Example 1: Effect of topotecan on VCN-01 replication in cell lines derived from lung cancer and ovarian cancer

[0225] VCN-01 replication + / - topotecan was evaluated in A549 adherent and suspension cells, as well as in two ovarian cancer cell lines, PA-1 and SW-626. Readings included viral particles (vp) obtained by qPCR at three time points: 4 h (injection), 48 h, and 72 h post-infection, and infection titers (transduction units, TU) obtained by hexagonal staining. See also Figure 1 .

[0226] The study design included evaluations of topoisomerase I inhibitors, topotecan, irinotecan, the irinotecan metabolite SN-38, cell cycle inhibitors, hydroxyurea, and other agents, including carboplatin and melphalan. Topotecan and SN-38 were expected to enhance VCN-01 replication in these cell lines. Gemcitabine was used as a negative control because it has been shown to block adenovirus replication in vitro.

[0227] Example 2: Pancreatic tumor cell lines' response to topoisomerase I (topoI) inhibitors, irinotecan (IRI), and their active ingredients. Sex metabolites, SN-38 and topotecan, (Topo) in vitro Sensitivity test

[0228] A diverse group of human pancreatic tumor cell lines were tested to evaluate the cytotoxicity of IRI and Topo as single agents. Human pancreatic cell lines (such as BxPC3 (ATCC-CRL1687), Panc-I (ATCC CRL 1469), NP-9, or NP-18) could be infected with human adenovirus, and VCN-01 virus replication was demonstrated in these cell lines. The HP-1 hamster pancreatic tumor cell line was also tested to assess its sensitivity to topoI inhibitors.

[0229] The concentrations (IC50 values) of SN-38, IRI, or Topo required to halve the cell culture viability of each cell line were determined. For this purpose, tumor cells (15,000 cells / well) were seeded in 96-well plates and maintained at 37°C and 5% CO2 in Dulbecco modified Eagle medium (DMEM) containing 5% fetal bovine serum. After 24 hours, serial dilutions of each chemotherapeutic agent (IRI, SN-38, or Topo) in DMEM + 5% FBS were prepared, and the chemotherapeutic medium was added to the wells in quadruplicates. Cells were incubated for an additional 5 days under standard culture conditions, followed by washing with PBS once and staining for total protein content using a commercial dioctanine assay (BCA; Pierce Biotechnology) (4 μl Reagent A + 196 μl Reagent B per well). Absorbance (A540 nm) was quantified after incubation at 37°C for 30 minutes. Using Prism software, standard nonlinear regression was employed to estimate the concentration (IC50 value) of each chemotherapeutic agent required to reduce protein mass by half compared to untreated wells from the dose-response curves. At least three replicates were performed for each cell line. Results were presented in... Figures 2A to 2E as well as Figures 3A to 3E As shown in Table 1.

[0230] Table 1. Mean IC50 values ​​for SN-38, IRI, and Topo pancreatic cancer cell lines. Final IC50 50 The value is calculated by averaging each repetition.

[0231]

[0232] Example 3: Analysis of NP-18 and NP-9 human pancreas exposed to VCN-01 plus topoI inhibitors SN-38, IRI, and Topo Assessment of viral (E1A) and cellular (E2F1) gene expression in cancer cell lines

[0233] Cells were loaded at 2 × 10 6 Cells were seeded at a density of 100 cells / well in 6-well plates. 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-fold of the previously determined IC50. The topoI concentrations used for NP-18 cells were: 0.1 μmol SN-38, 200 μmol IRI, and 2 μmol Topo. For NP-9 cells, these concentrations were 2 μmol SN-38, 1500 μmol IRI, and 0.5 μmol Topo. Cell pellets were collected 24 hours post-exposure, and RNA was isolated using the Qiagen RNAeasy Mini Kit and treated with DNase. RNA was quantified and reverse transcribed, followed by qPCR specific for E1A or E2F1. E1A and E2F1 RNA levels were normalized to human β-actin control levels.

