Oncolytic adenovirus and topoisomerase i inhibitors for use in treating a cancer

Abstract

The present disclosure relates, inter alia, to combination therapies of an oncolytic adenovirus with a topoisomerase I inhibitor or prodrug thereof, such as topotecan or SN-38, or a prodrug of a topoisomerase inhibitor, such as irinotecan, for treating or preventing ovarian, cervical, lung, colon or colorectal, and / or pancreatic cancer.

Description

[0001] ONCOLYTIC ADENOVIRUS AND TOPOISOMERASE I INHIBITORS OR PRODRUGS THEREOF

[0002] COMBINATION THERAPIES

[0003] TECHNICAL FIELD

[0004] The present disclosure relates, inter alia, to combination therapies of an oncolytic adenovirus with topotecan, irinotecan, or SN-38 for use in specific tumors.

[0005] CROSS-REFERENCE TO RELATED APPLICATIONS

[0006] This application claims the benefit of U.S. Provisional Application No. 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 in their entireties.

[0007] DESCRIPTION OF THE XML FILE SUBMITTED ELECTRONICALLY

[0008] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML file, created on September 19, 2024, is named "VCN-007_Sequence_Listing.xml” and is 53,098 bytes in size.

[0009] BACKGROUND

[0010] Combination therapy is a central tenet of cancer therapy. The combination of anti-cancer drugs may enhance efficacy compared to a monotherapy approach because it may target key pathways in a synergistic manner. This approach can potentially reduce resistance, while also providing therapeutic anti-cancer benefits, such as reducing tumor growth and limiting tumor metastatic potential.

[0011] New treatment strategies that provide efficient and effective results at an affordable cost, especially those that use current treatments in combination, are needed.

[0012] SUMMARY

[0013] Accordingly, in aspects, the disclosure provides methods for treating or preventing pancreatic, ovarian, cervical, lung, and / or colon or colorectal pancreatic cancer, in a patient in need thereof, comprising coadministering to the patient (i) an oncolytic adenovirus comprising replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and (ii) a topoisomerase I inhibitor or a prodrug of a topoisomerase inhibitor. In embodiments, the oncolytic adenovirus is administered by intravenous, intraarterial, intratumoral, and / or intraperitoneal administration, e.g. by injection. In embodiments, the topoisomerase I inhibitor or a prodrug thereof is administered by intraperitoneal, intravenous, intraarterial, or intratumoral administration, e.g. by injection and / or oral administration.

[0014] In embodiments, the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are formulated in separate compositions. In embodiments, the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are formulated in a single composition. In embodiments, the separate compositions are administered simultaneously or contemporaneously.

[0015] In embodiments, the oncolytic adenovirus is administered first and the topoisomerase I inhibitor or a prodrug thereof is administered within about 60 minutes of the administration of the oncolytic adenovirus. In embodiments, the topoisomerase I inhibitor or a prodrug thereof is administered within about 30 minutes, within about 20 minutes, within about 10 minutes, within about 5 minutes, or within about 1 minute of the administration of the oncolytic adenovirus. In embodiments, the topoisomerase I inhibitor or a prodrug thereof is administered first and the oncolytic adenovirus is administered within about 60 minutes of the administration of the oncolytic adenovirus. In embodiments, the oncolytic adenovirus is administered within about 30 minutes, within about 20 minutes, within about 10 minutes, within about 5 minutes, or within about 1 minute of the administration of the topoisomerase I inhibitor or a prodrug thereof.

[0016] In embodiments, the oncolytic adenovirus is administered first and the topoisomerase I inhibitor or a prodrug thereof is administered within about 1 day of the administration of the oncolytic adenovirus. In embodiments, the topoisomerase I inhibitor or a prodrug thereof is administered within about 14 days, within about 10 days, within about 7 days, within about 5 days, or within about 1 day of the administration of the oncolytic adenovirus. In embodiments, the topoisomerase I inhibitor or a prodrug thereof is administered first and the oncolytic adenovirus is administered within about 1 day of the administration of the oncolytic adenovirus. In embodiments, the oncolytic adenovirus is administered within about 14 days, within about 10 days, within about 7 days, within about 5 days, or within about 1 day of the administration of the topoisomerase I inhibitor or a prodrug thereof.

[0017] In embodiments, the topoisomerase I inhibitor is topotecan, or SN-38, or the SN-38 prodrug irinotecan, which is converted to SN-38 in vivo.

[0018] In embodiments, the oncolytic adenovirus optionally comprises a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome. In embodiments, the hyaluronidase enzyme is the human hyaluronidase enzyme PH20. In embodiments, the sequence that encodes a hyaluronidase enzyme 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 thereto.

[0019] In embodiments, the oncolytic adenovirus is generated from a human adenovirus serotype 5.

[0020] In embodiments, oncolytic adenoviral replication occurs in tumor cells with an aberrant Rb-E2F pathway (e.g. a solid tumor) and not in healthy, non-tumor, or normal cells. In embodiments, the oncolytic adenovirus is engineered to replicate in tumor cells and not healthy, non-tumor, or normal cells by deletion of the Rb binding domain in the sequence coding for the E1a protein and the insertion of four binding sites to E2F-1 and one binding site to Sp1 into the endogenous promoter of E1 a to control the expression of E1 a.

[0021] In embodiments, oncolytic adenoviral replication occurs in tumor cells with an aberrant Rb-E2F pathway (e.g. a solid tumor) and not in healthy, non-tumor, or normal cells. In embodiments, the oncolytic adenovirus is engineered to replicate in tumor cells and not healthy, non-tumor, or normal cells by deletion A24 in the sequence coding for the E1 a protein and the insertion of four binding sites to E2F-1 and one binding site to Sp1 into the endogenous promoter of E1 a to control the expression of E1a.

[0022] In embodiments, the oncolytic adenovirus has the capsid modified such that the binding domain91KKTK94(SEQ ID NO: 6) of the heparan sulfates present in the adenovirus fibre has been replaced.

[0023] In embodiments, the oncolytic adenovirus has the capsid modified such that the binding domain91KKTK94(SEQ ID NO: 6) of the heparan sulfates present in the adenovirus fibre has been replaced by the domain91RGDK94(SEQ ID NO: 7).

[0024] In embodiments, the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3), or a functional variant thereof.

[0025] In embodiments, the patient is a human patient. In embodiments, the human patient is a female human patient.

[0026] In embodiments, the pancreatic, ovarian, cervical, lung, and / or colon or colorectal cancer is resistant to conventional chemotherapy and / or radiotherapy treatment.

[0027] In embodiments, the method improves and / or increases and / or enhances antitumor efficacy compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus. In embodiments, the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with ovarian cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus. In embodiments, the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with cervical cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus. In embodiments, the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with lung cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus. In embodiments, the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with colon or colorectal cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus. In embodiments, the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with pancreatic cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0028] In aspects, the disclosure provides methods for treating or preventing pancreatic, ovarian, cervical, lung, and / or colon or colorectal, cancer, in a patient in need thereof, comprising administering to the patient an oncolytic adenovirus comprising replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is undergoing or has undergone treatment with a topoisomerase I inhibitor or a prodrug thereof.

[0029] In embodiments, the oncolytic adenovirus is administered by intravenous, intraarterial, intratumoral, and / or intraperitoneal administration, e.g. by injection. In embodiments, the topoisomerase I inhibitor or a prodrug thereof is administered by intraperitoneal, intravenous, intraarterial, intratumoral, and / or oral administration.

[0030] In embodiments, the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are formulated in separate compositions.

[0031] In embodiments, the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are formulated in a single composition.

[0032] In embodiments, the oncolytic adenovirus is administered first and the topoisomerase I inhibitor or a prodrug thereof is administered within about 12 weeks of the administration of the oncolytic adenovirus. In embodiments, the topoisomerase I inhibitor or a prodrug thereof is administered within about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or about 11 weeks of the administration of the oncolytic adenovirus.

[0033] In embodiments, the topoisomerase I inhibitor or a prodrug thereof is administered first and the oncolytic adenovirus is administered within about 12 weeks of the administration of the topoisomerase I inhibitor or a prodrug thereof. In embodiments, the oncolytic adenovirus is administered within about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or about 11 weeks of the administration of the topoisomerase I inhibitor or a prodrug thereof.

[0034] In embodiments, the topoisomerase I inhibitor or prodrug thereof is topotecan, SN-38, and / or irinotecan.

[0035] In embodiments, the hyaluronidase enzyme is the human hyaluronidase enzyme PH20. In embodiments, the sequence that encodes a hyaluronidase enzyme 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 thereto.

[0036] In embodiments, the oncolytic adenovirus is generated from a human adenovirus serotype 5.

[0037] In embodiments, oncolytic adenoviral replication occurs in tumor cells with an aberrant Rb-E2F pathway and not in healthy, non-tumor, or normal cells. In embodiments, the oncolytic adenovirus is engineered to replicate in tumor cells and not healthy, non-tumor, or normal cells by a deletion of the Rb binding domain or the deletion A24 in the sequence coding for the E1a protein and the insertion of four binding sites to E2F-1 and one binding site to Sp1 into the endogenous promoter of E1 a to control the expression of E1 a.

[0038] In embodiments, the oncolytic adenovirus has the capsid modified such that the binding domain91KKTK94(SEQ ID NO: 6) of the heparan sulfates present in the adenovirus fibre has been replaced. In embodiments, the oncolytic adenovirus has the capsid modified such that the binding domain91KKTK94(SEQ ID NO: 6) of the heparan sulfates present in the adenovirus fibre has been replaced by the domain91RGDK94(SEQ ID NO: 7).

[0039] In embodiments, the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3), or a functional variant thereof.

[0040] In embodiments, the patient is a human patient. In embodiments, the human patient is a female human patient. In embodiments, the pancreatic, ovarian, cervical, lung, and / or colon or colorectal cancer is resistant to conventional chemotherapy and / or radiotherapy treatment.

[0041] In embodiments, the method improves and / or increases and / or enhances antitumor efficacy compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus. In embodiments, the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with ovarian cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus. In embodiments, the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with cervical cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus. In embodiments, the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with lung cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus. In embodiments, the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with colon or colorectal cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus. In embodiments, the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with pancreatic cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0042] In embodiments, the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with colon or colorectal cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus. In embodiments, the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with pancreatic cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0043] In embodiments, the ovarian cancer is an epithelial ovarian carcinoma, germ cell tumor, and / or stromal cell tumor. In embodiments, the cervical cancer is squamous cell carcinoma, adenocarcinoma, and / or adenosquamous (mixed) carcinoma. In embodiments, the lung cancer is small-cell lung cancer (also called oat-cell cancer), non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, large cell carcinoma of the lung, mesothelioma, and / or lung carcinoid tumors. In embodiments, the colon or colorectal cancer is an adenocarcinoma, gastrointestinal stromal tumor (GIST), squamous cell carcinoma, carcinoid tumor, lymphoma, or sarcoma of the colon. In embodiments, the pancreatic cancer is an exocrine or endocrine cancer, or an adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, or a colloid carcinoma.

[0044] BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 shows a schematic of a non-limiting study design evaluating the effects of topotecan or irinotecan + VCN-01 in a lung cancer or ovarian cancer model.

[0046] Figures 2A-2E show cytotoxicity results of SN-38, irinotecan (IRI), topotecan (T°P°) in apanel of pancreatic cancer cell lines, specifically NP-18 (Figure 2A), NP-9 (Figure 2B), PANC-1 (Figure 2C), and BxPC3 (Figure 2D) and HP-2 (Figure 2E). Cell survival curves after exposure to serial dilutions with drugs are represented. Cell survival was measured on day 5 by BCA assay (mean+SEM is represented). Cell survival curves were adjusted by a non-linear regression fit: log(inhibitor) vs. normalized response - Variable slope. N=3 experiments were performed in each cell line. Graphs show one representative experiment.

[0047] Figures 3A-3E show average IC50 values (n=3) in a panel of pancreatic cancer cell lines, specifically NP-18 (Figure 3A), NP-9 (Figure 3B), PANC-1 (Figure 3C), BxPC3 (Figure 3D), and HP-1 (Figure 3E). IC50 values of SN-38, IR, and Topo in each cell line. IC50 values were estimated by applying a nonlinear regression fit in cell viability curves. Three replicates were performed.

[0048] Figure 4 shows relative E1A and E2F1 mRNA levels in NP-18 and NP-9 cells. The mRNA levels are displayed relative to VCN-01 alone infected cells (E1A) or untreated cells (E2F1).

[0049] Figure 5 shows a schematic diagram of a non-limiting example of a mouse NP-18 tumor study.

[0050] Figure 6 shows percent body weight gain / loss of NP-18 tumor-bearing animals of the study described in Example 4.

[0051] Figure 7 shows tumor growth (%) of NP-18 tumor-bearing animals of the study described in Example 4.

[0052] Figure 8 shows quantification of VCN-01 viral genomes (vgs) and E1 A mRNA in tumors of the study described in Example 4. Tumors were collected at end of study, Day 37. Tumor DNA was isolated and subjected to qPCR to measure vgs and tumor RNA was isolated and subjected to qPCR to measure E1 A mRNA levels.

[0053] Figure 9 shows a schematic diagram of a non-limiting example of a NP-9 tumor study. DETAILED DESCRIPTION

[0054] Treatment of Ovarian Cancer

[0055] In aspects, the present disclosure provides methods of treating or preventing ovarian cancer in a patient in need thereof, comprising administering to the patient (i) an oncolytic adenovirus comprising replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, and (ii) a topoisomerase I inhibitor or prodrug thereof, e.g, topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE).

[0056] In aspects, the present disclosure provides methods of preventing, removing or reducing metastases, secondary malignancies, or recurrent disease associated with ovarian cancer, in a patient in need thereof, comprising administering to the patient (I) an oncolytic adenovirus comprising replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and (ii) a topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE).

[0057] In aspects, the disclosure provides methods for treating or preventing ovarian, cervical, lung, colon or colorectal, and / or pancreatic cancer, in a patient in need thereof, comprising administering to the patient an oncolytic adenovirus comprising replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is undergoing or has undergone treatment with a topoisomerase I inhibitor or a prodrug thereof.

[0058] In aspects, the present disclosure provides methods of preventing, removing or reducing metastases, secondary malignancies, or recurrent disease associated with ovarian cancer, in a patient in need thereof, comprising administering to the patient an oncolytic adenovirus comprising replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is undergoing or has undergone treatment with a topoisomerase I inhibitor or a prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE).

[0059] In embodiments, the method reduces or eliminates a ovarian tumor or cancer, or cells thereof. In embodiments, the method slows the growth and / or progression and / or metastasis of the ovarian tumor or cancer, or cells thereof.

[0060] In embodiments, the ovarian cancer is a primary or metastatic tumor. Primary tumors or cancers that migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. A metastasis is a cancer cell or group of cancer cells, distinct from the primary tumor location, resulting from the dissemination of cancer cells from the primary tumor to other parts of the body. Metastases may eventually result in death of a subject. For example, the present ovarian cancers, in embodiments, include benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases.

[0061] In embodiments, the ovarian cancer is an epithelial ovarian carcinoma, germ cell tumor, or stromal cell tumor.

[0062] In embodiments, the ovarian cancer is serous, papillary serous, metastatic, borderline, mucinous or clear cell.

[0063] In embodiments, the ovarian cancer is a grade 1 ovarian cancer characterized by, e.g.: (i) well- differentiated tissue; or (ii) low grade nuclei with infrequent mitotic figures.

[0064] In embodiments, the ovarian cancer is a stage I ovarian cancer characterized by, e.g. (i) a tumor limited to one ovary, capsule intact, no tumor on ovarian surface and negative washings (Stage IA); (ii) a tumor involving both ovaries, capsule intact, no tumor on ovarian surface and negative washings (Stage IB); (iii) surgical spill (Stage IC1); (iv) capsule rupture before surgery or tumor on ovarian surface (Stage IC2); or (v) malignant cells in ascites or in peritoneal washings (Stage IC3).

[0065] In embodiments, the ovarian cancer is a stage II ovarian cancer characterized by, e.g.: (i) extension and / or implant of a tumor on uterus and / or Fallopian tubes (Stage 11 A); or (ii) extension of a tumor to other pelvic intraperitoneal tissues (Stage IIB).

[0066] In embodiments, the ovarian cancer is a stage III ovarian cancer characterized by, e.g., spread outside the pelvis to the peritoneum and / or lymph nodes, e.g. in the back of the abdomen. In embodiments, the ovarian cancer is a stage III ovarian cancer characterized by, e.g., spread to the lymph nodes in the back of the abdomen (Stage 3A1); cancer cells in tissue samples from the lining of the peritoneum and / or the lymph nodes (Stage 3A2); cancer growths that are 2 cm or smaller in size on the lining of the peritoneum and / or the lymph nodes (Stage 3B); or cancer growths larger than 2 cm on the lining of the peritoneum (Stage 3C).

[0067] In embodiments, the ovarian cancer is a stage IV ovarian cancer, characterized by, e.g. advanced (metastatic) cancer {e.g. spread to liver or lungs).

[0068] In embodiments, the method slows or prevents the progression from stage I {e.g. IA, IB or IC) to one of stage II {e.g. I IA or IIB), stage III {e.g. II IA, III B, or III IC), and stage IV. In embodiments, the method slows or prevents the progression from stage II {e.g. IIA or 11 B) to one of stage III {e.g. IIIA, IIIB, or IIIIC), and stage IV.

[0069] In embodiments, the method slows or prevents the progression from stage III {e.g. IIIA, IIIB, or IIIIC) to stage IV.

[0070] In embodiments, the method for treatment ovarian cancer is in further combination with one or more of surgery, radiation therapy, chemotherapy, targeted therapy, and immunotherapy. In embodiments, the chemotherapy is one or more of a platinum-based agent {e.g., without limitation, cisplatin or carboplatin) and a taxane {e.g., without limitation, paclitaxel or docetaxel). In embodiments, the chemotherapy is one or more of cisplatin, carboplatin paclitaxel, docetaxel, nab-paclitaxel, altretamine, capecitabine, cyclophosphamide, etoposide, gemcitabine, ifosfamide, doxorubicin, melphalan, pemetrexed, and vinorelbine. In embodiments, the targeted therapy is an anti-angiogenesis therapy {e.g. bevacizumab or ramucirumab). In embodiments, the immunotherapy is one or more of an agent that targets the PD-1 pathway, e.g. atezolizumab, durvalumab, cemiplimab, nivolumab, and pembrolizumab and / or an agent that targets the CTLA-4 pathway, e.g. ipilimumab.

[0071] In embodiments, the ovarian cancer is metastatic ovarian cancer. In embodiments, the ovarian cancer is metastatic ovarian cancer after disease progression on or after initial or subsequent chemotherapy.

[0072] Treatment of Cervical Cancer

[0073] In aspects, the present disclosure provides methods of treating or preventing cervical cancer in a patient in need thereof, comprising administering to the patient (i) an oncolytic adenovirus comprising replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and replication machinery specific for tumor cells, and (ii) a topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan.

[0074] In aspects, the present disclosure provides methods of treating or preventing cervical cancer in a patient in need thereof, comprising administering to the patient (i) an oncolytic adenovirus comprising replication machinery specific for tumor cells and a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and replication machinery specific for tumor cells, and (ii) a topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE).

[0075] In aspects, the present disclosure provides methods of treating or preventing cervical cancer in a patient in need thereof, comprising administering to the patient an oncolytic adenovirus comprising replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and replication machinery specific for tumor cells, wherein the patient is undergoing or has undergone treatment with a topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan.

[0076] In aspects, the present disclosure provides methods of treating or preventing cervical cancer in a patient in need thereof, comprising administering to the patient an oncolytic adenovirus comprising replication machinery specific for tumor cells and a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and replication machinery specific for tumor cells, wherein the patient is undergoing or has undergone treatment with a topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g, by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE).

[0077] In embodiments, the method reduces or eliminates a cervical tumor or cancer, or cells thereof. In embodiments, the method slows the growth and / or progression and / or metastasis of the cervical tumor or cancer, or cells thereof.

[0078] In embodiments, the cervical cancer is a primary or metastatic tumors. Primary tumors or cancers that migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. A metastasis is a cancer cell or group of cancer cells, distinct from the primary tumor location, resulting from the dissemination of cancer cells from the primary tumor to other parts of the body. Metastases may eventually result in death of a subject. For example, the present cervical cancers, in embodiments, include benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases.

[0079] In embodiments, the cervical cancer is squamous cell carcinoma, adenocarcinoma, and / or adenosquamous (mixed) carcinoma.