[0234] like Figure 4 As shown, in NP-18 cells, viral E1A mRNA expression was approximately 4-6 times higher with the use of a topoI inhibitor compared to cells infected with VCN-01 alone. In NP-9 cells, E1A expression was greater than 10 times higher with the use of SN-38 and IRI compared to cells infected with VCN-01, and approximately 2 times higher with the use of TopoI. Similarly, E2F1 mRNA levels were 2-3 times higher in NP-18 cells with a topoI inhibitor compared to untreated cells, and 1-3 times higher in NP-9 cells with a topoI inhibitor.

[0235] While we do not wish to be bound by theory, these data are consistent with the following view: topoI inhibitors increase the expression of the cellular transcription factor E2F1, thereby activating the modified VCN-01 E1A early viral promoter, leading to increased expression of viral genes.

[0236] Example 4: VCN-01+ liposomal irinotecan in an immunodeficient mouse model of human pancreatic cancer NP-18 Evaluation of its anti-tumor efficacy

[0237] The aim of this study was to evaluate the antitumor efficacy of the combination of VCN-01 and liposomal irinotecan (nal-IRI; ONIVYDE) against immunodeficient nude mice carrying human pancreatic NP-18 subcutaneous tumors. Liposome irinotecan is a component of the NALIRIFOX chemotherapy mixture, which consists of liposomal irinotecan + 5-fluorouracil (5-FU) + leucovorin + oxaliplatin.

[0238] Nude mice with athymus (n=54, 6 groups, 8 male mice + 6 additional animals per group) were injected subcutaneously with a 25G needle at a dose of 4×10⁻⁶ mg / L. 6 One NP-18 cell per side (0.2 ml cell suspension in PBS) was subcutaneously seeded into each side of the animal. After implantation, tumor growth was monitored twice weekly by palpation, and the length and width of the tumor nodules were measured using calipers. Once the tumor reached 150 mm... 3 Then treat the animals as instructed ( Figure 5 On day 1, six groups of mice (n=8 per group, 16 tumors in total) were treated with VCN-01 or PBS via intravenous injection via tail vein. On days 3, 10, and 17, animals received a specified dose of PBS or Na-IRI via intravenous injection. Animal body weight and tumor growth were measured weekly. At the end of the study (day 37), animals were sacrificed, blood was collected via cardiac puncture, and specified hematological and biochemical parameters in the serum were analyzed. Tumors were collected on day 37 for vial genomic (DNA) and E1A mRNA qPCR analysis.

[0239] The animals' weight was measured throughout the study. Figure 6 Animals treated with VCN-01 and VCN-01 + naI-IRI initially showed weight loss. One animal in the VCN-01 + naI-IRI (10 mg / kg) group was euthanized due to severe weight loss (>30%). All other animals recovered by the end of the study.

[0240] 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, respectively) showed significantly reduced tumor growth compared to the PBS group. Animals treated with combination therapy of VCN-01 + naI-IRI (at doses of 5 mg / kg and 10 mg / kg, respectively) showed significantly reduced tumor growth compared to either VCN-01 alone or naI-IRI alone. There was no significant difference in tumor growth between the two combination therapy groups: VCN-01 + naI-IRI (5 mg / kg) and VCN-01 + naI-IRI (10 mg / kg).

[0241] Hematological and biochemical results (data not shown). Blood was collected from each animal at the end of the study (day 37) for hematological and biochemical analysis. Hematological parameters assessed included white blood cells, red blood cells, platelets, hemoglobin, lymphocytes, monocytes, neutrophils, eosinophils, and basophils. High-dose naI-IRI treatment alone (10 mg / kg) or in combination with VCN-01 showed a significant reduction in platelets. Thrombocytopenia is a known side effect of irinotecan. Other hematological parameters did not differ significantly between the PBS group and the treatment group. Biochemical parameters tested included ALT, AST, alkaline phosphatase, albumin, amylase, GGT, lipase, total bilirubin, total protein, creatinine, and urea. Combination therapy (VCN-01 + naI-IRI, regardless of high or low naI-IRI dose) significantly increased alkaline phosphatase. Other biochemical parameters assessed did not differ significantly between the treatment groups.

[0242] Tumor samples were collected at the end of the study (day 37). The presence of VCN-01 viral genome (vgs) in isolated tumor DNA was assessed by qPCR, and the presence of viral E1A mRNA in tumor RNA was analyzed by reverse transcription and qPCR. Figure 8 Vgs were detected in tumors of animals treated with VCN-01, indicating that VCN-01 remained in the tumors throughout the study and suggesting persistent viral replication. Tumor vgs were significantly reduced in animals treated with VCN-01 + naI-IRI (5 mg / kg) compared to VCN-01 alone, and there was no significant difference in tumor vgs in animals treated with VCN-01 + naI-IRI 10 mg / kg compared to VCN-01 alone. E1A mRNA analysis revealed the presence of E1A mRNA in all animals treated with VCN-01, indicating persistent viral transcription. No differences in E1A mRNA levels were observed between the VCN-01 treatment groups.

[0243] While we do not wish to be bound by theory, these data are consistent with the following view: the topoI inhibitor irinotecan (naI-IRI) increases the expression of the cellular transcription factor E2F1, thereby activating the modified VCN-01 E1A early viral promoter, leading to increased expression of viral genes and improved anti-tumor efficacy.

[0244] Example 5: Effects of VCN-01+ liposomal irinotecan on the immunodeficient mouse model of human pancreatic cancer NP-9. Evaluation of anti-tumor efficacy

[0245] The aim of this study was to evaluate the antitumor efficacy of the combination of VCN-01 and liposomal irinotecan (nal-IRI; ONIVYDE) against immunodeficient mice carrying human pancreatic NP-9 subcutaneous tumors.

[0246] Athymic nude mice (n=54, 6 groups, 8 female mice + 6 additional animals per group) were injected subcutaneously with a 25G needle at 7.5×10⁻⁶ mg / L. 6 One NP-9 cell per side (0.2 ml cell suspension in PBS) was subcutaneously injected into both sides of the animal. Tumor growth was monitored twice weekly by palpation after implantation. Once the tumor reached 150 mm... 3 The animals will be treated as instructed. Figure 9 On day 1, six groups of mice (n=8 per group, totaling 16 tumors) were treated with VCN-01 or PBS via intravenous injection via tail vein. On days 3, 10, and 17, animals were administered a specified dose of PBS or Na-IRI via intravenous injection. Animal body weight and tumor growth were measured weekly. At the end of the study, animals were sacrificed, blood was collected via cardiac puncture, and serum hematological and biochemical parameters were analyzed. Tumors were collected at the end of the study for vial genomic (DNA) and E1A mRNA qPCR analysis.

[0247] Compared to PBS alone, antitumor efficacy is expected to be observed for each of the following treatments: VCN-01, 5 mg / kg naI-IRI, and 2.5 mg / kg naI-IRI. Improved antitumor efficacy is expected for the combination therapy of VCN-01 + naI-IRI. Efficacy is expected for the combination therapy of high-dose (5 mg / kg) naI-IRI and low-dose (2.5 mg / kg naI-IRI).

[0248] By incorporating via reference

[0249] All patents and publications cited in this article are hereby incorporated in their entirety by reference.

[0250] The publications discussed herein are provided only with respect to their public disclosure prior to the date of this application. Nothing herein should be construed as an admission that this disclosure is not authorized to precede such publications due to prior disclosures.

[0251] As used herein, all headings are for organization purposes only and are not intended to limit this disclosure in any way. The content of any single section is equally applicable to all sections.

[0252] Implementation Plan

[0253] The embodiments listed below provide various additional embodiments of this disclosure, which can be combined in any number and in any combination.