[0080] In embodiments, the cervical cancer is any one of the following stages:

[0081] Stage I, characterized by, e.g., the cancer cells having grown from the surface of the cervix into deeper tissues of the cervix but cancer has not spread to nearby lymph nodes or distant sites. Stage I can be subdivided as:

[0082] IA: cancer only be visualized under a microscope;

[0083] I A1 : cancer only be visualized under a microscope and is less than 3 mm deep;

[0084] IA2: cancer only be visualized under a microscope and is between 3 mm and 5 mm deep;

[0085] IB: stage I cancer that has spread deeper than 5 mm but is still limited to the cervix;

[0086] IB1 : cancer is deeper than 5 mm but not more than 2 cm in size; I B2: cancer is at least 2 cm in size but not larger than 4 cm;

[0087] I B3: cancer is at least 4 cm in size and limited to the cervix;

[0088] Stage II, characterized by, e.g. the cancer having grown beyond the cervix and uterus, but not spread to the walls of the pelvis or the lower part of the vagina but cancer has not spread to nearby lymph nodes or distant sites. Stage II can be subdivided as:

[0089] 11 A: cancer has grown beyond the cervix and uterus but has not spread into the tissues next to the cervix (parametria);

[0090] IIA1 : cancer is not larger than 4 cm;

[0091] I IA2: cancer is 4 cm or larger;

[0092] I IB: cancer has grown beyond the cervix and uterus and has spread into the tissues next to the cervix (the parametria);

[0093] Stage III, characterized by, e.g., the cancer having spread to the lower part of the vagina or the walls of the pelvis. The cancer may be blocking the ureters. Cancer may have spread to nearby lymph nodes but has not spread to distant sites. Stage III can be subdivided as:

[0094] I II A: cancer has spread to the lower part of the vagina but not the walls of the pelvis.

[0095] IIIB: cancer has grown into the walls of the pelvis and / or is blocking one or both ureters causing hydronephrosis

[0096] NIC: cancer can be any size, imaging tests or a biopsy show the cancer has spread to nearby pelvic lymph nodes (IIIC1 ) or para-aortic lymph nodes (IIIC2); and

[0097] Stage IV, characterized by, e.g. the cancer having grown into the bladder or rectum or having grown in further away organs like the lungs or bones. Stage IV can be subdivided as:

[0098] IVA: cancer has spread to the bladder or rectum or it is growing out of the pelvis.

[0099] IVB: cancer has spread to distant organs outside the pelvic area, such as distant lymph nodes, lungs or bones.

[0100] In embodiments, the method slows or prevents the progression from stage I {e.g. IA or IB) to one of stage II {e.g. I IA or I IB), stage III {e.g. II IA, IIIB, or III IC), and stage IV {e.g. IVA or IVB).

[0101] In embodiments, the method slows or prevents the progression from stage II {e.g. IIA or 11 B) to one of stage III {e.g. IIIA, IIIB, or IIIIC), and stage IV {e.g. IVA or IVB). In embodiments, the method slows or prevents the progression from stage III {e.g. IIIA, III B, or IIIIC) to stage IV {e.g. IVA or IVB).

[0102] In embodiments, the cervical cancer is Stage IV-B, recurrent, or persistent cervical cancer.

[0103] In embodiments, the cervical cancer is metastatic cervical cancer. In embodiments, the cervical cancer is treated in further combination with cisplatin.

[0104] In embodiments, the method for treatment cervical cancer is in further combination with one or more of surgery, radiation therapy, chemotherapy, targeted therapy, and immunotherapy. In embodiments, the chemotherapy is one or more of cisplatin, carboplatin, paclitaxel, docetaxel, ifosfamide, 5-fl uorouracil (5- FU), gemcitabine, and mitomycin. In embodiments, the targeted therapy is an anti-angiogenesis therapy {e.g. bevacizumab or ramucirumab). In embodiments, the immunotherapy is one or more of an agent that targets the PD-1 pathway, e.g. atezolizumab, durvalumab, cemiplimab, nivolumab, and pembrolizumab and / or an agent that targets the CTLA-4 pathway, e.g. ipilimumab.

[0105] Treatment of Lung Cancer

[0106] In aspects, the present disclosure provides methods of treating or preventing lung cancer in a patient in need thereof, comprising administering to the patient (i) an oncolytic adenovirus comprising replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and replication machinery specific for tumor cells, and (ii) a topoisomerase I inhibitor or prodrug thereof, e.g, topotecan, SN-38, and / or irinotecan {e.g, by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE).

[0107] In aspects, the present disclosure provides methods of treating or preventing lung cancer in a patient in need thereof, comprising administering to the patient (i) an oncolytic adenovirus comprising replication machinery specific for tumor cells and a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and replication machinery specific for tumor cells, and (ii) a topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE).

[0108] In aspects, the present disclosure provides methods of treating or preventing lung cancer in a patient in need thereof, comprising administering to the patient an oncolytic adenovirus comprising replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and replication machinery specific for tumor cells, wherein the patient is undergoing or has undergone treatment with a topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g, by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE). In aspects, the present disclosure provides methods of treating or preventing lung cancer in a patient in need thereof, comprising administering to the patient an oncolytic adenovirus comprising replication machinery specific for tumor cells and a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and replication machinery specific for tumor cells, wherein the patient is undergoing or has undergone treatment with a topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g, by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE).

[0109] In embodiments, the method reduces or eliminates a lung tumor or cancer, or cells thereof. In embodiments, the method slows the growth and / or progression and / or metastasis of the lung tumor or cancer, or cells thereof.

[0110] In embodiments, the lung cancer is a primary or metastatic tumor. Primary tumors or cancers that migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. A metastasis is a cancer cell or group of cancer cells, distinct from the primary tumor location, resulting from the dissemination of cancer cells from the primary tumor to other parts of the body. Metastases may eventually result in death of a subject. For example, the present lung cancers, in embodiments, include benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases.

[0111] In embodiments, the lung cancer is small-cell lung cancer. In embodiments, the lung cancer is non-small cell lung cancer. In embodiments, the lung cancer is adenocarcinoma of the lung. In embodiments, the lung cancer is squamous carcinoma of the lung. In embodiments, the lung cancer is large cell carcinoma of the lung. In embodiments, the lung cancer is mesothelioma. In embodiments, the lung cancer is a lung carcinoid tumor.

[0112] In embodiments, the lung cancer is one of the following stages of lung cancer, as optionally assessed by the TNM system:

[0113] • occult stage, characterized, e.g., by cancer cells being present in a subject's mucus you cough up;

[0114] • stage 0, characterized, e.g., by small tumors and a lack of spread into deeper lung tissues or outside the lungs;

[0115] • stage I {e.g. IA, e.g. IA1 , IA2, IA3 and IB), characterized, e.g., by presence of cancer in lung tissues but not lymph nodes; • stage II {e.g. IIA or IIB), characterized, e.g., by presence of cancer in lung tissues and lymph nodes near the lungs;

[0116] • stage III {e.g. IIIA, IIIB, or IIIC), characterized, e.g., by presence of cancer in lung tissues and spread further into the lymph nodes and the middle of the chest; and / or

[0117] • stage IV, characterized, e.g, by presence of cancer in lung tissues spread widely in the body {e.g. to brain, bones, and / or liver).

[0118] In embodiments, the method slows or prevents the progression from occult stage to one of stage 0, stage I {e.g. IA, e.g. IA1 , IA2, IA3 and IB), stage II {e.g. IIA or IIB), stage III {e.g. IIIA, IIIB, or IIIC), and stage IV.

[0119] In embodiments, the method slows or prevents the progression from stage 0 to one of stage I {e.g. I A, e.g. IA1 , IA2, IA3 and IB), stage II {e.g. IIA or IIB), stage III {e.g. IIIA, IIIB, or IIIC), and stage IV.

[0120] In embodiments, the method slows or prevents the progression from stage I {e.g. IA, e.g. IA1, IA2, IA3 and IB) to one of stage II {e.g. IIA or IIB), stage III {e.g. IIIA, IIIB, or IIIC), and stage IV.

[0121] In embodiments, the method slows or prevents the progression from stage II {e.g. IIA or IIB) to one of stage III {e.g. IIIA, IIIB, or IIIC), and stage IV.

[0122] In embodiments, the method slows or prevents the progression from stage III {e.g. IIIA, IIIB, or IIIC) to stage IV.

[0123] In embodiments, the lung cancer is small-cell lung cancer or non-small-cell lung cancer.

[0124] In embodiments, the lung cancer is small-cell lung cancer with platinum-sensitive disease. In embodiments, the lung cancer is small-cell lung cancer with platinum-sensitive disease in a subject who progressed at least about 60 days after initiation of first-line chemotherapy.

[0125] In embodiments, the lung cancer is non-small-cell lung cancer with platinum-sensitive disease. In embodiments, the lung cancer is small-cell lung cancer with platinum-sensitive disease in a subject who progressed at least about 60 days after initiation of first-line chemotherapy.

[0126] In embodiments, the method for treatment lung cancer is in further combination with one or more of surgery, radiation therapy, chemotherapy, targeted therapy, and immunotherapy. In embodiments, the chemotherapy is one or more of carboplatin, cisplatin, docetaxel, etoposide, gemcitabine, nab-paclitaxel, paclitaxel, pemetrexed, and vinorelbine. In embodiments, the targeted therapy is one or more of epidermal growth factor receptor (EGFR) inhibitors {e.g. afatinib, dacomitinib, entrectinib, erlotinib, gefitinib, and osimertinib), agents targeting the EGFR exon 20 insertion {e.g. amivantamab or mobocertinib), agents targeting HER2 mutations {e.g. Fam-trastuzumab deruxtecan-nxki), anaplastic lymphoma kinase (ALK) inhibitors {e.g. alectinib, brigatinib, ceritinib, crizotinib, and lorlatinib), agents targeting R0S1 fusion {e.g. ceritinib, crizotinib, and entrectinib), agents targeting KRAS G12C mutations {e.g. sotorasib), agents targeting NTRK fusion {e.g. larotrectinib), agents targeting BRAF V600E mutations {e.g. dabrafenib or trametinib), agents targeting MET exon 14 skipping {e.g. capmatinib or tepotinib), agents targeting RET fusion {e.g. pralsetinib or selpercatinib), and anti-angiogenesis therapy {e.g. bevacizumab or ramucirumab). In embodiments, the immunotherapy is one or more of an agent that targets the PD-1 pathway, e.g. atezolizumab, durvalumab, cemiplimab, nivolumab, and pembrolizumab and / or an agent that targets the CTLA-4 pathway, e.g. ipilimumab.

[0127] Treatment of Colon or Colorectal Cancer

[0128] In aspects, the present disclosure provides methods of treating or preventing colon or colorectal cancer in a patient in need thereof, comprising administering to the patient (i) an oncolytic adenovirus comprising replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and replication machinery specific for tumor cells, and (ii) a topoisomerase I inhibitor or prodrug thereof, e.g, topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE).

[0129] In aspects, the present disclosure provides methods of treating or preventing colon or colorectal cancer in a patient in need thereof, comprising administering to the patient (i) an oncolytic adenovirus comprising replication machinery specific for tumor cells and a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and replication machinery specific for tumor cells, and (ii) a topoisomerase I inhibitor or prodrug thereof, e.g, topotecan, SN-38, and / or irinotecan {e.g, by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE).

[0130] In aspects, the present disclosure provides methods of treating or preventing colon or colorectal cancer in a patient in need thereof, comprising administering to the patient an oncolytic adenovirus comprising replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and replication machinery specific for tumor cells, wherein the patient is undergoing or has undergone treatment with a topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE).

[0131] In aspects, the present disclosure provides methods of treating or preventing colon or colorectal cancer in a patient in need thereof, comprising administering to the patient an oncolytic adenovirus comprising replication machinery specific for tumor cells and a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and replication machinery specific for tumor cells, wherein the patient is undergoing or has undergone treatment with a topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g, by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE).

[0132] In embodiments, the method reduces or eliminates a colon or colorectal tumor or cancer, or cells thereof. In embodiments, the method slows the growth and / or progression and / or metastasis of the colon or colorectal tumor or cancer, or cells thereof.

[0133] In embodiments, the colon or colorectal cancer is a primary or metastatic tumor. Primary tumors or cancers that migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. A metastasis is a cancer cell or group of cancer cells, distinct from the primary tumor location, resulting from the dissemination of cancer cells from the primary tumor to other parts of the body. Metastases may eventually result in death of a subject. For example, the present colon or colorectal cancers, in embodiments, include benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases.

[0134] In embodiments, the colon or colorectal cancer is an adenocarcinoma, gastrointestinal stromal tumor (GIST), squamous cell carcinoma, carcinoid tumor, lymphoma, or sarcoma of the colon.

[0135] In embodiments, the colon or colorectal cancer one of the following stages of colon or colorectal cancer, as optionally assessed by the TNM system:

[0136] • stage 0, characterized, e.g, early cancer on the innermost layer of the intestine;

[0137] • stage I, characterized, e.g., by presence of cancer in the inner layers of the colon;

[0138] • stage II, characterized, e.g, by cancer which has spread through the muscle wall of the colon;

[0139] • stage III, characterized, e.g., by cancer which has spread to the lymph nodes; and / or

[0140] • stage IV, characterized, e.g., by cancer which has spread to other organs outside the colon.

[0141] In embodiments, the method slows or prevents the progression from stage 0 to one of stage I, stage II, stage III, and stage IV.

[0142] In embodiments, the method slows or prevents the progression from stage I to one of stage II, stage III, and stage IV.

[0143] In embodiments, the method slows or prevents the progression from stage II to one of stage III, and stage IV.

[0144] In embodiments, the method slows or prevents the progression from stage III to stage IV. In embodiments, the method for treatment colon or colorectal cancer is in further combination with one or more of surgery, radiation therapy, chemotherapy, targeted therapy, and immunotherapy. In embodiments, the chemotherapy is one or more of 5-FU, leucovorin, oxaliplatin, capecitabine, regorafenib, trifluridine and tipiracil. In embodiments, the chemotherapy is one or more of FOLFOX (5- FU, leucovorin, and oxaliplatin), CapeOx (capecitabine and oxaliplatin), or 5-FU with leucovorin or capecitabine regimens. In embodiments, the targeted therapy is one or more of an agent that targets VEGF (e.g. bevacizumab [Avastin], ziv-aflibercept [Zaltrap], or ramucirumab [Cyramza]), or an agent that targets EGFR (e.g. cetuximab [Erbitux] or panitumumab [Vectibix]). In embodiments, the immunotherapy is one or more of an agent that targets the PD-1 pathway, e.g. atezolizumab, durvalumab, cemiplimab, nivolumab, and pembrolizumab and / or an agent that targets the CTLA-4 pathway, e.g. ipilimumab.

[0145] In embodiments, the method for treatment colon or colorectal cancer is in further combination with an ataxia telangiectasia and Rad3-related (ATR) inhibitor, e.g. berzosertib. In embodiments, the method for treatment colon or colorectal cancer comprises the administration of topotecan and berzosertib.

[0146] Treatment of Pancreatic Cancer

[0147] In aspects, the present disclosure provides methods of treating or preventing pancreatic cancer in a patient in need thereof, comprising administering to the patient (i) an oncolytic adenovirus comprising replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and replication machinery specific for tumor cells, and (ii) a topoisomerase I inhibitor or prodrug thereof, e.g, topotecan, SN-38, and / or irinotecan (e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE).

[0148] In aspects, the present disclosure provides methods of treating or preventing pancreatic cancer in a patient in need thereof, comprising administering to the patient (i) an oncolytic adenovirus comprising replication machinery specific for tumor cells and a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and replication machinery specific for tumor cells, and (ii) a topoisomerase I inhibitor or prodrug thereof, e.g, topotecan, SN-38, and / or irinotecan (e.g, by way of non-limitation, liposomal irinotecan, e.g, by way of non-limitation ONIVYDE).

[0149] In aspects, the present disclosure provides methods of treating or preventing pancreatic cancer in a patient in need thereof, comprising administering to the patient an oncolytic adenovirus comprising replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and replication machinery specific for tumor cells, wherein the patient is undergoing or has undergone treatment with a topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan (e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE).

[0150] In aspects, the present disclosure provides methods of treating or preventing pancreatic cancer in a patient in need thereof, comprising administering to the patient an oncolytic adenovirus comprising replication machinery specific for tumor cells and a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and replication machinery specific for tumor cells, wherein the patient is undergoing or has undergone treatment with a topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g, by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE).

[0151] In embodiments, the method reduces or eliminates a pancreatic tumor or cancer, or cells thereof. In embodiments, the method slows the growth and / or progression and / or metastasis of the pancreatic tumor or cancer, or cells thereof.

[0152] In embodiments, the pancreatic cancer is a primary or metastatic tumor. Primary tumors or cancers that migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. A metastasis is a cancer cell or group of cancer cells, distinct from the primary tumor location, resulting from the dissemination of cancer cells from the primary tumor to other parts of the body. Metastases may eventually result in death of a subject. For example, the present pancreatic cancers, in embodiments, include benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases.

[0153] In embodiments, the pancreatic cancer is an exocrine or endocrine cancer, or an adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, or a colloid carcinoma.

[0154] In embodiments, the pancreatic cancer one of the following stages of pancreatic cancer, as optionally assessed by the TNM system:

[0155] • Stage 0: characterized by, e.g., cancer in situ, in which the cancer has not yet grown outside the duct in which it started (Tis, NO, MO);

[0156] • Stage IA: characterized by, e.g., a tumor which is 2 cm or smaller in the pancreas. It has not spread to lymph nodes or other parts of the body (T 1 , NO, MO);

[0157] • Stage IB: characterized by, e.g., a tumor which is larger than 2 cm is in the pancreas. It has not spread to lymph nodes or other parts of the body (T2, NO, MO); • Stage IIA: characterized by, e.g., a tumor which is larger than 4 cm and extends beyond the pancreas. It has not spread to nearby arteries, veins, lymph nodes, or other parts of the body (T3, NO, MO);

[0158] • Stage II B: characterized by, e.g., a tumor of any size has not spread to nearby arteries or veins. It has spread to 1 to 3 regional lymph nodes but not to other parts of the body (T1, T2, or T3; N1 ; MO);

[0159] • Stage III: characterized by, e.g., a tumor of any size that has spread to 4 or more regional lymph nodes but not to nearby arteries, veins, or other parts of the body (T1 , T2, or T3, N2, MO), or which is tumor that has spread to nearby arteries and veins and may have spread to regional lymph nodes. It has not spread to other parts of the body (T4, any N, MO); and

[0160] • Stage IV: characterized by, e.g., any tumor that has spread to other parts of the body (any T, any N, M1).

[0161] In embodiments, the method slows or prevents the progression from stage 0 to one of stage IA or IB, stage IIA or I IB, stage III, and stage IV.

[0162] In embodiments, the method slows or prevents the progression from stage IA or IB to one of stage IIA or I IB, stage III, and stage IV.

[0163] In embodiments, the method slows or prevents the progression from stage IIA or IIB to one of stage III, and stage IV.

[0164] In embodiments, the method slows or prevents the progression from stage III to stage IV.

[0165] In embodiments, the method for treatment pancreatic cancer is in further combination with one or more of surgery, radiation therapy, chemotherapy, targeted therapy, and immunotherapy. In embodiments, the chemotherapy is one or more of gemcitabine, 5-FU, oxaliplatin, paclitaxel, capecitabine, and cisplatin. In embodiments, the targeted therapy is one or more of an EGFR inhibitor {e.g. erlotinib), a PARP inhibitor {e.g. olaparib), a NTRK inhibitor {e.g. larotrectinib or entrectinib). In embodiments, the immunotherapy is one or more of an agent that targets the PD-1 pathway, e.g. atezolizumab, durvalumab, cemiplimab, nivolumab, and pembrolizumab and / or an agent that targets the CTLA-4 pathway, e.g. ipilimumab.