[0254] Implementation Scheme 1. A method for treating or preventing ovarian cancer, cervical cancer, lung cancer, colon or colorectal cancer and / or pancreatic cancer in a patient in need, the method comprising co-administering to the patient (i) an oncolytic adenovirus comprising a replication mechanism specific to tumor cells and, optionally, a polynucleotide sequence encoding hyaluronidase inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug thereof.

[0255] Implementation Scheme 2. The method according to Implementation Scheme 1, wherein the oncolytic adenovirus is administered intravenously, intra-arterially, intratumorally, and / or intraperitoneally, for example by injection.

[0256] Implementation Scheme 3. The method according to Implementation Scheme 1 or 2, wherein the topoisomerase I inhibitor or its prodrug is administered via intraperitoneal, intravenous, intraarterial, intratumoral, and / or oral administration.

[0257] Implementation Scheme 4. The method according to any one of Implementation Schemes 1 to 3, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug are formulated into separate compositions.

[0258] Implementation Scheme 5. The method according to any one of Implementation Schemes 1 to 3, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug are formulated into a single composition.

[0259] Implementation Scheme 6. The method according to any one of Implementation Schemes 1 to 5, wherein the individual compositions are applied simultaneously or concurrently.

[0260] Implementation Scheme 7. The method according to any one of Implementation Schemes 1 to 6, wherein the oncolytic adenovirus is first administered, 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.

[0261] Implementation Scheme 8. The method according to Implementation Scheme 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 the administration of the oncolytic adenovirus.

[0262] Implementation Scheme 9. The method according to any one of Implementation Schemes 1 to 6, wherein the topoisomerase I inhibitor or its prodrug is first administered, and 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 after the administration of the oncolytic adenovirus, and / or wherein the oncolytic adenovirus is administered within approximately 30 minutes, approximately 20 minutes, approximately 10 minutes, approximately 5 minutes, or approximately 1 minute after the administration of the topoisomerase I inhibitor or its prodrug.

[0263] Implementation Scheme 10. The method according to any one of Implementation Schemes 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 type); (c) the lung cancer is small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, large cell lung carcinoma, mesothelioma, and / or lung carcinoid; (d) the colon or colorectal cancer is colonic adenocarcinoma, gastrointestinal stromal tumor (GIST), squamous cell carcinoma, carcinoid, lymphoma, or sarcoma. In the implementation scheme, and / or (e) the pancreatic cancer is exocrine carcinoma or endocrine carcinoma, or adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, or colloid carcinoma.

[0264] Implementation Scheme 11. The method according to any one of Implementation Schemes 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, optionally ONIVYDE.

[0265] Implementation Scheme 12. The method according to any one of Implementation Schemes 1 to 11, wherein the hyaluronidase is human hyaluronidase PH20.

[0266] Implementation Scheme 13. The method according to Implementation Scheme 12, wherein the sequence encoding hyaluronidase is SEQ ID NO: 9 or a sequence having at least about 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 with it.

[0267] Implementation Scheme 14. The method according to any one of Implementation Schemes 1 to 13, wherein the oncolytic adenovirus is generated from human adenovirus serotype 5.

[0268] Implementation Scheme 15. The method according to any one of Implementation Schemes 1 to 14, wherein oncolytic adenovirus replication occurs in tumor cells with an abnormal Rb-E2F pathway, rather than in healthy, non-tumor, or normal cells.

[0269] Implementation Scheme 16. The method according to Implementation Scheme 15, wherein the oncolytic adenovirus is engineered to replicate in tumor cells, rather than in healthy, non-tumor, or normal cells, by deleting the Rb-binding domain or Δ24 deletion in the sequence encoding the E1a protein and inserting four binding sites with E2F-1 and one binding site with Sp1 into the endogenous promoter of E1a to control the expression of E1a.

[0270] Implementation Scheme 17. The method according to any one of Implementation Schemes 1 to 16, wherein the capsid of the oncolytic adenovirus is modified such that the heparin-binding domain present in the adenovirus fibers is... 91 KKTK 94 (SEQ ID NO: 6) Optional ground cover domain 91 RGDK 94 (SEQ ID NO: 7) Replacement.