[0166] In embodiments, the method for treatment pancreatic cancer comprises the FOLFIRINOX (leucovorin, fluorouracil / 5FU, irinotecan, and oxaliplatin) or the NALIRIFOX (irinotecan, 5-FU, leucovorin, oxaliplatin) regimen. Co-administration of Oncolytic Adenovirus and a Topoisomerase Inhibitor

[0167] In one aspect, the present disclosure provides methods for improving and / or increasing and / or enhancing antitumor efficacy in a patient in need thereof, comprising administering to the patient (i) an oncolytic adenovirus comprising replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and (ii) a topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan (e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), and (iii) observing improved antitumor efficacy compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0168] In one aspect, the present disclosure provides methods for improving and / or increasing and / or enhancing antitumor efficacy in a patient in need thereof, comprising administering to the patient (i) an oncolytic adenovirus comprising replication machinery specific for tumor cells and a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and (ii) a topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan (e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), and (iii) observing improved antitumor efficacy compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0169] In one aspect, the present disclosure provides methods for improving and / or increasing and / or enhancing antitumor efficacy in a patient in need thereof, comprising administering to the patient an oncolytic adenovirus comprising replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is undergoing or has undergone treatment with a topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan (e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), and observing improved antitumor efficacy compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0170] In one aspect, the present disclosure provides methods for improving and / or increasing and / or enhancing antitumor efficacy in a patient in need thereof, comprising administering to the patient an oncolytic adenovirus comprising replication machinery specific for tumor cells and a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is undergoing or has undergone treatment with a topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan (e.g., by way of non-limitation, liposomal irinotecan, e.g, by way of non-limitation ONIVYDE), and observing improved antitumor efficacy compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus. In embodiments, the oncolytic adenovirus, the topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g, by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), of the present disclosure are co-administered. In embodiments, the coadministration can occur simultaneously or sequentially.

[0171] In embodiments, the oncolytic adenovirus and the topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g, by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), are administered to a subject simultaneously. The term "simultaneously” as used herein, means that the oncolytic adenovirus and the topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g, by way of non-limitation ONIVYDE), are administered with a time separation of no more than about 60 minutes, such as no more than about 30 minutes, no more than about 20 minutes, no more than about 10 minutes, no more than about 5 minutes, or no more than about 1 minute. Administration of the oncolytic adenovirus and the topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g, by way of non-limitation ONIVYDE), can be by simultaneous administration of a single formulation {e.g., a formulation comprising the oncolytic adenovirus and the topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE)) or of separate formulations {e.g, a first formulation including the oncolytic adenovirus and a second formulation including the topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE)).

[0172] In embodiments, the oncolytic adenovirus and the topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g, by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), are administered to a subject simultaneously but the release of the oncolytic adenovirus and the topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), from their respective dosage forms (or single unit dosage form if co-formulated) may occur sequentially.

[0173] Co-administration does not require the oncolytic adenovirus and the topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), to be administered simultaneously, if, for example, the timing of their administration is such that the pharmacological activities of the oncolytic adenovirus and the topoisomerase I inhibitor or prodrug thereof, e.g, topotecan, SN-38, and / or irinotecan {e.g, by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), overlap in time. For example, the oncolytic adenovirus and the topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), can be administered sequentially or contemporaneously. The terms "sequentially” and "contemporaneously” as used herein mean that the oncolytic adenovirus and the topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), are administered with a time separation of more than about 60 minutes. For example, the time between the sequential administration of the oncolytic adenovirus and the topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), can be more than about 60 minutes, more than about 2 hours, more than about 5 hours, more than about 10 hours, more than about 1 day, more than about 2 days, more than about 3 days, or more than about 1 week apart. The administration times will depend on the rates of metabolism, excretion, and / or the pharmacodynamic activity of the oncolytic adenovirus and the topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), being administered. Either the oncolytic adenovirus or the topoisomerase I inhibitor or prodrug thereof, e.g., topotecan or SN-38, may be administered first.

[0174] In embodiments, the oncolytic adenovirus is administered first and the topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), is administered about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks later.

[0175] In embodiments, the topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE) is administered first and the oncolytic adenovirus is administered about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks later. In embodiments, co-administration also does not require the oncolytic adenovirus and the topoisomerase I inhibitor or prodrug thereof, e.g, topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), to be administered to the subject by the same route of administration. Rather, in embodiments, each therapeutic agent can be administered by any appropriate route, for example, intravenously, intraventricularly, intratumorally, intraperitoneally, orally, non-intraventricularly, non-intratumorally, non-intraperitoneally, or non-orally.

[0176] In embodiments, administration of the present compositions and formulations comprising the oncolytic adenovirus and / or topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), may be combined with additional agents, such as one or more additional anti-tumor agents, including, but not limited to, one or more chemotherapy drugs, and / or one or more additional therapy conventionally used for treating or preventing ovarian, cervical, lung, colon or colorectal, and / or pancreatic cancer. In embodiments, co-administration of the additional agent and the present compositions / formulations may be simultaneous or sequential. In embodiments, the additional agent can be included in the present compositions and formulations comprising the oncolytic adenovirus and / or topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE). In embodiments, the additional agent can be administered in a separate composition from the present compositions and formulations comprising the oncolytic adenovirus and / or topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g, by way of non-limitation ONIVYDE). In embodiments, the additional agent is administered at the same time as the present compositions and formulations comprising the oncolytic adenovirus and / or topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g, by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE). In embodiments, the additional agent is administered before the present compositions and formulations comprising the oncolytic adenovirus and / or topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g, by way of non-limitation ONIVYDE). In embodiments, the additional agent is administered after the present compositions and formulations comprising the oncolytic adenovirus and / or topoisomerase I inhibitor or prodrug thereof, e.g, topotecan, SN-38, and / or irinotecan {e.g, by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE). In embodiments, the present compositions and formulations comprising the oncolytic adenovirus and / or topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), can be used alone in a therapeutic regimen for treating or preventing ovarian, cervical, lung, colon or colorectal, and / or pancreatic cancer, i.e. without using other anti-tumor agents and / or other conventional therapies.

[0177] Further, the present compositions / formulations may comprise an additional agent (e.g. via coformulation). For example, the additional agent and the oncolytic adenovirus and / or topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan (e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), may be combined into a single formulation. Alternatively, the additional agent and the oncolytic adenovirus and / or topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan (e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), may be formulated separately.

[0178] Oncolytic Adenoviruses

[0179] The present disclosure is directed, in part, to pharmaceutical compositions, formulations, and uses of one or more oncolytic adenoviruses.

[0180] As used in the present document, "oncolytic adenovirus” and its plural refer to adenoviruses capable of replicating themselves or being replication-competent in the tumor cell. The oncolytic adenoviruses are differentiated from non-replicating adenoviruses because the latter are unable to replicate themselves in the target cell.

[0181] Adenoviruses

[0182] In embodiments, oncolytic adenoviruses used in the present disclosure are oncolytic adenoviruses with replication machinery and a capsid that allows infection and replication in human cancer cells. In embodiments, the oncolytic adenovirus is generated from an adenovirus that infects humans. Examples of adenoviruses that infect humans include, but are not limited to, a human adenovirus of serotype 1 to 51 , such as an adenovirus of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 of human adenoviruses, or combinations thereof, for example a hybrid recombinant of two or more different serotypes of human adenoviruses.

[0183] The 51 serotypes of human adenoviruses that have been identified are classified into six groups, differentiated from A to F. Human adenovirus serotype 5 (Ad5), which belongs to group C, is a virus formed by an icosahedral protein capsid containing a 36-kilobase linear deoxyribonucleic acid (DNA). In adults, Ad5 infection is usually asymptomatic, and in children it causes common cold and conjunctivitis. Ad5 generally infects epithelial cells, the cells of the bronchial epithelium during a natural infection. It enters the cell by means of the interaction of the fibre, a viral protein that extends like an antenna from the twelve vertices of the capsid, with a cell protein involved in intercellular adhesion, called the Coxsackie-Adenovirus Receptor (CAR). When viral DNA reaches the interior of the nucleus, it starts systematically transcribing the early genes (E1 to E4) of the virus. The first viral genes to be expressed correspond to early region genes 1A (E1A). E1A binds to the cell protein of the retinoblastoma to release E2F and thus activate the transcription of other viral genes such as E2, E3, E4, and the cellular genes that activate the cell cycle. E1 B, for its part, binds to the p53 protein, to activate the cell cycle and prevent apoptosis of the infected cell. E2 encodes for virus replication proteins; E3 encodes proteins that inhibit the antiviral immune response; E4 encodes for proteins that transport viral RNA. Expression of the early genes leads to replication of the viral DNA, and once this is replicated, the major late promoter is activated, leading to expression of a messenger ribonucleic acid (RNA) transcript which, by cutting and differential splicing, generates all the RNAs that encode for the structural proteins that form the capsid.

[0184] In embodiments, an oncolytic adenovirus used in the present disclosure is generated from a human adenovirus serotype 5.

[0185] Hyaluronidase

[0186] In embodiments, an oncolytic adenovirus used in the present disclosure comprises a sequence encoding a hyaluronidase enzyme inserted into its genome. In embodiments, an oncolytic adenovirus used in the present disclosure does not comprise a sequence encoding a hyaluronidase enzyme inserted into its genome.

[0187] Hyaluronidase enzymes are a family of enzymes responsible for degrading hyaluronic acid. In the human species, six genes that encode for hyaluronidase enzymes, having different properties and locations, have been located to date. The isoforms Hyall and Hyal2 are found in most tissues, Hyall being the predominant form in human plasma. Hyal3 is located in the bone marrow and testicles, but its function is not well characterized. The hyaluronidase enzyme PH20 is highly expressed in the testicles and is involved in the process by which the oocyte is fertilized by the spermatozoon. The hyaluronidase enzyme PH20 is anchored to the plasma membrane and the inner acrosomal membrane of the spermatozoa and gives the spermatozoon the ability to penetrate the extracellular matrix of the cumulus cells (which are rich in hyaluronic acid) and reach the zona pel I uci da of the oocyte. During the acrosome reaction, some of the hyaluronidase enzymes anchored in the spermatozoon membrane are processed enzymatically to give rise to a soluble form of the protein, which is released from the acrosomal membrane. The membrane protein PH20 is the only enzyme in the mammalian hyaluronidase family with activity at neutral pH.

[0188] Without wishing to be bound by theory, it is believed that expression of the hyaluronidase enzyme by an oncolytic adenovirus can help degrade the dense physical and immunosuppressive stroma barrier surrounding solid tumors, thereby ensuring greater tumor penetration by the oncolytic adenovirus and co-administered therapies. Without wishing to be bound by theory, this ensures that the adenovirus can reach and infect a larger number of tumor cells. Degrading the tumor stroma can also expose tumor neoantigens, thereby enabling an antitumor immune response by the patient's immune system.

[0189] In embodiments the hyaluronidase enzyme is a mammalian testicular hyaluronidase enzyme, optionally a human one (e.g, GenBank Gene ID: 6677), also known as SPAM1 or spermatozoon adhesion molecule 1 or PH20. In embodiments, the sequence of the hyaluronidase enzyme has the sequence corresponding to the enzyme having the membrane-binding carboxy-terminal domain deleted from it, such that the enzyme is soluble. When this carboxy-terminal domain is deleted, the resulting enzyme is secreted into the extracellular environment.

[0190] In embodiments, the sequence encoding the hyaluronidase enzyme inserted into an oncolytic adenovirus genome is SEQ ID NO: 1 , from which nucleotides 1471 to 1527, corresponding to the carboxy-terminal domain, have been deleted.

[0191] Also, in embodiments, the hyaluronidase enzyme is a hyaluronidase variant. A hyaluronidase variant has at least one or more amino acid modifications, generally amino acid substitutions, as compared to the parental wild-type sequence. In embodiments, a hyaluronidase of the 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 of the sequences disclosed herein. In addition, in embodiments, hyaluronidase variants retain most or all of their biochemical activity, measured by any suitable method known in the art.

[0192] In embodiments, a hyaluronidase of the 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

[0193] 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about

[0194] 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about

[0195] 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about

[0196] 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. In embodiments, a hyaluronidase of the 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

[0197] 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about

[0198] 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about

[0199] 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about

[0200] 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 , from which nucleotides 1471 to 1527, corresponding to the carboxy-terminal domain, have been deleted.

[0201] In embodiments, a hyaluronidase of the 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

[0202] 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about

[0203] 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about

[0204] 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about

[0205] 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.

[0206] In embodiments, a hyaluronidase of the 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

[0207] 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about

[0208] 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about

[0209] 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about

[0210] 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with SEQ ID NO: 8.

[0211] In embodiments, a hyaluronidase of the 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

[0212] 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about

[0213] 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about

[0214] 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about

[0215] 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with SEQ ID NO: 10. In embodiments, an oncolytic adenovirus used in the present disclosure comprises one or more regulatory elements, modifications, or variations that provide for tumor-specific enzyme expression or viral replication.

[0216] In embodiments, expression of the hyaluronidase enzyme is controlled by a promoter operational in tumor cells. In embodiments, the expression of the hyaluronidase enzyme is controlled by a promoter operational in the ovarian, cervical, lung, colon or colorectal, and / or pancreatic cancer cells to be treated. Optionally, in embodiments, the expression of the enzyme is controlled by a promoter operational in animal cells. Optionally, in embodiments, the promoter is selected from the cytomegalovirus promoter, the adenovirus major late promoter, the SV40 promoter, the herpes simplex virus thymidine kinase promoter, the RSV promoter, the EF1-a promoter, the beta-actin promoter, the human IL-2 promoter, the human IL-4 promoter, the IFN promoter, the E2F promoter, the human GM-CSF promoter, or combinations thereof.

[0217] In embodiments, the promoter that regulates the expression of the enzyme can be present naturally in the adenovirus, as is the case with the adenovirus major late promoter. In embodiments, the promoter can also be inserted together with the sequence encoding the enzyme. In embodiments, the promoter is the adenovirus major late promoter and this is already located in the oncolytic adenovirus genome. In embodiments, it is not necessary to introduce the promoter together with the hyaluronidase enzyme sequence, but rather the latter is introduced into the oncolytic adenovirus genome in such a way that it remains under the control of the promoter.

[0218] In embodiments, an oncolytic adenovirus used in the present disclosure comprises additional sequences that allow promotion or optimization of the protein translation of the sequence encoding the hyaluronidase enzyme. In embodiments, the sequences are inside or outside the hyaluronidase enzyme gene. For example, in embodiments, the additional sequences are selected from a sequence of cutting and splicing that allows RNA to be processed, IRES (internal ribosome entry site) sequences, the picornavirus sequence 2A, or combinations thereof.

[0219] Replication Machinery Specific for Tumor Cells

[0220] In embodiments, tumor cell-specific replication machinery comprised by an oncolytic virus used in the present disclosure is machinery that causes the oncolytic adenovirus to replicate itself in a specific form in tumor cells and not in healthy, non-tumor, or normal cells. In embodiments, the machinery can take different forms if, as a result, it provides oncolytic adenoviruses with a replicative capacity solely, or largely, in the tumor cells, e.g., ovarian, cervical, lung, colon or colorectal, and / or pancreatic cancer cells. In other words, in embodiments, adenoviruses with selective replication in which their replication machinery requires the virus to enter the cancer or tumor cells in order for the virus to replicate itself. In embodiments, the oncolytic adenovirus used can have modifications in its genome sequence that give it selective replication in tumor cells.

[0221] In embodiments, this is achieved by the incorporation of a specific tissue promoter or a specific tumor promoter, where the promoter controls the expression of one or more genes in the group E1 a, E1b, E2 and E4. In embodiments, the promoter is selected from the E2F promoter, the telomerase hTERT promoter, the tyrosinase promoter, the prostate-specific antigen (PSA) promoter, the alpha-fetoprotein promoter, the COX-2 promoter, as well as artificial promoters formed by various transcription factor binding sites such as binding sites for hypoxia-inducible factor (HI F-1 ), the ETS transcription factor, the tumor cytotoxic factor (TCF), the E2F transcription factor or the Sp1 transcription factor. In embodiments, the promoter controls the expression of E1a.

[0222] In embodiments, the treatment is of tumors with an aberrant Rb-E2F pathway. In healthy, non-tumor, or normal cells, E1A protein expressed by the adenovirus, dissociates the host cell Rb-E2F complex and allows free E2F protein to drive both cell division and viral replication. In tumor cells with aberrant Rb- E2F machinery, free E2F allows the cells to divide (and virus to replicate) continuously without being initiated by virus E1A. Genetic modifications that suppress normal viral E1 A expression thus prevent viral replication in healthy, non-tumor, or normal cells while permitting replication in tumor cells. Almost all solid tumors have aberrant Rb-E2F pathways, including, but not limited to ovarian, cervical, lung, colon or colorectal, and / or pancreatic cancer. In embodiments, an oncolytic adenovirus used in the present disclosure is characterized by a deletion of the Rb binding domain or the A24 deletion, which affects the interaction of E1 a with the retinoblastoma protein, and the insertion of four sites binding to E2F-1 and one site binding to Sp1 into the endogenous E1 a promoter to control the expression of E1a. Said DNA sequence corresponds to SEQ ID NO: 2.

[0223] Furthermore, it is envisaged that an oncolytic adenovirus used in the present disclosure, in embodiments, comprises one or more modifications in the capsid allowing improved biodistribution and reduced clearance of the oncolytic adenovirus from the body. In embodiments, an oncolytic adenovirus used in the present disclosure has the capsid modified to reduce its sequestration and destruction in the liver, such that the binding domain KKTK of the heparan sulfates present in the adenovirus fibre has been replaced, e.g. by the domain RGDK. The modification relates to positions 91 to 94 of the adenovirus fibre, taking the standard sequence of the adenovirus serotype 5 fibre as the reference. The sequence SEQ ID NO: 4 shows the complete sequence of the adenovirus type 5 fibre protein with the modified version in its heparan sulfate binding domain (modification RGDK). Therefore, in embodiments, an oncolytic adenovirus used in the present disclosure is generated from a human adenovirus serotype 5 and comprises:

[0224] • a sequence encoding a hyaluronidase enzyme inserted into its genome, optionally the human testicular hyaluronidase enzyme sequence (PH20, SEQ ID NO: 1 ) from which the sequence corresponding to the membrane-binding carboxy-terminal domain has been deleted in order for the enzyme to be soluble, optionally the sequence encodes a hyaluronidase enzyme having the amino acid sequence of SEQ ID NO: 10;

[0225] • oncolytic adenoviral replication occurs in tumor cells with an aberrant Rb-E2F pathway and not in healthy, non-tumor, or normal cells; optionally the oncolytic adenovirus a deletion of the Rb binding domain or the deletion A24, which affects the interaction of E1 a with the Rb protein, and the insertion of four binding sites to E2F-1 and one binding site to Sp1 into the endogenous E1a promoter to control the expression of E1 a; and / or

[0226] • modification of the adenovirus capsid to improve biodistribution and reduce clearance of the oncolytic adenovirus from the body; optionally the binding domain KKTK of the heparan sulfates present in the adenovirus fibre has been replaced, e.g. by the domain RGDK.

[0227] In embodiments, an oncolytic adenovirus of the present 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 embodiments, the oncolytic adenovirus has a nucleotide sequence of SEQ ID NO: 3.

[0228] Methods of Making Oncolytic Adenoviruses of the Disclosure

[0229] In embodiments, to construct an oncolytic adenovirus to be used in the present disclosure, use is made of any of the methods of construction of genetically modified adenovirus known in the field of gene therapy and virotherapy using adenoviruses. The most commonly used method is based on first constructing the desired genetic modification in a plasmid containing the adenoviral region to be modified, and then carrying out homologous recombination in bacteria with a plasmid containing the rest of the viral genome. As will be appreciated by those in the art, the protein sequences depicted herein can be encoded by any number of possible nucleic acid sequences, due to the degeneracy of the genetic code. In embodiments, the nucleic acids encoding the components of the disclosure can be incorporated into oncolytic adenoviruses as is known in the art, and depending on the host cells, used to produce the hyaluronidase of the disclosure. Generally, the nucleic acids are operably linked to any number of regulatory elements (promoters, origin of replication, selectable markers, ribosomal binding sites, inducers, etc.).

[0230] In embodiments, an oncolytic adenovirus used in the present disclosure is propagated and amplified in cell lines normally used in the field of gene therapy and virotherapy such as the lines HEK-293 (Reference number: ATCC CRL-1573) and A549 (Reference number: ATCC CCL185). In embodiments, a method of propagation of the adenovirus is by infection of a cell line permitting the replication of the adenovirus. The lung adenocarcinoma line A549 is an example of a line with such characteristics. In embodiments, propagation takes place, for example, as follows: the A549 cells are grown on plastic cell culture plates and are infected using 100 virus particles per cell. Two days later, the cytopathic effect reflecting virus production can be observed as a clustering of the cells. In embodiments, the cells are collected and stored in tubes. In embodiments, after centrifuging at about 1 ,000 g for about 5 minutes, the cell pellet is frozen and thawed three times, to lyse the cells. In embodiments, the resulting cell extract is centrifuged at about 1,000 g for about 5 minutes and the supernatant, in which the viruses are located, is loaded over a cesium chloride gradient and centrifuged for about 1 hour at about 35,000 g. In embodiments, the virus band obtained from the gradient is loaded again over another cesium chloride gradient and centrifuged for about 16 hours at about 35,000 g. In embodiments, the virus band is collected and dialyzed opposite PBS-10% glycerol. In embodiments, the virus dialysate is aliquoted and stored at about -80° C. The number of plaque-forming units and particles may be quantified following standard protocols known in the prior art. Phosphate-buffered saline (PBS) with 5% glycerol is a standard formulation for storing adenoviruses and is used in embodiments. However, new formulations improving virus stability have been described and are used in embodiments.