[0271] Implementation Scheme 18. The method according to any one of Implementation Schemes 1 to 17, wherein the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3).

[0272] Implementation Scheme 19. The method according to any one of Implementation Schemes 1 to 18, wherein the patient is a human patient.

[0273] Implementation Scheme 20. The method according to Implementation Scheme 19, wherein the human patient is a female human patient.

[0274] Implementation Scheme 21. The method according to any one of Implementation Schemes 1 to 20, wherein the ovarian cancer, cervical cancer, lung cancer, colon or colorectal cancer and / or pancreatic cancer is resistant to conventional chemotherapy and / or radiotherapy.

[0275] Implementation Scheme 22. The method according to any one of Implementation Schemes 1 to 21, wherein the method improves and / or increases and / or enhances the antitumor efficacy compared with treatment using the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

[0276] Implementation Scheme 23. The method according to any one of Implementation Schemes 1 to 22, wherein, compared with treatment using the topoisomerase I inhibitor or its prodrug without using the oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies or recurrent diseases associated with ovarian cancer.

[0277] Implementation Scheme 24. The method according to any one of Implementation Schemes 1 to 22, wherein, compared with treatment using the topoisomerase I inhibitor or its prodrug without using the oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies or recurrent diseases associated with cervical cancer.

[0278] Implementation Scheme 25. The method according to any one of Implementation Schemes 1 to 22, wherein, compared with treatment using the topoisomerase I inhibitor or its prodrug without using the oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of lung cancer-related metastases, secondary malignancies or recurrent diseases.

[0279] Implementation Scheme 26. The method according to any one of Implementation Schemes 1 to 22, wherein, compared with treatment using the topoisomerase I inhibitor or its prodrug without using the oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies or recurrent diseases associated with colon or colorectal cancer.

[0280] Implementation Scheme 27. The method according to any one of Implementation Schemes 1 to 22, wherein, compared with treatment using the topoisomerase I inhibitor or its prodrug without using the oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies or recurrent diseases associated with pancreatic cancer.

[0281] Implementation Scheme 28. A method for treating or preventing ovarian cancer, cervical cancer, lung cancer, colon or colorectal cancer and / or pancreatic cancer in a patient in need, the method comprising administering to the patient an oncolytic adenovirus comprising a replication mechanism specific to tumor cells and, optionally, an intercalation into the patient’s genome of a polynucleotide sequence encoding hyaluronidase, wherein the patient is receiving or has received treatment with a topoisomerase I inhibitor or a prodrug thereof.

[0282] Implementation Scheme 29. The method according to Implementation Scheme 28, wherein the oncolytic adenovirus is administered intravenously, intra-arterially, intratumorally, and / or intraperitoneally, for example by injection.

[0283] Implementation Scheme 30. The method according to Implementation Scheme 28 or 29, wherein the topoisomerase I inhibitor or its prodrug is administered via intraperitoneal, intravenous, intraarterial, intratumoral, and / or oral administration.

[0284] Implementation Scheme 31. The method according to any one of Implementation Schemes 28 to 30, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug are formulated into separate compositions.

[0285] Implementation Scheme 32. The method according to any one of Implementation Schemes 28 to 30, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug are formulated into a single composition.

[0286] Implementation Scheme 33. The method according to any one of Implementation Schemes 28 to 32, wherein the oncolytic adenovirus is first administered, and the topoisomerase I inhibitor or its prodrug is administered within approximately 12 weeks after the administration of the oncolytic adenovirus, or wherein the topoisomerase I inhibitor or its prodrug is first administered, and the oncolytic adenovirus is administered within approximately 12 weeks after the administration of the topoisomerase I inhibitor or its prodrug.

[0287] Implementation Scheme 34. The method according to Implementation Scheme 33, wherein 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 after administration.