[0231] Topoisomerase I Inhibitors

[0232] Topoisomerase inhibitors can inhibit cell proliferation by, e.g., preventing DNA replication, stimulating DNA damage and inducing cell cycle arrest.

[0233] Topotecan is a semisynthetic derivative of the cytotoxic alkaloid, camptothecin. SN-38 is the active topoisomerase l-inhibiting metabolite of the prodrug irinotecan. Generally, topoisomerase I inhibitors cause cell cycle arrest in the S-phase by stabilizing the complex between topoisomerase I and DNA, thereby inhibiting re-ligation of topoisomerase l-mediated single-strand DNA breaks and producing potentially lethal double-strand DNA breaks. In embodiments, topoisomerase I inhibitors or prodrugs thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), are administered in various ways to treat ovarian, cervical, lung, colon or colorectal, and / or pancreatic cancers, including systemic, intra-arterial, intratumoral, intraperitoneal and / or oral administration.

[0234] In embodiments, for systemic administration, the topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g, by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), is injected intravenously or administered orally. The topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), then enters the bloodstream and is distributed throughout the body. Irinotecan is converted to the active topoisomerase I inhibitor SN-38 by liver and tumor carboxylesterase enzymes.

[0235] In embodiments, the topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g, by way of non-limitation ONIVYDE), is administered intravenously, intraarterially, intraperitoneally, or orally.

[0236] In embodiments, the liposomal irinotecan {e.g., by way of non-limitation ONIVYDE), is administered intravenous infusion.

[0237] In embodiments, the methods of the present disclosure improve and / or increase and / or enhance antitumor efficacy compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus. In embodiments, the method makes the patient suitable for treatment with a combination therapy of more than one cancer therapies.

[0238] Formulations

[0239] The present disclosure provides the described oncolytic adenovirus and / or topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), composition in various formulations. Any oncolytic adenovirus and / or topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), described herein can take 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 use.

[0240] It will be understood that the compositions used in the present disclosure are used in a pharmaceutically acceptable presentation. This means that any oncolytic adenovirus and / or topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), described herein can be administered to a subject as a component of a composition that comprises a pharmaceutically acceptable carrier or vehicle. Such compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration.

[0241] Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In embodiments, the pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any agent described herein is administered by intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, specifically for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, cellulose, hypromellose, lactose, sucrose, trehalose, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, povidone, crosspovidone, water, ethanol and the like. Any agent described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents. Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), incorporated herein by reference.

[0242] Administration and Dosages

[0243] It will be appreciated that the actual dose of the oncolytic adenovirus and / or topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), to be administered according to the present disclosure will vary according to different parameters, for example the size and stage of the ovarian, cervical, lung, colon or colorectal, and / or pancreatic cancer, tumor to be treated, the age and weight of the patient to be treated, the particular dosage form, and the mode of administration. It will be understood that the doses of oncolytic adenovirus and of co-administered topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), must be sufficient to ensure the combination of the method produces a positive therapeutic effect on the ovarian, cervical, lung, colon or colorectal, and / or pancreatic cancers. In embodiments, the positive therapeutic effect for ovarian, cervical, lung, colon or colorectal, and / or pancreatic cancer, refers to halted tumor growth, or reductions in tumor volume, or prevention of tumor penetration into surrounding tissues, or prevention of metastases. In embodiments, e.g. for ovarian cancer, the dose of topotecan is about 1.5 mg / m2e.g. by intravenous infusion over about 30 minutes daily for about 5 consecutive days, starting on Day 1 of a 21 -day cycle until disease progression or unacceptable toxicity.

[0244] In embodiments, e.g. for cervical cancer, the dose of topotecan is about 0.75 mg / m2e.g. by intravenous infusion over about 30 minutes daily on Days 1 , 2, and 3, e.g. in combination with cisplatin of about 50 mg / m2on Day 1, of a 21 -day cycle until disease progression or unacceptable toxicity.

[0245] In embodiments, e.g. for lung cancer, the dose of topotecan is about 1.5 mg / m2e.g. by intravenous infusion over about 30 minutes daily for about 5 consecutive days, starting on Day 1 of a 21 -day cycle until disease progression or unacceptable toxicity.

[0246] In embodiments, topotecan may be administered orally for the treatment of lung cancer, e.g. topotecan oral capsule doses of 2.3 mg / m2(rounded to the nearest 0.25 mg) administer once daily for about 5 consecutive days, starting on Day 1 of a 21 -day cycle until disease progression or unacceptable toxicity.

[0247] In embodiments, e.g. for colon or colorectal cancer, the dose of topotecan is about 1 .25 mg / m2about 1 .5 mg / m2administered intravenously by infusion, e.g. for about 5 days, e.g. in about 21 -day cycles.

[0248] In embodiments, e.g. for pancreatic cancer, the dose of topotecan is about 1.25 mg / m2about 1.5 mg / m2administered intravenously by infusion, e.g. for about 5 days, e.g. in about 21 -day cycles. In embodiments, e.g. for pancreatic cancer, topotecan is infused at about 350 mg / m2over about 30 minutes about once every 3 weeks. In embodiments, e.g. for pancreatic cancer, topotecan is infused at about 100 mg / m2weekly or 150mg / m2every 2 weeks.

[0249] Many factors that may modify the action of the oncolytic adenovirus and / or topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g., by way of non-limitation, liposomal irinotecan, e.g, by way of non-limitation ONIVYDE), {e.g., body weight, gender, diet, time of administration, route of administration, rate of excretion, condition of the subject, drug combinations, genetic disposition and reaction sensitivities) can be considered by those skilled in the art. Administration can be carried out continuously or in one or more discrete doses up to the maximum tolerated dose. Optimal administration rates for a given set of conditions can be ascertained by those skilled in the art using conventional evaluation of dosage administration and dose tolerability.

[0250] Compositions of the present disclosure, e.g, compositions comprising an oncolytic adenovirus disclosed herein, are administered by any route ensuring that the necessary amount of the oncolytic adenovirus reaches the tumor site. In the case of ovarian, cervical, lung, colon or colorectal, and / or pancreatic cancer, compositions of the present disclosure, e.g., compositions comprising an oncolytic adenovirus disclosed herein, are injected by any route ensuring that the necessary amount of the oncolytic adenovirus reaches the tumor site. Optional routes of administration include, but are not limited to, intravenous injection, intraarterial injection, intratumoral injection, and / or intraperitoneal injection. Therefore, in embodiments, the preferred compositions comprising an oncolytic adenovirus disclosed herein take an appropriate form for intravenous, intraarterial, intratumoral, and / or intraperitoneal administration or injection.

[0251] In embodiments, the oncolytic adenovirus and / or topoisomerase I inhibitor or prodrug thereof, e.g., topotecan, SN-38, and / or irinotecan {e.g, by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONI VYDE), may be administered, for example, more than once daily {e.g., about two, about three, about four, about five, about six, about seven, about eight, about nine, or about ten times per day), about once per day, about every other day, about every third day, about once a week, about once every two weeks, about once every month, about once every two months, about once every three months, about once every six months, or about once every year.

[0252] Definitions

[0253] As used herein, "a,” "an,” or "the” can mean one or more than one.

[0254] Further, the term "about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language "about 50%” covers the range of 45% to 55%.

[0255] An "effective amount,” when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disorder of interest.

[0256] As used herein, something is "decreased” if a read-out of activity and / or effect is reduced by a significant amount, such as by 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%, in the presence of an agent or stimulus relative to the absence of such modulation. As will be understood by one of ordinary skill in the art, in embodiments, activity is decreased and some downstream read-outs will decrease but others can increase.

[0257] Conversely, activity is "increased” if a read-out of activity and / or effect is increased by a significant amount, for example by 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% or more, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, in the presence of an agent or stimulus, relative to the absence of such agent or stimulus.

[0258] As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word "include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms "can” and "may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.

[0259] Although the open-ended term "comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the disclosure, the present disclosure, or embodiments thereof, may alternatively be described using alternative terms such as "consisting of' or "consisting essentially of.”

[0260] As used herein, the words "preferred” and "preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation 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 technology.

[0261] In embodiments, the terms "patient” and "subject” are used interchangeably. In embodiments, the subject and / or animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon. In embodiments, the subject and / or animal is a non-mammal, such, for example, a zebrafish.

[0262] In embodiments, methods of the invention are useful in treatment a human subject. In embodiments, the human is an adult human. In embodiments, the human is a geriatric human. In embodiments, the human may be referred to as a patient. In embodiments, the human is a female. In embodiments, the human is a male.

[0263] In embodiments, the human has an age in a range of from about 1 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.

[0264] The amount of each component in the compositions described herein needed for achieving a therapeutic effect may be determined empirically in accordance with conventional procedures for the particular purpose. Generally, for administering therapeutic agents (e.g., oncolytic adenoviruses and / or topoisomerase I inhibitor or prodrug thereof, e.g, topotecan, SN-38, and / or irinotecan (e.g., by way of non-limitation, liposomal irinotecan, e.g., by way of non-limitation ONIVYDE), compositions described herein) for therapeutic purposes, the therapeutic agents are given at a pharmacologically effective dose. A "pharmacologically effective amount,” "pharmacologically effective dose,” "therapeutically effective amount,” or "effective amount” refers to an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating or preventing the disorder or disease. An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease {e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.

[0265] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures, tissue samples, tissue homogenates or experimental animals, e.g., for determining the LD50 (the dose lethal to about 50% of the population) and the ED50 (the dose therapeutically effective in about 50% of the population) or the maximum tolerated dose. The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. In embodiments, compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from in vitro assays, including, for example, cell culture assays or measurements or methane production in stool samples. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture, or in an appropriate animal model. Levels of the described compositions in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0266] In embodiments, 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 embodiments, the effect will result in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.

[0267] As used herein, "methods of treatment” are equally applicable to use of a composition for treating the diseases or disorders described herein and / or compositions for use and / or uses in the manufacture of a medicaments for treating the diseases or disorders described herein.

[0268] EXAMPLES

[0269] Example 1: Effect of Topotecan on VCN-01 Replication in Lung and Ovarian Cancer-Derived Cell Lines

[0270] VCN-01 replication + / - topotecan is evaluated in A549 adherent and suspension cells, and in two ovarian cancer cell lines, PA-1 and SW-626. Readouts include viral particles (vp) via qPCR and infectious titer (transducing units, TU) via hexon staining, at three time points, 4 hrs (input), 48 hrs, and 72 hrs after infection. See FIG. 1.

[0271] The study design includes evaluation of the topoisomerase I inhibitors, topotecan, irinotecan, the irinotecan metabolite SN-38, the cell-cycle inhibitor, hydroxyurea, and other agents including carboplatin and melphalan. It is expected that topotecan and SN-38 will enhance VCN-01 replication in these cell lines. As gemcitabine was demonstrated to block adenovirus replication in vitro, gemcitabine will be used as a negative control.

[0272] Example 2 In vitro sensitivity testing of pancreatic tumor cell lines to topoisomerase I (topol) inhibitors, irinotecan (IRI), its active metabolite, SN-38, and topotecan (Topo)

[0273] A panel of different human pancreatic tumor cell lines was tested to evaluate the cytotoxicity of IRI and Topo as a single agent. Human pancreatic cell lines such as BxPC3 (ATCC-CRL1687), Panc-I (ATCC CRL 1469), NP-9 or NP-18 are infectable by human adenovirus, and VCN-01 viral replication has been demonstrated in these cell lines. The HP-1 hamster pancreatic tumor cell line was also tested to evaluate the sensitivity of this cell line to the topol inhibitors.

[0274] The concentration of SN-38, IRI, or Topo required to reduce cell culture viability by half was determined for each of the cell lines (IC50 value). For this purpose, tumor cells (15000 cells / well) were seeded in 96- well plates and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum at 37°C and 5% CO2. 24 hours later, serial 1 / 10 dilutions of each chemotherapy, IRI, SN-38, or Topo in DMEM + 5% FBS were prepared, and chemotherapeutic-containing media was added to the wells in quadruplicate. Cells were incubated at standard culture conditions for 5 additional days, after which cells were washed once with PBS and stained for total protein content using a commercial bicinchoninic acid assay (BCA; Pierce Biotechnology) (4 l of reagent A + 196 pl Reagent B per well). After 30 minutes incubation at 37°C, absorbance (A540 nm) was quantified. Concentration of each chemotherapy required to reduce to half the amount of protein compared to non-treated wells (IC50 value) was estimated in dose-response curves by standard nonlinear regression, using Prism software. At least three replicates were performed for each cell line. The results are shown in Figures 2A-2E and Figures 3A-3E and Table 1.

[0275] Table 1. Average IC50 values of SN-38, IRI, and Topo in pancreatic cancer cell lines. Final IC50 values were calculated by the average of each replicate.

[0276] Example 3 Evaluation of viral (E1A) and cellular (E2F1) gene expression in NP-18 and NP-9 human pancreatic cancer cell lines exposed to VCN-01 plus the topol inhibitors, SN-38, IRI, and Topo

[0277] Cells were plated in 6-well culture plates at a density of 2 x 106cells / well. Cells were infected with VCN- 01 at an MOI of 1. At 24 hrs after infection, cells were treated with SN-38, IRI, or Topo at 100X the previously determined IC50. The topol concentrations used for NP-18 cells were SN-38 at 0.1 microM, IRI at 200 microM and Topo at 2 microM. For NP-9 cells these concentrations were SN-38 at 2 microM, IRI at 1500 microM, and Topo at 0.5 microM. Cell pellets were collected 24 hrs after drug exposure, and RNA was isolated using the Qiagen RNAeasy Mini Kit and treated with DNAse. RNA was quantified and subjected to reverse transcription followed by qPCR specific for E1 A or E2F1. ElA and E2F1 RNA levels were normalized to that of a human beta-actin control.

[0278] As shown in Figure 4, in NP-18 cells, viral E1A mRNA expression was increased approximately 4-6 fold with the topol inhibitors compared to VCN-01 alone infected cells, while in NP-9 cells, E1A was increased greater than 10-fold with SN-38 and IRI, and approximately 2-fold with Topo compared to VCN-01 infected cells. Likewise, E2F1 mRNA levels were increased 2-3 fold in NP-18 cells with topol inhibitors and 1-3 fold in NP-9 cells with topol inhibitors compared to untreated cells. Without wishing to be bound by theory, these data are consistent with the idea that topol inhibitors increase expression of the cellular transcription factor E2F1 to activate the modified VCN-01 E1 A early viral promoter to result in increased expression of viral genes.

[0279] Example 4: Evaluation of anti-tumor efficacy of VCN-01 + liposomal irinotecan in an immunodeficient mouse model of human pancreatic cancer, NP-18

[0280] The objective of the study was to evaluate the anti-tumor efficacy of the combination of VCN-01 with liposomal irinotecan (nal-IRI; ONIVYDE) in immunodeficient nude mice bearing human pancreatic NP- 18 subcutaneous tumors. Liposomal irinotecan is a component of the NALIRI FOX chemotherapy cocktail which consists of liposomal irinotecan + 5-fluorouracil (5-FU) + leucovorin + oxaliplatin).

[0281] Athymic nude mice (n=54, 6 cohorts @ 8 male mice per cohort + 6 extra animals) were subcutaneously inoculated with 4 x 106NP-18 cells / flank (0.2 ml cell suspension in PBS) into two flanks of the animals with a hypodermic (25G) needle. After implantation, tumor growth was monitored two times a week by palpation and the length and width of the tumor nodules were measured with a vernier caliper. Once tumors reached 150mm3, animals were treated as indicated (Figure 5). Six groups of 8 mice each (16 total tumors) were treated on Day 1 with VCN-01 or PBS IV via tail vein. On Day 3, Day 10 and Day 17, animals received PBS or Na-IRI, IP at the indicated doses. Animal body weight and tumor growth was measured weekly. At end of study, Day 37, animals were sacrificed, blood collected via cardiac puncture, and serum analyzed for the indicated hematologic and biochemical parameters. Tumors were collected on Day 37 and were subjected to vial genome (DNA) and E1A mRNA qPCR analyses.

[0282] Animal body weights were measured throughout the study (Figure 6). Animals treated with VCN-01 and VCN-01 + nal-IRI showed an initial loss of body weight. One animal in the VCN-01 + nal-IRI (10 mg / kg) cohort was sacrificed due to extreme body weight loss (>30%). All other animals recovered by the end of study.

[0283] Tumor growth was followed (Figure 7). Animals treated with VCN-01 alone, or nal-IRI alone at the 5 and 10 mg / kg doses displayed a significantly decreased tumor growth compared to the PBS cohort. Animals treated with the combination therapy of VCN-01 +nal-IRI at both the 5 mg / kg and 10 mg / kg doses showed significantly decreased tumor growth compared to each treatment alone. There was no significant difference in tumor growth between the two combination treatment cohorts, VCN-01 + nal-IRI (5 mg / kg) and VCN-01 + nal-IRI (10 mg / kg).

[0284] Hematology and biochemistry results (data not shown). Blood was collected from each animal at end of study, Day 37, and subjected to hematological and biochemical analyses. The hematological parameters evaluated included white blood cells, red blood cells, platelets, hemoglobin, lymphocytes, monocytes, neutrophils, eosinophils, and basophils. Platelets showed a significant reduction with the high dose nal- IRI treatment (10 mg / kg) alone or combined with VCN-01. Thrombocytopenia is a known side effect of irinotecan. The other hematological parameters were not significantly different between PBS or treatment cohorts. Biochemical parameters tested included ALT, AST, alkaline phosphatase, albumin, amylase, GGT, lipase, total bilirubin, total protein, creatinine and urea. Alkaline phosphatase was significantly increased with combination treatment, VCN-01 + nal-IRI at both the high and low nal-IRI doses. The other evaluated biochemical parameters were not significantly different between treatment cohorts.

[0285] Tumors were collected at end of study, Day 37. Isolated tumor DNA was assessed by qPCR for the presence of VCN-01 viral genomes (vgs), and tumor RNA was analyzed by reverse transcription and qPCR for the presence of viral E1 A mRNA (Figure 8). Vgs were detected in tumors from animals treated with VCN-01 demonstrating the retention of VCN-01 in the tumors throughout the study and suggesting ongoing viral replication. There was a significant reduction in tumor vgs in animals treated with VCN-01 + nal-IRI (5 mg / kg) compared to VCN-01 alone and no significant difference in tumor vgs in animals treated with VCN-01 + nal-IRI 10 mg / kg compared to VCN-01 alone. E1 A mRNA analysis revealed the presence of E1A mRNA in all animals treated with VCN-01 , indicating ongoing viral transcription. No difference in E1A mRNA levels were observed between VCN-01 -treated cohorts.

[0286] Without wishing to be bound by theory, these data are consistent with the idea that the topol inhibitor, irinotecan (nal-IRI) increases expression of the cellular transcription factor E2F1 to activate the modified VCN-01 E1 A early viral promoter to result in increased expression of viral genes and improve anti-tumor efficacy.

[0287] Example 5: Evaluation of anti-tumor efficacy of VCN-01 + liposomal irinotecan in an immunodeficient mouse model of human pancreatic cancer, NP-9

[0288] The objective of this study is to evaluate the anti-tumor efficacy of the combination of VCN-01 with liposomal irinotecan (nal-IRI; ONIVYDE) in immunodeficient mice bearing human pancreatic NP-9 subcutaneous tumors.

[0289] Athymic nude mice (n=54, 6 cohorts @ 8 female mice per cohort + 6 extra animals) were subcutaneously inoculated with 7.5 x 106NP-9 cells / flank (0.2 ml cell suspension in PBS) into two flanks of the animals with a hypodermic (25G) needle. After implantation, tumor growth is being monitored two times a week by palpation. Once tumors reach 150mm3, animals will be treated as indicated (Figure 9). Six groups of 8 mice each (16 total tumors) will be treated on Day 1 with VCN-01 or PBS IV via tail vein. On Day 3, Day 10 and Day 17, animals will receive PBS or Na-IRI, IP at the indicated doses. Animal body weight and tumor growth will be measured weekly. At end of study, animals will be sacrificed, blood collected via cardiac puncture, and serum analyzed for hematologic and biochemical parameters. Tumors will be collected at end of study and will be subjected to vial genome (DNA) and E1 A mRNA qPCR analyses.