[0288] Implementation Scheme 35. The method according to any one of Implementation Schemes 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.

[0289] Implementation Scheme 36. The method according to any one of Implementation Schemes 28 to 35, wherein the hyaluronidase is human hyaluronidase PH20.

[0290] Implementation Scheme 37. The method according to Implementation Scheme 36, wherein the sequence encoding hyaluronidase is SEQ ID NO: 9 or a sequence having at least about 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 with it.

[0291] Implementation Scheme 38. The method according to any one of Implementation Schemes 28 to 37, wherein the oncolytic adenovirus is generated from human adenovirus serotype 5.

[0292] Implementation Scheme 39. The method according to any one of Implementation Schemes 28 to 38, wherein oncolytic adenovirus replication occurs in tumor cells with an abnormal Rb-E2F pathway, rather than in healthy, non-tumor, or normal cells.

[0293] Implementation Scheme 40. The method according to Implementation Scheme 39, wherein the oncolytic adenovirus is engineered to replicate in tumor cells, rather than in healthy, non-tumor, or normal cells, by deleting the Rb-binding domain or Δ24 deletion in the sequence encoding the E1a protein and inserting four binding sites with E2F-1 and one binding site with Sp1 into the endogenous promoter of E1a to control the expression of E1a.

[0294] Implementation Scheme 41. The method according to any one of Implementation Schemes 28 to 40, wherein the capsid of the oncolytic adenovirus is modified such that heparin sulfate binding domains present in the adenovirus fibers are... 91 KKTK 94 (SEQ ID NO: 6) Optional ground cover domain 91 RGDK 94 (SEQ ID NO: 7) Replacement.

[0295] Implementation Scheme 42. The method according to any one of Implementation Schemes 28 to 41, wherein the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3).

[0296] Implementation Scheme 43. The method according to any one of Implementation Schemes 28 to 42, wherein the patient is a human patient.

[0297] Implementation Scheme 44. The method according to Implementation Scheme 43, wherein the human patient is a female human patient.

[0298] Implementation Scheme 45. The method according to any one of Implementation Schemes 28 to 44, wherein the ovarian cancer, cervical cancer, lung cancer, colon or colorectal cancer and / or pancreatic cancer is resistant to conventional chemotherapy and / or radiotherapy.

[0299] Implementation Scheme 46. The method according to any one of Implementation Schemes 28 to 45, wherein the method improves and / or increases and / or enhances antitumor efficacy compared to treatment using the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

[0300] Implementation Scheme 47. The method according to any one of Implementation Schemes 28 to 46, wherein, compared with treatment using the topoisomerase I inhibitor or its prodrug without using the oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies or recurrent diseases associated with ovarian cancer.

[0301] Implementation Scheme 48. The method according to any one of Implementation Schemes 28 to 46, wherein, compared with treatment using the topoisomerase I inhibitor or its prodrug without using the oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies or recurrent diseases associated with cervical cancer.

[0302] Implementation Scheme 49. The method according to any one of Implementation Schemes 28 to 46, wherein, compared with treatment using the topoisomerase I inhibitor or its prodrug without using the oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of lung cancer-related metastases, secondary malignancies or recurrent diseases.

[0303] Implementation Scheme 50. The method according to any one of Implementation Schemes 28 to 46, wherein, compared with treatment using the topoisomerase I inhibitor or its prodrug without using the oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies or recurrent diseases associated with colon or colorectal cancer.

[0304] Implementation Scheme 51. The method according to any one of Implementation Schemes 28 to 46, wherein, compared with treatment using the topoisomerase I inhibitor or its prodrug without using the oncolytic adenovirus, the method results in reduced or maintained tumor size and / or prevention or reduction of metastases, secondary malignancies or recurrent diseases associated with pancreatic cancer.

[0305] Implementation Scheme 52. The method according to any one of Implementation Schemes 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 a NALIRIFOX (irinotecan, 5-FU, leucovorin, oxaliplatin) regimen.