[0290] Anti-tumor efficacy is expected to be seen with each treatment alone, VCN-01 , nal-IRI at 5 mg / kg, and nal-IRI at 2.5 mg / kg compared to PBS alone. Improved anti-tumor efficacy is expected to be seen with the combination therapy of VCN-01 + nal-IRI. Efficacy is expected to be seen at both the higher dose (5 mg / kg) nal-IRI and lower dose (2.5 mg / kg nal-IRI) combination treatments.

[0291] INCORPORATION BY REFERENCE

[0292] All patents and publications referenced herein are hereby incorporated by reference in their entireties.

[0293] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure.

[0294] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

[0295] EMBODIMENTS

[0296] Various additional embodiments of the disclosure are provided by the following enumerated embodiments, which can be combined in any number and in any combination.

[0297] Embodiment 1. A method for treating or preventing ovarian, cervical, lung, colon or colorectal, and / or pancreatic cancer in a patient in need thereof, comprising co-administering to the patient (I) an oncolytic adenovirus comprising replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome and (II) a topoisomerase I inhibitor or a prodrug thereof.

[0298] Embodiment 2. The method according to embodiment 1 , wherein the oncolytic adenovirus is administered by intravenous, intraarterial, intratumoral, and / or intraperitoneal administration, e.g. by injection.

[0299] Embodiment 3. The method according to embodiment 1 or 2, wherein the topoisomerase I inhibitor or a prodrug thereof is administered by intraperitoneal, intravenous, intraarterial, intratumoral, and / or oral administration.

[0300] Embodiment 4. The method according to any one of embodiments 1-3, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are formulated in separate compositions. Embodiment s. The method according to any one of embodiments 1-3, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are formulated in a single composition.

[0301] Embodiment 6. The method according to any one of embodiments 1-5, wherein the separate compositions are administered simultaneously or contemporaneously.

[0302] Embodiment 7. The method according to any one of embodiments 1-6, wherein the oncolytic adenovirus is administered first and the topoisomerase I inhibitor or a prodrug thereof is administered within about 14 days, within about 10 days, within about 7 days, within about 5 days, within about 1 day, or within about 60 minutes of the administration of the oncolytic adenovirus.

[0303] Embodiment 8. The method according to embodiment 7, wherein the topoisomerase I inhibitor or a prodrug thereof is administered within about 30 minutes, within about 20 minutes, within about 10 minutes, within about 5 minutes, or within about 1 minute of the administration of the oncolytic adenovirus.

[0304] Embodiment 9. The method according to any one of embodiments 1-6, wherein the topoisomerase I inhibitor or a prodrug thereof is administered first and the oncolytic adenovirus is administered within about 14 days, within about 10 days, within about 7 days, within about 5 days, within about 1 day, or within about 60 minutes of the administration of the oncolytic adenovirus and / or wherein the oncolytic adenovirus is administered within about 30 minutes, within about 20 minutes, within about 10 minutes, within about 5 minutes, or within about 1 minute of the administration of the topoisomerase I inhibitor or a prodrug thereof.

[0305] Embodiment 10. The method according to any one of embodiments 1-9, wherein the: (a) ovarian cancer is an epithelial ovarian carcinoma, germ cell tumor, or stromal cell tumor; (b) cervical cancer is squamous cell carcinoma, adenocarcinoma and / or adenosquamous (mixed) carcinoma; (c) lung cancer is small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, large cell carcinoma of the lung, mesothelioma, and / or lung carcinoid tumor; (d) colon or colorectal cancer is an adenocarcinoma, gastrointestinal stromal tumor (GIST), squamous cell carcinoma, carcinoid tumor, lymphoma, or sarcoma of the colon. In embodiments, and / or (e) pancreatic cancer is an exocrine or endocrine cancer, or an adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, or a colloid carcinoma.

[0306] Embodiment 11. The method according to any one of embodiments 1-10, wherein the topoisomerase I inhibitor is topotecan or SN-38 and / or the topoisomerase I inhibitor prodrug is irinotecan, optionally a liposomal irinotecan, optionally ONIVYDE.

[0307] Embodiment 12. The method according to any one of embodiments 1-11 , wherein the hyaluronidase enzyme is the human hyaluronidase enzyme PH20. Embodiment 13. The method according to embodiment 12, wherein the sequence that encodes a hyaluronidase enzyme 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 thereto.

[0308] Embodiment 14. The method according to any one of embodiments 1-13, wherein the oncolytic adenovirus is generated from a human adenovirus serotype 5.

[0309] Embodiment 15. The method according to any one of embodiments 1-14, wherein the oncolytic adenoviral replication occurs in tumor cells with an aberrant Rb-E2F pathway and not in healthy, nontumor, or normal cells.

[0310] Embodiment 16. The method according to embodiment 15, wherein the oncolytic adenovirus is engineered to replicate in tumor cells and not healthy, non-tumor, or normal cells by a deletion of a Rb binding domain or a A24 deletion in the sequence coding for the E1a protein and the insertion of four binding sites to E2F-1 and one binding site to Sp1 into the endogenous promoter of E1 a to control the expression of E1 a.

[0311] Embodiment 17. The method according to any one of embodiments 1-16, wherein the oncolytic adenovirus has the capsid modified such that the binding domain91KKTK94(SEQ ID NO: 6) of the heparan sulfates present in the adenovirus fibre has been replaced, optionally by the domain91RGDK94(SEQ ID NO: 7).

[0312] Embodiment 18. The method according to any one of embodiments 1-17, wherein the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3).

[0313] Embodiment 19. The method according to any one of embodiments 1-18, wherein the patient is a human patient.

[0314] Embodiment 20. The method according to embodiment 19, wherein the human patient is a female human patient.

[0315] Embodiment 21. The method according to any one of embodiments 1-20, wherein the ovarian, cervical, lung, colon or colorectal, and / or pancreatic cancer is resistant to conventional chemotherapy and / or radiotherapy treatment.

[0316] Embodiment 22. The method according to any one of embodiments 1-21 , wherein the method improves and / or increases and / or enhances antitumor efficacy compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus. Embodiment 23. The method according to any one of embodiments 1-22, wherein the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with ovarian cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0317] Embodiment 24. The method according to any one of embodiments 1-22, wherein the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with cervical cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0318] Embodiment 25. The method according to any one of embodiments 1-22, wherein the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with lung cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0319] Embodiment 26. The method according to any one of embodiments 1-22, wherein the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with colon or colorectal cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0320] Embodiment 27. The method according to any one of embodiments 1-22, wherein the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with pancreatic cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0321] Embodiment 28. A method for treating or preventing ovarian, cervical, lung, colon or colorectal, and / or pancreatic cancer, in a patient in need thereof, comprising administering to the patient an oncolytic adenovirus comprising replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase enzyme inserted into its genome, wherein the patient is undergoing or has undergone treatment with a topoisomerase I inhibitor or a prodrug thereof.

[0322] Embodiment 29. The method according to embodiment 28, wherein the oncolytic adenovirus is administered by intravenous, intraarterial, intratumoral, and / or intraperitoneal administration, e.g. by injection.

[0323] Embodiment 30. The method according to embodiment 28 or 29, wherein the topoisomerase I inhibitor or a prodrug thereof is administered by intraperitoneal, intravenous, intraarterial, intratumoral, and / or oral administration. Embodiment 31. The method according to any one of embodiments 28-30, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are formulated in separate compositions.

[0324] Embodiment 32. The method according to any one of embodiments 28-30, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are formulated in a single composition.

[0325] Embodiment 33. The method according to any one of embodiments 28-32, wherein the oncolytic adenovirus is administered first and the topoisomerase I inhibitor or a prodrug thereof is administered within about 12 weeks of the administration of the oncolytic adenovirus or wherein the topoisomerase I inhibitor or a prodrug thereof is administered first and the oncolytic adenovirus is administered within about 12 weeks of the administration of the topoisomerase I inhibitor or a prodrug thereof.

[0326] Embodiment 34. The method according to embodiment 33, wherein the topoisomerase I inhibitor or a prodrug thereof is administered within about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or about 11 weeks of the administration of the oncolytic adenovirus.

[0327] Embodiment 35. The method according to any one of embodiments 28-34, wherein the topoisomerase I inhibitor is topotecan or SN-38 and / or the prodrug of the topoisomerase I inhibitor is irinotecan, optionally a liposomal irinotecan, optionally ONIVYDE.

[0328] Embodiment 36. The method according to any one of embodiments 28-35, wherein the hyaluronidase enzyme is the human hyaluronidase enzyme PH20.

[0329] Embodiment 37. The method according to embodiment 36, wherein the sequence that encodes a hyaluronidase enzyme 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 thereto.

[0330] Embodiment 38. The method according to any one of embodiments 28-37, wherein the oncolytic adenovirus is generated from a human adenovirus serotype 5. Embodiment 39. The method according to any one of embodiments 28-38, wherein the oncolytic adenoviral replication occurs in tumor cells with an aberrant Rb-E2F pathway and not in healthy, nontumor, or normal cells.

[0331] Embodiment 40. The method according to embodiment 39, wherein the oncolytic adenovirus is engineered to replicate in tumor cells and not healthy, non-tumor, or normal cells by a deletion of a Rb binding domain or a A24 deletion in the sequence coding for the E1a protein and the insertion of four binding sites to E2F-1 and one binding site to Sp1 into the endogenous promoter of E1 a to control the expression of E1 a.

[0332] Embodiment 41. The method according to any one of embodiments 28-40, wherein the oncolytic adenovirus has the capsid modified such that the binding domain91KKTK94(SEQ ID NO: 6) of the heparan sulfates present in the adenovirus fibre has been replaced, optionally by the domain91RGDK94(SEQ ID NO: 7).

[0333] Embodiment 42. The method according to any one of embodiments 28-41 , wherein the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3).

[0334] Embodiment 43. The method according to any one of embodiments 28-42, wherein the patient is a human patient.

[0335] Embodiment 44. The method according to embodiment 43, wherein the human patient is a female human patient.

[0336] Embodiment 45. The method according to any one of embodiments 28-44, wherein the ovarian, cervical, lung, colon or colorectal, and / or pancreatic cancer is resistant to conventional chemotherapy and / or radiotherapy treatment.

[0337] Embodiment 46. The method according to any one of embodiments 28-45, wherein the method improves and / or increases and / or enhances antitumor efficacy compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0338] Embodiment 47. The method according to any one of embodiments 28-46, wherein the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with ovarian cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0339] Embodiment 48. The method according to any one of embodiments 28-46, wherein the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with cervical cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0340] Embodiment 49. The method according to any one of embodiments 28-46, wherein the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with lung cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0341] Embodiment 50. The method according to any one of embodiments 28-46, wherein the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with colon or colorectal cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0342] Embodiment 51. The method according to any one of embodiments 28-46, wherein the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with pancreatic cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0343] Embodiment 52. The method according to any one of embodiments 1-22, 27, 28-46, or 51 , wherein the prodrug of the topoisomerase I inhibitor is irinotecan, and the irinotecan is administered as part of the NALIRIFOX (irinotecan, 5-FU, leucovorin, oxaliplatin) regimen.

[0344] Embodiment 53. The method according to any one of embodiments 1-22, 27, 28-46, or 51 , wherein the prodrug of the topoisomerase I inhibitor is a liposomal irinotecan, optionally ONIVYDE, which is administered as part of the NALIRIFOX (irinotecan, 5-FU, leucovorin, oxaliplatin) regimen.

[0345] Embodiment 54. The method according to any one of embodiments 1-22, 27, 28-46, 51 or 53, wherein the prodrug of the topoisomerase I inhibitor is a liposomal irinotecan, optionally ONIVYDE, is administered by intravenous infusion.

[0346] Embodiment 55. The method according to any one of embodiments 1-54, wherein the method results in increased expression of the cellular transcription factor E2F1 , compared to treatment with the oncolytic virus without the topoisomerase I inhibitor or a prodrug thereof.

[0347] Embodiment 56. The method according to any one of embodiments 1-55, wherein the method results in increased expression of one or more viral genes, compared to treatment with the oncolytic virus without the topoisomerase I inhibitor or a prodrug thereof. SEQUENCES

[0348] SEQ ID NO: 1

[0349] Complete PH20 cDNA sequence comprising the carboxy-terminal domain, (from ATG until stop codon, both included, and with nt 1471-1527 underlined and bolded) atgggagtgctaaaattcaagcacatctttttcagaagctttgttaaatcaagtggagtatcccagatagttttcaccttccttctgattccatgttgcttg actctgaatttcagagcacctcctgttattccaaatgtgcctttcctctgggcctggaatgccccaagtgaattttgtcttggaaaatttgatgagccact agatatgagcctcttctctttcataggaagcccccgaataaacgccaccgggcaaggtgttacaatattttatgttgatagacttggctactatcctta catagattcaatcacaggagtaactgtgaatggaggaatcccccagaagatttccttacaagaccatctggacaaagctaagaaagacattac attttatatgccagtagacaatttgggaatggctgttattgactgggaagaatggagacccacttgggcaagaaactggaaacctaaagatgttta caagaataggtctattgaattggttcagcaacaaaatgtacaacttagtctcacagaggccactgagaaagcaaaacaagaatttgaaaaggc agggaaggatttcctggtagagactataaaattgggaaaattacttcggccaaatcacttgtggggttattatctttttccggattgttacaaccatcac tataagaaacccggttacaatggaagttgcttcaatgtagaaataaaaagaaatgatgatctcagctggttgtggaatgaaagcactgctctttac ccatccatttatttgaacactcagcagtctcctgtagctgctacactctatgtgcgcaatcgagttcgggaagccatcagagtttccaaaatacctgat gcaaaaagtccacttccggtttttgcatatacccgcatagtttttactgatcaagttttgaaattcctttctcaagatgaacttgtgtatacatttggcgaa actgttgctctgggtgcttctggaattgtaatatggggaaccctcagtataatgcgaagtatgaaatcttgcttgctcctagacaattacatggagact atactgaatccttacataatcaacgtcacactagcagccaaaatgtgtagccaagtgctttgccaggagcaaggagtgtgtataaggaaaaact ggaattcaagtgactatcttcacctcaacccagataattttgctattcaacttgagaaaggtggaaagttcacagtacgtggaaaaccgacacttg aagacctggagcaattttctgaaaaattttattgcagctgttatagcaccttgagttgtaaggagaaagctgatgtaaaagacactgatgctgttgat gtgtgtattgctgatggtgtctgtatagatgcttttctaaaacctcccatggagacagaagaacctcaaattttctacaatgcttcaccctccacactat ctqccacaatqtcatqtaqtatttqtttctatcattcttctqtaqcqaqttqtaa

[0350] SEQ ID NO: 2

[0351] Sequence of the modified endogenous promoter of E1a + encoding E1a-Delta24 region in VCN-01 (comprising the four binding sites to E2F-1, the binding site to Sp1 and the encoding region of E1 a- Delta24) aataagaggaagtgaaatctgaataattttgtgttactcatagcgcgtaatatttgtctagggccgcggggactttgaccgtttacgtggagactcgc ccaggtgtttttctcaggtgttttccgcgtacgtcggcggctcgtggctctttcgcggcaaaaaggatttggcgcgtaaaagtggttcgagtacgtcgg cggctcgtggctctttcgcggcaaaaaggatttggcgcgtaaaagtggttcgaagtacgtcgaccacaaaccccgcccagcgtcttgtcattggc gtcgacgctgtacggggtcaaagttggcgttttattattatagtcagctgacgtgtagtgtatttatacccggtgagttcctcaagaggccactcttgag tgccagcgagtagagttttctcctccgagccgctccgacaccgggactgaaaatgagacatattatctgccacggaggtgttattaccgaagaaa tggccgccagtcttttggaccagctgatcgaagaggtactggctgataatcttccacctcctagccattttgaaccacctacccttcacgaactgtat gatttagacgtgacggcccccgaagatcccaacgaggaggcggtttcgcagatttttcccgactctgtaatgttggcggtgcaggaagggattga cttactcacttttccgccggcgcccggttctccggagccgcctcacctttcccggcagcccgagcagccggagcagagagccttgggtccggtttc tatgccaaaccttgtaccggaggtgatcgatccacccagtgacgacgaggatgaagagggtgaggagtttgtgttagattatgtggagcacccc gggcacggttgcaggtcttgtcattatcaccggaggaatacgggggacccagatattatgtgttcgctttgctatatgaggacctgtggcatgtttgtc tacagtaagtgaaaattatgggcagtgggtgatagagtggtgggtttggtgtggtaattttttttttaatttttacagttttgtggtttaaagaattttgtattgt gatttttttaaaaggtcctgtgtctgaacctgagcctgagcccgagccagaaccggagcctgcaagacctacccgccgtcctaaaatggcgcctg ctatcctgagacgcccgacatcacctgtgtctagagaatgcaatagtagtacggatagctgtgactccggtccttctaacacacctcctgagatac acccggtggtcccgctgtgccccattaaaccagttgccgtgagagttggtgggcgtcgccaggctgtggaatgtatcgaggacttgcttaacgag cctgggcaacctttggacttgagctgtaaacgccccaggccataa