[0306] Implementation Scheme 53. The method according to any one of Implementation Schemes 1 to 22, 27, 28 to 46 or 51, wherein the prodrug of the topoisomerase I inhibitor is a liposomal irinotecan, optionally ONIVYDE, which is administered as part of a NALIRIFOX (irinotecan, 5-FU, leucovorin, oxaliplatin) regimen.

[0307] Implementation Scheme 54. The method according to any one of Implementation Schemes 1 to 22, 27, 28 to 46, 51 or 53, wherein the prodrug of the topoisomerase I inhibitor is a liposomal irinotecan, optionally ONIVYDE, which is administered by intravenous infusion.

[0308] Implementation Scheme 55. The method according to any one of Implementation Schemes 1 to 54, wherein, compared with treatment using the oncolytic adenovirus without using the topoisomerase I inhibitor or its prodrug, the method results in increased expression of the cellular transcription factor E2F1.

[0309] Implementation Scheme 56. The method according to any one of Implementation Schemes 1 to 55, wherein, compared with treatment using the oncolytic adenovirus without using the topoisomerase I inhibitor or its prodrug, the method results in increased expression of one or more viral genes.

[0310] sequence

[0311] SEQ ID NO: 1

[0312] The complete PH20 cDNA sequence containing the C-terminal domain. (From ATG up to the stop codon, both are included, with nt 1471-1527 underlined and bolded.)

[0313] gccacaatgttcattgttagtattttgtttcttatcatttct tctgtagcgagtttg taa

[0314] SEQ ID NO: 2

[0315] The endogenous promoter modified with E1a in VCN-01 + the sequence encoding the E1a-δ24 region (containing four binding sites with E2F-1, a binding site with Sp1, and the coding region of E1a-δ24).

[0316]

[0317] SEQ ID NO: 3

[0318] The complete sequence of VCN-01

[0319]

[0320] SEQ ID NO: 4

[0321] The modified amino acid sequence of the adenovirus serotype fibrils, in which RGDK modification was introduced.

[0322] MKRARPSEDTFNPVYPYDTETGPPTVPFLTPPFVSPNGFQESPPGVLSLRLSEPLVTSNGMLALKMGNGLSLDEAGNLTSQNVTTVSPPLRGDKSNINLEISAPLTVTSEALTVAAAAPLMVAGNTLTMGSQAPLTVHDSKLSI ATQGPLTVSEGKLALQTSGPLTTTDSSTLTITASPPLTTATGSLGIDLKEPIYTQNGKLGLKYGAPLHVTDDLNTLTVATGPGVTINNTSLGTKVTGALGFDSQGNMQLNVAGGLRIDSQNRRLILDVSYPFDAQNQLNLRLGQ GPLFINSAHNLDINYNKGLYLFTASNNSKKLEVNLSTAKGLMFDATAIAINAGDGLEFGSPNAPNTNPLKTKIIGHGLEFDSNKAMVPKLGTGLSFDSTGAITVGNKNNDKLTLWTTPAPSPNCDLNAEKDAKLTLVLTKCGSQ ILATVSVLAVKGSLAPISGTVQSAHLIIRFDENGVLLNNSFLDPEYWNFRNGDLTEGTAYTNAVGFMPNLSAYPKSHGKTAKSNIVSQVYLNGDKTKPVTLTITLNGTQETGDTTPSAYSMSFSWDWSGHNYINEIFATSSYTF

[0323] SEQ ID NO: 5

[0324] RGD-C peptide

[0325] CDCRGDCFC

[0326] SEQ ID NO: 6

[0327] Combined structural domain

[0328] KKTK

[0329] SEQ ID NO: 7

[0330] Combined structural domain

[0331] RGDK

[0332] SEQ ID NO: 8

[0333] Translation of SEQ ID NO: 1 (PH20 with carboxyl-terminal domain present)

[0334] MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKA KKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNE STALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQ EQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATMFIVSILFLIISSVASL-

[0335] SEQ ID NO: 9

[0336] cDNA encoding PH20 with a deleted C-terminal domain

[0337]

[0338] SEQ ID NO: 10

[0339] Translation of SEQ ID NO: 9 (PH20 with carboxyl-terminal domain deletion)

[0340] MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS-

Claims

1. 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, the method comprising co-administering to the patient (i) an oncolytic adenovirus comprising a replication mechanism specific to tumor cells and, optionally, a polynucleotide sequence encoding hyaluronidase inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug thereof.