[0352] SEQ ID N0: 3

[0353] Complete sequence of VCN-01 catcatcaataatataccttattttggattgaagccaatatgataatgagggggtggagtttgtgacgtggcgcggggcgtgggaacggggcgggt gacgtagtagtgtggcggaagtgtgatgttgcaagtgtggcggaacacatgtaagcgacggatgtggcaaaagtgacgtttttggtgtgcgccgg tgtacacaggaagtgacaattttcgcgcggttttaggcggatgttgtagtaaatttgggcgtaaccgagtaagatttggccattttcgcgggaaaact gaataagaggaagtgaaatctgaataattttgtgttactcatagcgcgtaatatttgtctagggccgcggggactttgaccgtttacgtggagactcg cccaggtgtttttctcaggtgttttccgcgtacgtcggcggctcgtggctctttcgcggcaaaaaggatttggcgcgtaaaagtggttcgagtacgtcg gcggctcgtggctctttcgcggcaaaaaggatttggcgcgtaaaagtggttcgaagtacgtcgaccacaaaccccgcccagcgtcttgtcattgg cgtcgacgctgtacggggtcaaagttggcgttttattattatagtcagctgacgtgtagtgtatttatacccggtgagttcctcaagaggccactcttga gtgccagcgagtagagttttctcctccgagccgctccgacaccgggactgaaaatgagacatattatctgccacggaggtgttattaccgaagaa atggccgccagtcttttggaccagctgatcgaagaggtactggctgataatcttccacctcctagccattttgaaccacctacccttcacgaactgta tgatttagacgtgacggcccccgaagatcccaacgaggaggcggtttcgcagatttttcccgactctgtaatgttggcggtgcaggaagggattga cttactcacttttccgccggcgcccggttctccggagccgcctcacctttcccggcagcccgagcagccggagcagagagccttgggtccggtttc tatgccaaaccttgtaccggaggtgatcgatccacccagtgacgacgaggatgaagagggtgaggagtttgtgttagattatgtggagcacccc gggcacggttgcaggtcttgtcattatcaccggaggaatacgggggacccagatattatgtgttcgctttgctatatgaggacctgtggcatgtttgtc tacagtaagtgaaaattatgggcagtgggtgatagagtggtgggtttggtgtggtaattttttttttaatttttacagttttgtggtttaaagaattttgtattgt gatttttttaaaaggtcctgtgtctgaacctgagcctgagcccgagccagaaccggagcctgcaagacctacccgccgtcctaaaatggcgcctg ctatcctgagacgcccgacatcacctgtgtctagagaatgcaatagtagtacggatagctgtgactccggtccttctaacacacctcctgagatac acccggtggtcccgctgtgccccattaaaccagttgccgtgagagttggtgggcgtcgccaggctgtggaatgtatcgaggacttgcttaacgag cctgggcaacctttggacttgagctgtaaacgccccaggccataaggtgtaaacctgtgattgcgtgtgtggttaacgcctttgtttgctgaatgagtt gatgtaagtttaataaagggtgagataatgtttaacttgcatggcgtgttaaatggggcggggcttaaagggtatataatgcgccgtgggctaatctt ggttacatctgacctcatggaggcttgggagtgtttggaagatttttctgctgtgcgtaacttgctggaacagagctctaacagtacctcttggttttgga ggtttctgtggggctcatcccaggcaaagttagtctgcagaattaaggaggattacaagtgggaatttgaagagcttttgaaatcctgtggtgagct gtttgattctttgaatctgggtcaccaggcgcttttccaagagaaggtcatcaagactttggatttttccacaccggggcgcgctgcggctgctgttgct tttttgagttttataaaggataaatggagcgaagaaacccatctgagcggggggtacctgctggattttctggccatgcatctgtggagagcggttgt gagacacaagaatcgcctgctactgttgtcttccgtccgcccggcgataataccgacggaggagcagcagcagcagcaggaggaagccagg cggcggcggcaggagcagagcccatggaacccgagagccggcctggaccctcgggaatgaatgttgtacaggtggctgaactgtatccaga actgagacgcattttgacaattacagaggatgggcaggggctaaagggggtaaagagggagcggggggcttgtgaggctacagaggaggct aggaatctagcttttagcttaatgaccagacaccgtcctgagtgtattacttttcaacagatcaaggataattgcgctaatgagcttgatctgctggcg cagaagtattccatagagcagctgaccacttactggctgcagccaggggatgattttgaggaggctattagggtatatgcaaaggtggcacttag gccagattgcaagtacaagatcagcaaacttgtaaatatcaggaattgttgctacatttctgggaacggggccgaggtggagatagatacggag gatagggtggcctttagatgtagcatgataaatatgtggccgggggtgcttggcatggacggggtggttattatgaatgtaaggtttactggcccca attttagcggtacggttttcctggccaataccaaccttatcctacacggtgtaagcttctatgggtttaacaatacctgtgtggaagcctggaccgatgt aagggttcggggctgtgccttttactgctgctggaagggggtggtgtgtcgccccaaaagcagggcttcaattaagaaatgcctctttgaaaggtgt accttgggtatcctgtctgagggtaactccagggtgcgccacaatgtggcctccgactgtggttgcttcatgctagtgaaaagcgtggctgtgattaa gcataacatggtatgtggcaactgcgaggacagggcctctcagatgctgacctgctcggacggcaactgtcacctgctgaagaccattcacgta gccagccactctcgcaaggcctggccagtgtttgagcataacatactgacccgctgttccttgcatttgggtaacaggaggggggtgttcctacctt accaatgcaatttgagtcacactaagatattgcttgagcccgagagcatgtccaaggtgaacctgaacggggtgtttgacatgaccatgaagatc tggaaggtgctgaggtacgatgagacccgcaccaggtgcagaccctgcgagtgtggcggtaaacatattaggaaccagcctgtgatgctggat gtgaccgaggagctgaggcccgatcacttggtgctggcctgcacccgcgctgagtttggctctagcgatgaagatacagattgaggtactgaaat gtgtgggcgtggcttaagggtgggaaagaatatataaggtgggggtcttatgtagttttgtatctgttttgcagcagccgccgccgccatgagcacc aactcgtttgatggaagcattgtgagctcatatttgacaacgcgcatgcccccatgggccggggtgcgtcagaatgtgatgggctccagcattgat ggtcgccccgtcctgcccgcaaactctactaccttgacctacgagaccgtgtctggaacgccgttggagactgcagcctccgccgccgcttcagc cgctgcagccaccgcccgcgggattgtgactgactttgctttcctgagcccgcttgcaagcagtgcagcttcccgttcatccgcccgcgatgacaa gttgacggctcttttggcacaattggattctttgacccgggaacttaatgtcgtttctcagcagctgttggatctgcgccagcaggtttctgccctgaag gcttcctcccctcccaatgcggtttaaaacataaataaaaaaccagactctgtttggatttggatcaagcaagtgtcttgctgtctttatttaggggttttg cgcgcgcggtaggcccgggaccagcggtctcggtcgttgagggtcctgtgtattttttccaggacgtggtaaaggtgactctggatgttcagataca tgggcataagcccgtctctggggtggaggtagcaccactgcagagcttcatgctgcggggtggtgttgtagatgatccagtcgtagcaggagcgc tgggcgtggtgcctaaaaatgtctttcagtagcaagctgattgccaggggcaggcccttggtgtaagtgtttacaaagcggttaagctgggatgggt gcatacgtggggatatgagatgcatcttggactgtatttttaggttggctatgttcccagccatatccctccggggattcatgttgtgcagaaccacca gcacagtgtatccggtgcacttgggaaatttgtcatgtagcttagaaggaaatgcgtggaagaacttggagacgcccttgtgacctccaagattttc catgcattcgtccataatgatggcaatgggcccacgggcggcggcctgggcgaagatatttctgggatcactaacgtcatagttgtgttccaggat gagatcgtcataggccatttttacaaagcgcgggcggagggtgccagactgcggtataatggttccatccggcccaggggcgtagttaccctca cagatttgcatttcccacgctttgagttcagatggggggatcatgtctacctgcggggcgatgaagaaaacggtttccggggtaggggagatcag ctgggaagaaagcaggttcctgagcagctgcgacttaccgcagccggtgggcccgtaaatcacacctattaccggctgcaactggtagttaag agagctgcagctgccgtcatccctgagcaggggggccacttcgttaagcatgtccctgactcgcatgttttccctgaccaaatccgccagaaggc gctcgccgcccagcgatagcagttcttgcaaggaagcaaagtttttcaacggtttgagaccgtccgccgtaggcatgcttttgagcgtttgaccaa gcagttccaggcggtcccacagctcggtcacctgctctacggcatctcgatccagcatatctcctcgtttcgcgggttggggcggctttcgctgtacg gcagtagtcggtgctcgtccagacgggccagggtcatgtctttccacgggcgcagggtcctcgtcagcgtagtctgggtcacggtgaaggggtg cgctccgggctgcgcgctggccagggtgcgcttgaggctggtcctgctggtgctgaagcgctgccggtcttcgccctgcgcgtcggccaggtagc atttgaccatggtgtcatagtccagcccctccgcggcgtggcccttggcgcgcagcttgcccttggaggaggcgccgcacgaggggcagtgca gacttttgagggcgtagagcttgggcgcgagaaataccgattccggggagtaggcatccgcgccgcaggccccgcagacggtctcgcattcca cgagccaggtgagctctggccgttcggggtcaaaaaccaggtttcccccatgctttttgatgcgtttcttacctctggtttccatgagccggtgtccac gctcggtgacgaaaaggctgtccgtgtccccgtatacagacttgagaggcctgtcctcgagcggtgttccgcggtcctcctcgtatagaaactcgg accactctgagacaaaggctcgcgtccaggccagcacgaaggaggctaagtgggaggggtagcggtcgttgtccactagggggtccactcg ctccagggtgtgaagacacatgtcgccctcttcggcatcaaggaaggtgattggtttgtaggtgtaggccacgtgaccgggtgttcctgaagggg ggctataaaagggggtgggggcgcgttcgtcctcactctcttccgcatcgctgtctgcgagggccagctgttggggtgagtactccctctgaaaag cgggcatgacttctgcgctaagattgtcagtttccaaaaacgaggaggatttgatattcacctggcccgcggtgatgcctttgagggtggccgcatc catctggtcagaaaagacaatctttttgttgtcaagcttggtggcaaacgacccgtagagggcgttggacagcaacttggcgatggagcgcagg gtttggtttttgtcgcgatcggcgcgctccttggccgcgatgtttagctgcacgtattcgcgcgcaacgcaccgccattcgggaaagacggtggtgc gctcgtcgggcaccaggtgcacgcgccaaccgcggttgtgcagggtgacaaggtcaacgctggtggctacctctccgcgtaggcgctcgttggt ccagcagaggcggccgcccttgcgcgagcagaatggcggtagggggtctagctgcgtctcgtccggggggtctgcgtccacggtaaagaccc cgggcagcaggcgcgcgtcgaagtagtctatcttgcatccttgcaagtctagcgcctgctgccatgcgcgggcggcaagcgcgcgctcgtatgg gttgagtgggggaccccatggcatggggtgggtgagcgcggaggcgtacatgccgcaaatgtcgtaaacgtagaggggctctctgagtattcc aagatatgtagggtagcatcttccaccgcggatgctggcgcgcacgtaatcgtatagttcgtgcgagggagcgaggaggtcgggaccgaggtt gctacgggcgggctgctctgctcggaagactatctgcctgaagatggcatgtgagttggatgatatggttggacgctggaagacgttgaagctgg cgtctgtgagacctaccgcgtcacgcacgaaggaggcgtaggagtcgcgcagcttgttgaccagctcggcggtgacctgcacgtctagggcgc agtagtccagggtttccttgatgatgtcatacttatcctgtcccttttttttccacagctcgcggttgaggacaaactcttcgcggtctttccagtactcttgg atcggaaacccgtcggcctccgaacggtaagagcctagcatgtagaactggttgacggcctggtaggcgcagcatcccttttctacgggtagcg cgtatgcctgcgcggccttccggagcgaggtgtgggtgagcgcaaaggtgtccctgaccatgactttgaggtactggtatttgaagtcagtgtcgtc gcatccgccctgctcccagagcaaaaagtccgtgcgctttttggaacgcggatttggcagggcgaaggtgacatcgttgaagagtatctttcccgc gcgaggcataaagttgcgtgtgatgcggaagggtcccggcacctcggaacggttgttaattacctgggcggcgagcacgatctcgtcaaagcc gttgatgttgtggcccacaatgtaaagttccaagaagcgcgggatgcccttgatggaaggcaattttttaagttcctcgtaggtgagctcttcagggg agctgagcccgtgctctgaaagggcccagtctgcaagatgagggttggaagcgacgaatgagctccacaggtcacgggccattagcatttgca ggtggtcgcgaaaggtcctaaactggcgacctatggccattttttctggggtgatgcagtagaaggtaagcgggtcttgttcccagcggtcccatcc aaggttcgcggctaggtctcgcgcggcagtcactagaggctcatctccgccgaacttcatgaccagcatgaagggcacgagctgcttcccaaa ggcccccatccaagtataggtctctacatcgtaggtgacaaagagacgctcggtgcgaggatgcgagccgatcgggaagaactggatctccc gccaccaattggaggagtggctattgatgtggtgaaagtagaagtccctgcgacgggccgaacactcgtgctggcttttgtaaaaacgtgcgca gtactggcagcggtgcacgggctgtacatcctgcacgaggttgacctgacgaccgcgcacaaggaagcagagtgggaatttgagcccctcgc ctggcgggtttggctggtggtcttctacttcggctgcttgtccttgaccgtctggctgctcgaggggagttacggtggatcggaccaccacgccgcgc gagcccaaagtccagatgtccgcgcgcggcggtcggagcttgatgacaacatcgcgcagatgggagctgtccatggtctggagctcccgcgg cgtcaggtcaggcgggagctcctgcaggtttacctcgcatagacgggtcagggcgcgggctagatccaggtgatacctaatttccaggggctgg ttggtggcggcgtcgatggcttgcaagaggccgcatccccgcggcgcgactacggtaccgcgcggcgggcggtgggccgcgggggtgtcctt ggatgatgcatctaaaagcggtgacgcgggcgagcccccggaggtagggggggctccggacccgccgggagagggggcaggggcacgt cggcgccgcgcgcgggcaggagctggtgctgcgcgcgtaggttgctggcgaacgcgacgacgcggcggttgatctcctgaatctggcgcctct gcgtgaagacgacgggcccggtgagcttgaacctgaaagagagttcgacagaatcaatttcggtgtcgttgacggcggcctggcgcaaaatct cctgcacgtctcctgagttgtcttgataggcgatctcggccatgaactgctcgatctcttcctcctggagatctccgcgtccggctcgctccacggtgg cggcgaggtcgttggaaatgcgggccatgagctgcgagaaggcgttgaggcctccctcgttccagacgcggctgtagaccacgcccccttcgg catcgcgggcgcgcatgaccacctgcgcgagattgagctccacgtgccgggcgaagacggcgtagtttcgcaggcgctgaaagaggtagttg agggtggtggcggtgtgttctgccacgaagaagtacataacccagcgtcgcaacgtggattcgttgatatcccccaaggcctcaaggcgctccat ggcctcgtagaagtccacggcgaagttgaaaaactgggagttgcgcgccgacacggttaactcctcctccagaagacggatgagctcggcga cagtgtcgcgcacctcgcgctcaaaggctacaggggcctcttcttcttcttcaatctcctcttccataagggcctccccttcttcttcttctggcggcggt gggggaggggggacacggcggcgacgacggcgcaccgggaggcggtcgacaaagcgctcgatcatctccccgcggcgacggcgcatgg tctcggtgacggcgcggccgttctcgcgggggcgcagttggaagacgccgcccgtcatgtcccggttatgggttggcggggggctgccatgcgg cagggatacggcgctaacgatgcatctcaacaattgttgtgtaggtactccgccgccgagggacctgagcgagtccgcatcgaccggatcgga aaacctctcgagaaaggcgtctaaccagtcacagtcgcaaggtaggctgagcaccgtggcgggcggcagcgggcggcggtcggggttgtttc tggcggaggtgctgctgatgatgtaattaaagtaggcggtcttgagacggcggatggtcgacagaagcaccatgtccttgggtccggcctgctga atgcgcaggcggtcggccatgccccaggcttcgttttgacatcggcgcaggtctttgtagtagtcttgcatgagcctttctaccggcacttcttcttctcc ttcctcttgtcctgcatctcttgcatctatcgctgcggcggcggcggagtttggccgtaggtggcgccctcttcctcccatgcgtgtgaccccgaagcc cctcatcggctgaagcagggctaggtcggcgacaacgcgctcggctaatatggcctgctgcacctgcgtgagggtagactggaagtcatccatg tccacaaagcggtggtatgcgcccgtgttgatggtgtaagtgcagttggccataacggaccagttaacggtctggtgacccggctgcgagagctc ggtgtacctgagacgcgagtaagccctcgagtcaaatacgtagtcgttgcaagtccgcaccaggtactggtatcccaccaaaaagtgcggcgg cggctggcggtagaggggccagcgtagggtggccggggctccgggggcgagatcttccaacataaggcgatgatatccgtagatgtacctgg acatccaggtgatgccggcggcggtggtggaggcgcgcggaaagtcgcggacgcggttccagatgttgcgcagcggcaaaaagtgctccat ggtcgggacgctctggccggtcaggcgcgcgcaatcgttgacgctctagaccgtgcaaaaggagagcctgtaagcgggcactcttccgtggtct ggtggataaattcgcaagggtatcatggcggacgaccggggttcgagccccgtatccggccgtccgccgtgatccatgcggttaccgcccgcgt gtcgaacccaggtgtgcgacgtcagacaacgggggagtgctccttttggcttccttccaggcgcggcggctgctgcgctagcttttttggccactgg ccgcgcgcagcgtaagcggttaggctggaaagcgaaagcattaagtggctcgctccctgtagccggagggttattttccaagggttgagtcgcg ggacccccggttcgagtctcggaccggccggactgcggcgaacgggggtttgcctccccgtcatgcaagaccccgcttgcaaattcctccgga aacagggacgagccccttttttgcttttcccagatgcatccggtgctgcggcagatgcgcccccctcctcagcagcggcaagagcaagagcagc ggcagacatgcagggcaccctcccctcctcctaccgcgtcaggaggggcgacatccgcggttgacgcggcagcagatggtgattacgaaccc ccgcggcgccgggcccggcactacctggacttggaggagggcgagggcctggcgcggctaggagcgccctctcctgagcggcacccaagg gtgcagctgaagcgtgatacgcgtgaggcgtacgtgccgcggcagaacctgtttcgcgaccgcgagggagaggagcccgaggagatgcgg gatcgaaagttccacgcagggcgcgagctgcggcatggcctgaatcgcgagcggttgctgcgcgaggaggactttgagcccgacgcgcgaa ccgggattagtcccgcgcgcgcacacgtggcggccgccgacctggtaaccgcatacgagcagacggtgaaccaggagattaactttcaaaa aagctttaacaaccacgtgcgtacgcttgtggcgcgcgaggaggtggctataggactgatgcatctgtgggactttgtaagcgcgctggagcaa aacccaaatagcaagccgctcatggcgcagctgttccttatagtgcagcacagcagggacaacgaggcattcagggatgcgctgctaaacat agtagagcccgagggccgctggctgctcgatttgataaacatcctgcagagcatagtggtgcaggagcgcagcttgagcctggctgacaaggt ggccgccatcaactattccatgcttagcctgggcaagttttacgcccgcaagatataccataccccttacgttcccatagacaaggaggtaaagat cgaggggttctacatgcgcatggcgctgaaggtgcttaccttgagcgacgacctgggcgtttatcgcaacgagcgcatccacaaggccgtgagc gtgagccggcggcgcgagctcagcgaccgcgagctgatgcacagcctgcaaagggccctggctggcacgggcagcggcgatagagaggc cgagtcctactttgacgcgggcgctgacctgcgctgggccccaagccgacgcgccctggaggcagctggggccggacctgggctggcggtgg cacccgcgcgcgctggcaacgtcggcggcgtggaggaatatgacgaggacgatgagtacgagccagaggacggcgagtactaagcggtg atgtttctgatcagatgatgcaagacgcaacggacccggcggtgcgggcggcgctgcagagccagccgtccggccttaactccacggacgac tggcgccaggtcatggaccgcatcatgtcgctgactgcgcgcaatcctgacgcgttccggcagcagccgcaggccaaccggctctccgcaatt ctggaagcggtggtcccggcgcgcgcaaaccccacgcacgagaaggtgctggcgatcgtaaacgcgctggccgaaaacagggccatccg gcccgacgaggccggcctggtctacgacgcgctgcttcagcgcgtggctcgttacaacagcggcaacgtgcagaccaacctggaccggctgg tgggggatgtgcgcgaggccgtggcgcagcgtgagcgcgcgcagcagcagggcaacctgggctccatggttgcactaaacgccttcctgagt acacagcccgccaacgtgccgcggggacaggaggactacaccaactttgtgagcgcactgcggctaatggtgactgagacaccgcaaagtg aggtgtaccagtctgggccagactattttttccagaccagtagacaaggcctgcagaccgtaaacctgagccaggctttcaaaaacttgcaggg gctgtggggggtgcgggctcccacaggcgaccgcgcgaccgtgtctagcttgctgacgcccaactcgcgcctgttgctgctgctaatagcgccct tcacggacagtggcagcgtgtcccgggacacatacctaggtcacttgctgacactgtaccgcgaggccataggtcaggcgcatgtggacgagc atactttccaggagattacaagtgtcagccgcgcgctggggcaggaggacacgggcagcctggaggcaaccctaaactacctgctgaccaac cggcggcagaagatcccctcgttgcacagtttaaacagcgaggaggagcgcattttgcgctacgtgcagcagagcgtgagccttaacctgatg cgcgacggggtaacgcccagcgtggcgctggacatgaccgcgcgcaacatggaaccgggcatgtatgcctcaaaccggccgtttatcaacc gcctaatggactacttgcatcgcgcggccgccgtgaaccccgagtatttcaccaatgccatcttgaacccgcactggctaccgccccctggtttcta caccgggggattcgaggtgcccgagggtaacgatggattcctctgggacgacatagacgacagcgtgttttccccgcaaccgcagaccctgct agagttgcaacagcgcgagcaggcagaggcggcgctgcgaaaggaaagcttccgcaggccaagcagcttgtccgatctaggcgctgcggc cccgcggtcagatgctagtagcccatttccaagcttgatagggtctcttaccagcactcgcaccacccgcccgcgcctgctgggcgaggaggag tacctaaacaactcgctgctgcagccgcagcgcgaaaaaaacctgcctccggcatttcccaacaacgggatagagagcctagtggacaagat gagtagatggaagacgtacgcgcaggagcacagggacgtgccaggcccgcgcccgcccacccgtcgtcaaaggcacgaccgtcagcgg ggtctggtgtgggaggacgatgactcggcagacgacagcagcgtcctggatttgggagggagtggcaacccgtttgcgcaccttcgccccagg ctggggagaatgttttaaaaaaaaaaaaagcatgatgcaaaataaaaaactcaccaaggccatggcaccgagcgttggttttcttgtattcccctt agtatgcggcgcgcggcgatgtatgaggaaggtcctcctccctcctacgagagtgtggtgagcgcggcgccagtggcggcggcgctgggttctc ccttcgatgctcccctggacccgccgtttgtgcctccgcggtacctgcggcctaccggggggagaaacagcatccgttactctgagttggcacccc tattcgacaccacccgtgtgtacctggtggacaacaagtcaacggatgtggcatccctgaactaccagaacgaccacagcaactttctgaccac ggtcattcaaaacaatgactacagcccgggggaggcaagcacacagaccatcaatcttgacgaccggtcgcactggggcggcgacctgaaa accatcctgcataccaacatgccaaatgtgaacgagttcatgtttaccaataagtttaaggcgcgggtgatggtgtcgcgcttgcctactaaggac aatcaggtggagctgaaatacgagtgggtggagttcacgctgcccgagggcaactactccgagaccatgaccatagaccttatgaacaacgc gatcgtggagcactacttgaaagtgggcagacagaacggggttctggaaagcgacatcggggtaaagtttgacacccgcaacttcagactgg ggtttgaccccgtcactggtcttgtcatgcctggggtatatacaaacgaagccttccatccagacatcattttgctgccaggatgcggggtggacttc acccacagccgcctgagcaacttgttgggcatccgcaagcggcaacccttccaggagggctttaggatcacctacgatgatctggagggtggta acattcccgcactgttggatgtggacgcctaccaggcgagcttgaaagatgacaccgaacagggcgggggtggcgcaggcggcagcaaca gcagtggcagcggcgcggaagagaactccaacgcggcagccgcggcaatgcagccggtggaggacatgaacgatcatgccattcgcggc gacacctttgccacacgggctgaggagaagcgcgctgaggccgaagcagcggccgaagctgccgcccccgctgcgcaacccgaggtcga gaagcctcagaagaaaccggtgatcaaacccctgacagaggacagcaagaaacgcagttacaacctaataagcaatgacagcaccttcac ccagtaccgcagctggtaccttgcatacaactacggcgaccctcagaccggaatccgctcatggaccctgctttgcactcctgacgtaacctgcg gctcggagcaggtctactggtcgttgccagacatgatgcaagaccccgtgaccttccgctccacgcgccagatcagcaactttccggtggtgggc gccgagctgttgcccgtgcactccaagagcttctacaacgaccaggccgtctactcccaactcatccgccagtttacctctctgacccacgtgttca atcgctttcccgagaaccagattttggcgcgcccgccagcccccaccatcaccaccgtcagtgaaaacgttcctgctctcacagatcacgggac gctaccgctgcgcaacagcatcggaggagtccagcgagtgaccattactgacgccagacgccgcacctgcccctacgtttacaaggccctgg gcatagtctcgccgcgcgtcctatcgagccgcactttttgagcaagcatgtccatccttatatcgcccagcaataacacaggctggggcctgcgctt cccaagcaagatgtttggcggggccaagaagcgctccgaccaacacccagtgcgcgtgcgcgggcactaccgcgcgccctggggcgcgca caaacgcggccgcactgggcgcaccaccgtcgatgacgccatcgacgcggtggtggaggaggcgcgcaactacacgcccacgccgccac cagtgtccacagtggacgcggccattcagaccgtggtgcgcggagcccggcgctatgctaaaatgaagagacggcggaggcgcgtagcac gtcgccaccgccgccgacccggcactgccgcccaacgcgcggcggcggccctgcttaaccgcgcacgtcgcaccggccgacgggcggcc atgcgggccgctcgaaggctggccgcgggtattgtcactgtgccccccaggtccaggcgacgagcggccgccgcagcagccgcggccatta gtgctatgactcagggtcgcaggggcaacgtgtattgggtgcgcgactcggttagcggcctgcgcgtgcccgtgcgcacccgccccccgcgca actagattgcaagaaaaaactacttagactcgtactgttgtatgtatccagcggcggcggcgcgcaacgaagctatgtccaagcgcaaaatcaa agaagagatgctccaggtcatcgcgccggagatctatggccccccgaagaaggaagagcaggattacaagccccgaaagctaaagcgggt caaaaagaaaaagaaagatgatgatgatgaacttgacgacgaggtggaactgctgcacgctaccgcgcccaggcgacgggtacagtggaa aggtcgacgcgtaaaacgtgttttgcgacccggcaccaccgtagtctttacgcccggtgagcgctccacccgcacctacaagcgcgtgtatgatg aggtgtacggcgacgaggacctgcttgagcaggccaacgagcgcctcggggagtttgcctacggaaagcggcataaggacatgctggcgttg ccgctggacgagggcaacccaacacctagcctaaagcccgtaacactgcagcaggtgctgcccgcgcttgcaccgtccgaagaaaagcgc ggcctaaagcgcgagtctggtgacttggcacccaccgtgcagctgatggtacccaagcgccagcgactggaagatgtcttggaaaaaatgac cgtggaacctgggctggagcccgaggtccgcgtgcggccaatcaagcaggtggcgccgggactgggcgtgcagaccgtggacgttcagata cccactaccagtagcaccagtattgccaccgccacagagggcatggagacacaaacgtccccggttgcctcagcggtggcggatgccgcggt gcaggcggtcgctgcggccgcgtccaagacctctacggaggtgcaaacggacccgtggatgtttcgcgtttcagccccccggcgcccgcgcc gttcgaggaagtacggcgccgccagcgcgctactgcccgaatatgccctacatccttccattgcgcctacccccggctatcgtggctacacctac cgccccagaagacgagcaactacccgacgccgaaccaccactggaacccgccgccgccgtcgccgtcgccagcccgtgctggccccgattt ccgtgcgcagggtggctcgcgaaggaggcaggaccctggtgctgccaacagcgcgctaccaccccagcatcgtttaaaagccggtctttgtgg ttcttgcagatatggccctcacctgccgcctccgtttcccggtgccgggattccgaggaagaatgcaccgtaggaggggcatggccggccacgg cctgacgggcggcatgcgtcgtgcgcaccaccggcggcggcgcgcgtcgcaccgtcgcatgcgcggcggtatcctgcccctccttattccactg atcgccgcggcgattggcgccgtgcccggaattgcatccgtggccttgcaggcgcagagacactgattaaaaacaagttgcatgtggaaaaat caaaataaaaagtctggactctcacgctcgcttggtcctgtaactattttgtagaatggaagacatcaactttgcgtctctggccccgcgacacggct cgcgcccgttcatgggaaactggcaagatatcggcaccagcaatatgagcggtggcgccttcagctggggctcgctgtggagcggcattaaaa atttcggttccaccgttaagaactatggcagcaaggcctggaacagcagcacaggccagatgctgagggataagttgaaagagcaaaatttcc aacaaaaggtggtagatggcctggcctctggcattagcggggtggtggacctggccaaccaggcagtgcaaaataagattaacagtaagcttg atccccgccctcccgtagaggagcctccaccggccgtggagacagtgtctccagaggggcgtggcgaaaagcgtccgcgccccgacaggg aagaaactctggtgacgcaaatagacgagcctccctcgtacgaggaggcactaaagcaaggcctgcccaccacccgtcccatcgcgcccat ggctaccggagtgctgggccagcacacacccgtaacgctggacctgcctccccccgccgacacccagcagaaacctgtgctgccaggcccg accgccgttgttgtaacccgtcctagccgcgcgtccctgcgccgcgccgccagcggtccgcgatcgttgcggcccgtagccagtggcaactggc aaagcacactgaacagcatcgtgggtctgggggtgcaatccctgaagcgccgacgatgcttctgatagctaacgtgtcgtatgtgtgtcatgtatg cgtccatgtcgccgccagaggagctgctgagccgccgcgcgcccgctttccaagatggctaccccttcgatgatgccgcagtggtcttacatgca catctcgggccaggacgcctcggagtacctgagccccgggctggtgcagtttgcccgcgccaccgagacgtacttcagcctgaataacaagttt agaaaccccacggtggcgcctacgcacgacgtgaccacagaccggtcccagcgtttgacgctgcggttcatccctgtggaccgtgaggatact gcgtactcgtacaaggcgcggttcaccctagctgtgggtgataaccgtgtgctggacatggcttccacgtactttgacatccgcggcgtgctggac aggggccctacttttaagccctactctggcactgcctacaacgccctggctcccaagggtgccccaaatccttgcgaatgggatgaagctgctact gctcttgaaataaacctagaagaagaggacgatgacaacgaagacgaagtagacgagcaagctgagcagcaaaaaactcacgtatttggg caggcgccttattctggtataaatattacaaaggagggtattcaaataggtgtcgaaggtcaaacacctaaatatgccgataaaacatttcaacct gaacctcaaataggagaatctcagtggtacgaaacagaaattaatcatgcagctgggagagtcctaaaaaagactaccccaatgaaaccatg ttacggttcatatgcaaaacccacaaatgaaaatggagggcaaggcattcttgtaaagcaacaaaatggaaagctagaaagtcaagtggaaa tgcaatttttctcaactactgaggcagccgcaggcaatggtgataacttgactcctaaagtggtattgtacagtgaagatgtagatatagaaacccc agacactcatatttcttacatgcccactattaaggaaggtaactcacgagaactaatgggccaacaatctatgcccaacaggcctaattacattgc ttttagggacaattttattggtctaatgtattacaacagcacgggtaatatgggtgttctggcgggccaagcatcgcagttgaatgctgttgtagatttg caagacagaaacacagagctttcataccagcttttgcttgattccattggtgatagaaccaggtacttttctatgtggaatcaggctgttgacagctat gatccagatgttagaattattgaaaatcatggaactgaagatgaacttccaaattactgctttccactgggaggtgtgattaatacagagactcttac caaggtaaaacctaaaacaggtcaggaaaatggatgggaaaaagatgctacagaattttcagataaaaatgaaataagagttggaaataattt tgccatggaaatcaatctaaatgccaacctgtggagaaatttcctgtactccaacatagcgctgtatttgcccgacaagctaaagtacagtccttcc aacgtaaaaatttctgataacccaaacacctacgactacatgaacaagcgagtggtggctcccgggctagtggactgctacattaaccttggag cacgctggtcccttgactatatggacaacgtcaacccatttaaccaccaccgcaatgctggcctgcgctaccgctcaatgttgctgggcaatggtc gctatgtgcccttccacatccaggtgcctcagaagttctttgccattaaaaacctccttctcctgccgggctcatacacctacgagtggaacttcagg aaggatgttaacatggttctgcagagctccctaggaaatgacctaagggttgacggagccagcattaagtttgatagcatttgcctttacgccacctt cttccccatggcccacaacaccgcctccacgcttgaggccatgcttagaaacgacaccaacgaccagtcctttaacgactatctctccgccgcc aacatgctctaccctatacccgccaacgctaccaacgtgcccatatccatcccctcccgcaactgggcggctttccgcggctgggccttcacgcg ccttaagactaaggaaaccccatcactgggctcgggctacgacccttattacacctactctggctctataccctacctagatggaaccttttacctca accacacctttaagaaggtggccattacctttgactcttctgtcagctggcctggcaatgaccgcctgcttacccccaacgagtttgaaattaagcg ctcagttgacggggagggttacaacgttgcccagtgtaacatgaccaaagactggttcctggtacaaatgctagctaactataacattggctacca gggcttctatatcccagagagctacaaggaccgcatgtactccttctttagaaacttccagcccatgagccgtcaggtggtggatgatactaaata caaggactaccaacaggtgggcatcctacaccaacacaacaactctggatttgttggctaccttgcccccaccatgcgcgaaggacaggccta ccctgctaacttcccctatccgcttataggcaagaccgcagttgacagcattacccagaaaaagtttctttgcgatcgcaccctttggcgcatcccat tctccagtaactttatgtccatgggcgcactcacagacctgggccaaaaccttctctacgccaactccgcccacgcgctagacatgacttttgaggt ggatcccatggacgagcccacccttctttatgttttgtttgaagtctttgacgtggtccgtgtgcaccagccgcaccgcggcgtcatcgaaaccgtgt acctgcgcacgcccttctcggccggcaacgccacaacataaagaagcaagcaacatcaacaacagctgccgccatgggctccagtgagca ggaactgaaagccattgtcaaagatcttggttgtgggccatattttttgggcacctatgacaagcgctttccaggctttgtttctccacacaagctcgc ctgcgccatagtcaatacggccggtcgcgagactgggggcgtacactggatggcctttgcctggaacccgcactcaaaaacatgctacctctttg agccctttggcttttctgaccagcgactcaagcaggtttaccagtttgagtacgagtcactcctgcgccgtagcgccattgcttcttcccccgaccgct gtataacgctggaaaagtccacccaaagcgtacaggggcccaactcggccgcctgtggactattctgctgcatgtttctccacgcctttgccaact ggccccaaactcccatggatcacaaccccaccatgaaccttattaccggggtacccaactccatgctcaacagtccccaggtacagcccaccc tgcgtcgcaaccaggaacagctctacagcttcctggagcgccactcgccctacttccgcagccacagtgcgcagattaggagcgccacttcttttt gtcacttgaaaaacatgtaaaaataatgtactagagacactttcaataaaggcaaatgcttttatttgtacactctcgggtgattatttacccccaccct tgccgtctgcgccgtttaaaaatcaaaggggttctgccgcgcatcgctatgcgccactggcagggacacgttgcgatactggtgtttagtgctccac ttaaactcaggcacaaccatccgcggcagctcggtgaagttttcactccacaggctgcgcaccatcaccaacgcgtttagcaggtcgggcgccg atatcttgaagtcgcagttggggcctccgccctgcgcgcgcgagttgcgatacacagggttgcagcactggaacactatcagcgccgggtggtg cacgctggccagcacgctcttgtcggagatcagatccgcgtccaggtcctccgcgttgctcagggcgaacggagtcaactttggtagctgccttcc caaaaagggcgcgtgcccaggctttgagttgcactcgcaccgtagtggcatcaaaaggtgaccgtgcccggtctgggcgttaggatacagcgc ctgcataaaagccttgatctgcttaaaagccacctgagcctttgcgccttcagagaagaacatgccgcaagacttgccggaaaactgattggccg gacaggccgcgtcgtgcacgcagcaccttgcgtcggtgttggagatctgcaccacatttcggccccaccggttcttcacgatcttggccttgctaga ctgctccttcagcgcgcgctgcccgttttcgctcgtcacatccatttcaatcacgtgctccttatttatcataatgcttccgtgtagacacttaagctcgcc ttcgatctcagcgcagcggtgcagccacaacgcgcagcccgtgggctcgtgatgcttgtaggtcacctctgcaaacgactgcaggtacgcctgc aggaatcgccccatcatcgtcacaaaggtcttgttgctggtgaaggtcagctgcaacccgcggtgctcctcgttcagccaggtcttgcatacggcc gccagagcttccacttggtcaggcagtagtttgaagttcgcctttagatcgttatccacgtggtacttgtccatcagcgcgcgcgcagcctccatgcc cttctcccacgcagacacgatcggcacactcagcgggttcatcaccgtaatttcactttccgcttcgctgggctcttcctcttcctcttgcgtccgcata ccacgcgccactgggtcgtcttcattcagccgccgcactgtgcgcttacctcctttgccatgcttgattagcaccggtgggttgctgaaacccaccat ttgtagcgccacatcttctctttcttcctcgctgtccacgattacctctggtgatggcgggcgctcgggcttgggagaagggcgcttctttttcttcttggg cgcaatggccaaatccgccgccgaggtcgatggccgcgggctgggtgtgcgcggcaccagcgcgtcttgtgatgagtcttcctcgtcctcggact cgatacgccgcctcatccgcttttttgggggcgcccggggaggcggcggcgacggggacggggacgacacgtcctccatggttgggggacgt cgcgccgcaccgcgtccgcgctcgggggtggtttcgcgctgctcctcttcccgactggccatttccttctcctataggcagaaaaagatcatggagt cagtcgagaagaaggacagcctaaccgccccctctgagttcgccaccaccgcctccaccgatgccgccaacgcgcctaccaccttccccgtc gaggcacccccgcttgaggaggaggaagtgattatcgagcaggacccaggttttgtaagcgaagacgacgaggaccgctcagtaccaacag aggataaaaagcaagaccaggacaacgcagaggcaaacgaggaacaagtcgggcggggggacgaaaggcatggcgactacctagatg tgggagacgacgtgctgttgaagcatctgcagcgccagtgcgccattatctgcgacgcgttgcaagagcgcagcgatgtgcccctcgccatagc ggatgtcagccttgcctacgaacgccacctattctcaccgcgcgtaccccccaaacgccaagaaaacggcacatgcgagcccaacccgcgc ctcaacttctaccccgtatttgccgtgccagaggtgcttgccacctatcacatctttttccaaaactgcaagatacccctatcctgccgtgccaaccgc agccgagcggacaagcagctggccttgcggcagggcgctgtcatacctgatatcgcctcgctcaacgaagtgccaaaaatctttgagggtcttg gacgcgacgagaagcgcgcggcaaacgctctgcaacaggaaaacagcgaaaatgaaagtcactctggagtgttggtggaactcgagggtg acaacgcgcgcctagccgtactaaaacgcagcatcgaggtcacccactttgcctacccggcacttaacctaccccccaaggtcatgagcacag tcatgagtgagctgatcgtgcgccgtgcgcagcccctggagagggatgcaaatttgcaagaacaaacagaggagggcctacccgcagttggc gacgagcagctagcgcgctggcttcaaacgcgcgagcctgccgacttggaggagcgacgcaaactaatgatggccgcagtgctcgttaccgt ggagcttgagtgcatgcagcggttctttgctgacccggagatgcagcgcaagctagaggaaacattgcactacacctttcgacagggctacgta cgccaggcctgcaagatctccaacgtggagctctgcaacctggtctcctaccttggaattttgcacgaaaaccgccttgggcaaaacgtgcttcat tccacgctcaagggcgaggcgcgccgcgactacgtccgcgactgcgtttacttatttctatgctacacctggcagacggccatgggcgtttggca gcagtgcttggaggagtgcaacctcaaggagctgcagaaactgctaaagcaaaacttgaaggacctatggacggccttcaacgagcgctccg tggccgcgcacctggcggacatcattttccccgaacgcctgcttaaaaccctgcaacagggtctgccagacttcaccagtcaaagcatgttgcag aactttaggaactttatcctagagcgctcaggaatcttgcccgccacctgctgtgcacttcctagcgactttgtgcccattaagtaccgcgaatgccct ccgccgctttggggccactgctaccttctgcagctagccaactaccttgcctaccactctgacataatggaagacgtgagcggtgacggtctactg gagtgtcactgtcgctgcaacctatgcaccccgcaccgctccctggtttgcaattcgcagctgcttaacgaaagtcaaattatcggtacctttgagct gcagggtccctcgcctgacgaaaagtccgcggctccggggttgaaactcactccggggctgtggacgtcggcttaccttcgcaaatttgtacctg aggactaccacgcccacgagattaggttctacgaagaccaatcccgcccgcctaatgcggagcttaccgcctgcgtcattacccagggccaca ttcttggccaattgcaagccatcaacaaagcccgccaagagtttctgctacgaaagggacggggggtttacttggacccccagtccggcgagga gctcaacccaatccccccgccgccgcagccctatcagcagcagccgcgggcccttgcttcccaggatggcacccaaaaagaagctgcagct gccgccgccacccacggacgaggaggaatactgggacagtcaggcagaggaggttttggacgaggaggaggaggacatgatggaagact gggagagcctagacgaggaagcttccgaggtcgaagaggtgtcagacgaaacaccgtcaccctcggtcgcattcccctcgccggcgcccca gaaatcggcaaccggttccagcatggctacaacctccgctcctcaggcgccgccggcactgcccgttcgccgacccaaccgtagatgggaca ccactggaaccagggccggtaagtccaagcagccgccgccgttagcccaagagcaacaacagcgccaaggctaccgctcatggcgcggg cacaagaacgccatagttgcttgcttgcaagactgtgggggcaacatctccttcgcccgccgctttcttctctaccatcacggcgtggccttcccccg taacatcctgcattactaccgtcatctctacagcccatactgcaccggcggcagcggcagcaacagcagcggccacacagaagcaaaggcg accggatagcaagactctgacaaagcccaagaaatccacagcggcggcagcagcaggaggaggagcgctgcgtctggcgcccaacgaa cccgtatcgacccgcgagcttagaaacaggatttttcccactctgtatgctatatttcaacagagcaggggccaagaacaagagctgaaaataa aaaacaggtctctgcgatccctcacccgcagctgcctgtatcacaaaagcgaagatcagcttcggcgcacgctggaagacgcggaggctctct tcagtaaatactgcgcgctgactcttaaggactagtttcgcgccctttctcaaatttaagcgcgaaaactacgtcatctccagcggccacacccggc gccagcacctgttgtcagcgccattatgagcaaggaaattcccacgccctacatgtggagttaccagccacaaatgggacttgcggctggagct gcccaagactactcaacccgaataaactacatgagcgcgggaccccacatgatatcccgggtcaacggaatacgcgcccaccgaaaccga attctcctggaacaggcggctattaccaccacacctcgtaataaccttaatccccgtagttggcccgctgccctggtgtaccaggaaagtcccgct cccaccactgtggtacttcccagagacgcccaggccgaagttcagatgactaactcaggggcgcagcttgcgggcggctttcgtcacagggtg cggtcgcccgggcagggtataactcacctgacaatcagagggcgaggtattcagctcaacgacgagtcggtgagctcctcgcttggtctccgtc cggacgggacatttcagatcggcggcgccggccgctcttcattcacgcctcgtcaggcaatcctaactctgcagacctcgtcctctgagccgcgct ctggaggcattggaactctgcaatttattgaggagtttgtgccatcggtctactttaaccccttctcgggacctcccggccactatccggatcaatttatt cctaactttgacgcggtaaaggactcggcggacggctacgactgaatgttaagtggagaggcagagcaactgcgcctgaaacacctggtcca ctgtcgccgccacaagtgctttgcccgcgactccggtgagttttgctactttgaattgcccgaggatcatatcgagggcccggcgcacggcgtccg gcttaccgcccagggagagcttgcccgtagcctgattcgggagtttacccagcgccccctgctagttgagcgggacaggggaccctgtgttctca ctgtgatttgcaactgtcctaaccctggattacatcaagatctttgttgccatctctgtgctgagtataataaatacagaaattaaaatatactggggct cctatcgccatcctgtaaacgccaccgtcttcacccgcccaagcaaaccaaggcgaaccttacctggtacttttaacatctctccctctgtgatttac aacagtttcaacccagacggagtgagtctacgagagaacctctccgagctcagctactccatcagaaaaaacaccaccctccttacctgccgg gaacgtacgagtgcgtcaccggccgctgcaccacacctaccgcctgaccgtaaaccagactttttccggacagacctcaataactctgtttacca gaacaggaggtgagcttagaaaacccttagggtattaggccaaaggcgcagctactgtggggtttatgaacaattcaagcaactctacgggcta ttctaattcaggtttctctagaatcggggttggggttattctctgtcttgtgattctctttattcttatactaacgcttctctgcctaaggctcgccgcctgctgt gtgcacatttgcatttattgtcagctttttaaacgctggggtcgccacccaagatgattaggtacataatcctaggtttactcacccttgcgtcagccca cggtaccacccaaaaggtggattttaaggagccagcctgtaatgttacattcgcagctgaagctaatgagtgcaccactcttataaaatgcacca cagaacatgaaaagctgcttattcgccacaaaaacaaaattggcaagtatgctgtttatgctatttggcagccaggtgacactacagagtataatg ttacagttttccagggtaaaagtcataaaacttttatgtatacttttccattttatgaaatgtgcgacattaccatgtacatgagcaaacagtataagttgt ggcccccacaaaattgtgtggaaaacactggcactttctgctgcactgctatgctaattacagtgctcgctttggtctgtaccctactctatattaaata caaaagcagacgcagctttattgaggaaaagaaaatgccttaatttactaagttacaaagctaatgtcaccactaactgctttactcgctgcttgca aaacaaattcaaaaagttagcattataattagaataggatttaaaccccccggtcatttcctgctcaataccattcccctgaacaattgactctatgtg ggatatgctccagcgctacaaccttgaagtcaggcttcctggatgtcagcatctgactttggccagcacctgtcccgcggatttgttccagtccaact acagcgacccaccctaacagagatgaccaacacaaccaacgcggccgccgctaccggacttacatctaccacaaatacaccccaagtttctg cctttgtcaataactgggataacttgggcatgtggtggttctccatagcgcttatgtttgtatgccttattattatgtggctcatctgctgcctaaagcgcaa acgcgcccgaccacccatctatagtcccatcattgtgctacacccaaacaatgatggaatccatagattggacggactgaaacacatgttcttttct cttacagtatgattaaatgagacatgattcctcgagtttttatattactgacccttgttgcgcttttttgtgcgtgctccacattggctgcggtttctcacatcg aagtagactgcattccagccttcacagtctatttgctttacggatttgtcaccctcacgctcatctgcagcctcatcactgtggtcatcgcctttatccagt gcattgactgggtctgtgtgcgctttgcatatctcagacaccatccccagtacagggacaggactatagctgagcttcttagaattctttaattatgaa atttactgtgacttttctgctgattatttgcaccctatctgcgttttgttccccgacctccaagcctcaaagacatatatcatgcagattcactcgtatatgg aatattccaagttgctacaatgaaaaaagcgatctttccgaagcctggttatatgcaatcatctctgttatggtgttctgcagtaccatcttagccctag ctatatatccctaccttgacattggctggaacgcaatagatgccatgaaccacccaactttccccgcgcccgctatgcttccactgcaacaagttgtt gccggcggctttgtcccagccaatcagcctcgcccaccttctcccacccccactgaaatcagctactttaatctaacaggaggagatgactgaca ccctagatctagaaatggacggaattattacagagcagcgcctgctagaaagacgcagggcagcggccgagcaacagcgcatgaatcaag agctccaagacatggttaacttgcaccagtgcaaaaggggtatcttttgtctcgtaaagcaggccaaagtcacctacgacagtaataccaccgg acaccgccttagctacaagttgccaaccaagcgtcagaaattggtggtcatggtgggagaaaagcccattaccataactcagcactcggtaga aaccgaaggctgcattcactcaccttgtcaaggacctgaggatctctgcacccttattaagaccctgtgcggtctcaaagatcttattccctttaacta ataaaaaaaaataataaagcatcacttacttaaaatcagttagcaaatttctgtccagtttattcagcagcacctccttgccctcctcccagctctggt attgcagcttcctcctggctgcaaactttctccacaatctaaatggaatgtcagtttcctcctgttcctgtccatccgcacccactatcttcatgttgttgca gatgaagcgcgcaagaccgtctgaagataccttcaaccccgtgtatccatatgacacggaaaccggtcctccaactgtgccttttcttactcctccc tttgtatcccccaatgggtttcaagagagtccccctggggtactctctttgcgcctatccgaacctctagttacctccaatggcatgcttgcgctcaaaa tgggcaacggcctctctctggacgaggccggcaaccttacctcccaaaatgtaaccactgtgagcccacctctccgaggagacaagtcaaaca taaacctggaaatatctgcacccctcacagttacctcagaagccctaactgtggctgccgccgcacctctaatggtcgcgggcaacacactcac catgcaatcacaggccccgctaaccgtgcacgactccaaacttagcattgccacccaaggacccctcacagtgtcagaaggaaagctagccc tgcaaacatcaggccccctcaccaccaccgatagcagtacccttactatcactgcctcaccccctctaactactgccactggtagcttgggcattg acttgaaagagcccatttatacacaaaatggaaaactaggactaaagtacggggctcctttgcatgtaacagacgacctaaacactttgaccgta gcaactggtccaggtgtgactattaataatacttccttgcaaactaaagttactggagccttgggttttgattcacaaggcaatatgcaacttaatgta gcaggaggactaaggattgattctcaaaacagacgccttatacttgatgttagttatccgtttgatgctcaaaaccaactaaatctaagactaggac agggccctctttttataaactcagcccacaacttggatattaactacaacaaaggcctttacttgtttacagcttcaaacaattccaaaaagcttgag gttaacctaagcactgccaaggggttgatgtttgacgctacagccatagccattaatgcaggagatgggcttgaatttggttcacctaatgcaccaa acacaaatcccctcaaaacaaaaattggccatggcctagaatttgattcaaacaaggctatggttcctaaactaggaactggccttagttttgaca gcacaggtgccattacagtaggaaacaaaaataatgataagctaactttgtggaccacaccagctccatctcctaactgtagactaaatgcaga gaaagatgctaaactcactttggtcttaacaaaatgtggcagtcaaatacttgctacagtttcagttttggctgttaaaggcagtttggctccaatatct ggaacagttcaaagtgctcatcttattataagatttgacgaaaatggagtgctactaaacaattccttcctggacccagaatattggaactttagaaa tggagatcttactgaaggcacagcctatacaaacgctgttggatttatgcctaacctatcagcttatccaaaatctcacggtaaaactgccaaaagt aacattgtcagtcaagtttacttaaacggagacaaaactaaacctgtaacactaaccattacactaaacggtacacaggaaacaggagacaca actccaagtgcatactctatgtcattttcatgggactggtctggccacaactacattaatgaaatatttgccacatcctcttacactttttcatacattgcc caagaataaagaatcgtttgtgttatgtttcaacgtgtttatttttcaattgtactaagcggtgatgtttctgatcagccaccatgggagtgctaaaattca agcacatctttttcagaagctttgttaaatcaagtggagtatcccagatagttttcaccttccttctgattccatgttgcttgactctgaatttcagagcacc tcctgttattccaaatgtgcctttcctctgggcctggaatgccccaagtgaattttgtcttggaaaatttgatgagccactagatatgagcctcttctctttc ataggaagcccccgaataaacgccaccgggcaaggtgttacaatattttatgttgatagacttggctactatccttacatagattcaatcacaggag taactgtgaatggaggaatcccccagaagatttccttacaagaccatctggacaaagctaagaaagacattacattttatatgccagtagacaatt tgggaatggctgttattgactgggaagaatggagacccacttgggcaagaaactggaaacctaaagatgtttacaagaataggtctattgaattg gttcagcaacaaaatgtacaacttagtctcacagaggccactgagaaagcaaaacaagaatttgaaaaggcagggaaggatttcctggtaga gactataaaattgggaaaattacttcggccaaatcacttgtggggttattatctttttccggattgttacaaccatcactataagaaacccggttacaat ggaagttgcttcaatgtagaaataaaaagaaatgatgatctcagctggttgtggaatgaaagcactgctctttacccatccatttatttgaacactca gcagtctcctgtagctgctacactctatgtgcgcaatcgagttcgggaagccatcagagtttccaaaatacctgatgcaaaaagtccacttccggttt ttgcatatacccgcatagtttttactgatcaagttttgaaattcctttctcaagatgaacttgtgtatacatttggcgaaactgttgctctgggtgcttctgga attgtaatatggggaaccctcagtataatgcgaagtatgaaatcttgcttgctcctagacaattacatggagactatactgaatccttacataatcaa cgtcacactagcagccaaaatgtgtagccaagtgctttgccaggagcaaggagtgtgtataaggaaaaactggaattcaagtgactatcttcac ctcaacccagataattttgctattcaacttgagaaaggtggaaagttcacagtacgtggaaaaccgacacttgaagacctggagcaattttctgaa aaattttattgcagctgttatagcaccttgagttgtaaggagaaagctgatgtaaaagacactgatgctgttgatgtgtgtattgctgatggtgtctgtat agatgcttttctaaaacctcccatggagacagaagaacctcaaattttctacaatgcttcaccctccacactatcttaataaactttatttttcaattgca gaaaatttcaagtcatttttcattcagtagtatagccccaccaccacatagcttatacagatcaccgtaccttaatcaaactcacagaaccctagtatt caacctgccacctccctcccaacacacagagtacacagtcctttctccccggctggccttaaaaagcatcatatcatgggtaacagacatattctt aggtgttatattccacacggtttcctgtcgagccaaacgctcatcagtgatattaataaactccccgggcagctcacttaagttcatgtcgctgtccag ctgctgagccacaggctgctgtccaacttgcggttgcttaacgggcggcgaaggagaagtccacgcctacatgggggtagagtcataatcgtgc atcaggatagggcggtggtgctgcagcagcgcgcgaataaactgctgccgccgccgctccgtcctgcaggaatacaacatggcagtggtctcc tcagcgatgattcgcaccgcccgcagcataaggcgccttgtcctccgggcacagcagcgcaccctgatctcacttaaatcagcacagtaactgc agcacagcaccacaatattgttcaaaatcccacagtgcaaggcgctgtatccaaagctcatggcggggaccacagaacccacgtggccatca taccacaagcgcaggtagattaagtggcgacccctcataaacacgctggacataaacattacctcttttggcatgttgtaattcaccacctcccggt accatataaacctctgattaaacatggcgccatccaccaccatcctaaaccagctggccaaaacctgcccgccggctatacactgcagggaac cgggactggaacaatgacagtggagagcccaggactcgtaaccatggatcatcatgctcgtcatgatatcaatgttggcacaacacaggcaca cgtgcatacacttcctcaggattacaagctcctcccgcgttagaaccatatcccagggaacaacccattcctgaatcagcgtaaatcccacactg cagggaagacctcgcacgtaactcacgttgtgcattgtcaaagtgttacattcgggcagcagcggatgatcctccagtatggtagcgcgggtttct gtctcaaaaggaggtagacgatccctactgtacggagtgcgccgagacaaccgagatcgtgttggtcgtagtgtcatgccaaatggaacgccg gacgtagtcatatttcctgaagcaaaaccaggtgcgggcgtgacaaacagatctgcgtctccggtctcgccgcttagatcgctctgtgtagtagttg tagtatatccactctctcaaagcatccaggcgccccctggcttcgggttctatgtaaactccttcatgcgccgctgccctgataacatccaccaccgc agaataagccacacccagccaacctacacattcgttctgcgagtcacacacgggaggagcgggaagagctggaagaaccatgtttttttttttatt ccaaaagattatccaaaacctcaaaatgaagatctattaagtgaacgcgctcccctccggtggcgtggtcaaactctacagccaaagaacaga taatggcatttgtaagatgttgcacaatggcttccaaaaggcaaacggccctcacgtccaagtggacgtaaaggctaaacccttcagggtgaatc tcctctataaacattccagcaccttcaaccatgcccaaataattctcatctcgccaccttctcaatatatctctaagcaaatcccgaatattaagtccg gccattgtaaaaatctgctccagagcgccctccaccttcagcctcaagcagcgaatcatgattgcaaaaattcaggttcctcacagacctgtataa gattcaaaagcggaacattaacaaaaataccgcgatcccgtaggtcccttcgcagggccagctgaacataatcgtgcaggtctgcacggacca gcgcggccacttccccgccaggaaccatgacaaaagaacccacactgattatgacacgcatactcggagctatgctaaccagcgtagccccg atgtaagcttgttgcatgggcggcgatataaaatgcaaggtgctgctcaaaaaatcaggcaaagcctcgcgcaaaaaagaaagcacatcgta gtcatgctcatgcagataaaggcaggtaagctccggaaccaccacagaaaaagacaccatttttctctcaaacatgtctgcgggtttctgcataa acacaaaataaaataacaaaaaaacatttaaacattagaagcctgtcttacaacaggaaaaacaacccttataagcataagacggactacgg ccatgccggcgtgaccgtaaaaaaactggtcaccgtgattaaaaagcaccaccgacagctcctcggtcatgtccggagtcataatgtaagactc ggtaaacacatcaggttgattcacatcggtcagtgctaaaaagcgaccgaaatagcccgggggaatacatacccgcaggcgtagagacaac attacagcccccataggaggtataacaaaattaataggagagaaaaacacataaacacctgaaaaaccctcctgcctaggcaaaatagcac cctcccgctccagaacaacatacagcgcttccacagcggcagccataacagtcagccttaccagtaaaaaagaaaacctattaaaaaaaca ccactcgacacggcaccagctcaatcagtcacagtgtaaaaaagggccaagtgcagagcgagtatatataggactaaaaaatgacgtaacg gttaaagtccacaaaaaacacccagaaaaccgcacgcgaacctacgcccagaaacgaaagccaaaaaacccacaacttcctcaaatcgtc acttccgttttcccacgttacgtcacttcccattttaagaaaactacaattcccaacacatacaagttactccgccctaaaacctacgtcacccgccc cgttcccacgccccgcgccacgtcacaaactccaccccctcattatcatattggcttcaatccaaaataaggtatattattgatgatg