2. The method of claim 1, wherein the oncolytic adenovirus is administered intravenously, intra-arterially, intratumorally, and / or intraperitoneally, for example by injection.

3. The method according to claim 1 or 2, wherein the topoisomerase I inhibitor or its prodrug is administered via intraperitoneal, intravenous, intraarterial, 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 into 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 into a single composition.

6. The method according to any one of claims 1 to 5, wherein the individual compositions are applied simultaneously or concurrently.

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

8. The method of claim 7, wherein the topoisomerase I inhibitor or its prodrug is administered within about 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes or about 1 minute after the 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 first administered, and the oncolytic adenovirus is administered within about 14 days, about 10 days, about 7 days, about 5 days, about 1 day, or about 60 minutes after the administration of the oncolytic adenovirus, and / or wherein the oncolytic adenovirus is administered within about 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes, or about 1 minute after the administration of the topoisomerase I inhibitor or its prodrug.

10. The method according to any one of claims 1 to 9, wherein: The pancreatic cancer is an exocrine or endocrine carcinoma, or an adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, or colloid carcinoma; The ovarian cancer mentioned refers to epithelial ovarian cancer, germ cell tumors, or stromal cell tumors. The cervical cancer mentioned is squamous cell carcinoma, adenocarcinoma, and / or adenosquamous carcinoma (mixed type); 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; and / or The colon or colorectal cancer mentioned is colonic adenocarcinoma, gastrointestinal stromal tumor (GIST), squamous cell carcinoma, carcinoid, lymphoma, or 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, optionally ONIVYDE.

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

13. The method of claim 12, wherein the sequence encoding hyaluronidase is SEQ ID NO: 9 or a sequence having at least about 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 with it.

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

15. The method according to any one of claims 1 to 14, wherein the oncolytic adenovirus replication occurs in tumor cells with an abnormal Rb-E2F pathway, rather than in healthy, non-tumor, or normal cells.

16. The method of claim 15, wherein the oncolytic adenovirus is engineered to replicate in tumor cells, rather than in healthy, non-tumor, or normal cells, by deleting the Rb-binding domain or Δ24 deletion in the sequence encoding the E1a protein and inserting four binding sites with E2F-1 and one binding site with Sp1 into the endogenous promoter of E1a to control the expression of E1a.

17. The method according to any one of claims 1 to 16, wherein the capsid of the oncolytic adenovirus is modified such that heparin-binding domains present in the adenovirus fibers are... 91 KKTK 94 (SEQ ID NO: 6) Optional ground cover domain 91 RGDK 94 (SEQ ID NO: 7) Replacement.

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 of 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, the ovarian cancer, the cervical cancer, the lung cancer and / or the colon 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 increases and / or enhances the antitumor efficacy compared to treatment using the topoisomerase I inhibitor or its prodrug without the oncolytic adenovirus.

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

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

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

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

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

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 a 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 a liposomal irinotecan, optionally ONIVYDE, which is administered as part of a 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 a liposomal irinotecan, optionally ONIVYDE, which is administered by intravenous infusion.

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

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

33. A method for treating or preventing pancreatic cancer in a patient in need, the method 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 wherein the prodrug of the topoisomerase I inhibitor is a liposomal irinotecan, optionally ONIVYDE, which is administered as part of a NALIRIFOX (irinotecan, 5-FU, leucovorin, oxaliplatin) regimen.

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

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