[0354] SEQ ID N0: 4

[0355] Amino acid sequence of the modified version of the fibre of adenovirus serotype in which RGDK modification has been introduced

[0356] MKRARPSEDTFNPVYPYDTETGPPTVPFLTPPFVSPNGFQESPPGVLSLRLSEPLVTSNGMLALKMGN

[0357] GLSLDEAGNLTSQNVTTVSPPLRGDKSNINLEISAPLTVTSEALTVAAAAPLMVAGNTLTMGSQAPLTV

[0358] HDSKLSIATQGPLTVSEGKLALQTSGPLTTTDSSTLTITASPPLTTATGSLGIDLKEPIYTQNGKLGLKYG

[0359] APLHVTDDLNTLTVATGPGVTINNTSLGTKVTGALGFDSQGNMQLNVAGGLRIDSQNRRLILDVSYPFD

[0360] AQNQLNLRLGQGPLFINSAHNLDINYNKGLYLFTASNNSKKLEVNLSTAKGLMFDATAIAINAGDGLEFG

[0361] SPNAPNTNPLKTKIIGHGLEFDSNKAMVPKLGTGLSFDSTGAITVGNKNNDKLTLWTTPAPSPNCDLNA

[0362] EKDAKLTLVLTKCGSQILATVSVLAVKGSLAPISGTVQSAHLIIRFDENGVLLNNSFLDPEYWNFRNGDL TEGTAYTNAVGFMPNLSAYPKSHGKTAKSNIVSQVYLNGDKTKPVTLTITLNGTQETGDTTPSAYSMSF

[0363] SWDWSGHNYINEIFATSSYTF

[0364] SEQ ID NO: 5

[0365] RGD-C peptide

[0366] CDCRGDCFC

[0367] SEQ ID NO: 6 binding domain

[0368] KKTK

[0369] SEQ ID NO: 7 binding domain

[0370] RGDK SEQ ID NO: 8

[0371] Translation of SEQ ID NO: 1 (PH20 with carboxy-terminal domain present)

[0372] MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDM SLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLG MAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKL LRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLY VRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSM KSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVR GKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPS TLSATMFIVSILFLIISSVASL-

[0373] SEQ ID NO: 9 cDNA encoding PH20 with carboxy-terminal domain deleted atgggagtgctaaaattcaagcacatctttttcagaagctttgttaaatcaagtggagtatcccagatagttttcaccttccttctgattccatgttgcttg actctgaatttcagagcacctcctgttattccaaatgtgcctttcctctgggcctggaatgccccaagtgaattttgtcttggaaaatttgatgagccact agatatgagcctcttctctttcataggaagcccccgaataaacgccaccgggcaaggtgttacaatattttatgttgatagacttggctactatcctta catagattcaatcacaggagtaactgtgaatggaggaatcccccagaagatttccttacaagaccatctggacaaagctaagaaagacattac attttatatgccagtagacaatttgggaatggctgttattgactgggaagaatggagacccacttgggcaagaaactggaaacctaaagatgttta caagaataggtctattgaattggttcagcaacaaaatgtacaacttagtctcacagaggccactgagaaagcaaaacaagaatttgaaaaggc agggaaggatttcctggtagagactataaaattgggaaaattacttcggccaaatcacttgtggggttattatctttttccggattgttacaaccatcac tataagaaacccggttacaatggaagttgcttcaatgtagaaataaaaagaaatgatgatctcagctggttgtggaatgaaagcactgctctttac ccatccatttatttgaacactcagcagtctcctgtagctgctacactctatgtgcgcaatcgagttcgggaagccatcagagtttccaaaatacctgat gcaaaaagtccacttccggtttttgcatatacccgcatagtttttactgatcaagttttgaaattcctttctcaagatgaacttgtgtatacatttggcgaa actgttgctctgggtgcttctggaattgtaatatggggaaccctcagtataatgcgaagtatgaaatcttgcttgctcctagacaattacatggagact atactgaatccttacataatcaacgtcacactagcagccaaaatgtgtagccaagtgctttgccaggagcaaggagtgtgtataaggaaaaact ggaattcaagtgactatcttcacctcaacccagataattttgctattcaacttgagaaaggtggaaagttcacagtacgtggaaaaccgacacttg aagacctggagcaattttctgaaaaattttattgcagctgttatagcaccttgagttgtaaggagaaagctgatgtaaaagacactgatgctgttgat gtgtgtattgctgatggtgtctgtatagatgcttttctaaaacctcccatggagacagaagaacctcaaattttctacaatgcttcaccctccacactat cttaa

[0374] SEQ ID NO: 10 Translation of SEQ ID NO: 9 (PH20 with carboxy-terminal domain deleted)

[0375] MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDM

[0376] SLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLG

[0377] MAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKL

[0378] LRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLY

[0379] VRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSM

[0380] KSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVR

[0381] GKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPS TLS-

Claims

CLAIMSWhat is claimed is:

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

2. The method according to claim 1 , wherein the oncolytic adenovirus is administered by intravenous, intraarterial, intratumoral, and / or intraperitoneal administration, e.g. by injection.

3. The method according to claim 1 or 2, wherein the topoisomerase I inhibitor or a prodrug thereof is administered by intraperitoneal, intravenous, intraarterial, intratumoral, and / or oral administration.

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

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

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

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

8. The method of claim 7, wherein the topoisomerase I inhibitor or a prodrug thereof is administered within about 30 minutes, within about 20 minutes, within about 10 minutes, within about 5 minutes, or within about 1 minute of the administration of the oncolytic adenovirus.

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

10. The method according to any one of claims 1-9, wherein the: pancreatic cancer is an exocrine or endocrine cancer, or an adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, or a colloid carcinoma; ovarian cancer is an epithelial ovarian carcinoma, germ cell tumor, or stromal cell tumor; cervical cancer is squamous cell carcinoma, adenocarcinoma and / or adenosquamous (mixed) carcinoma; lung cancer is small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, large cell carcinoma of the lung, mesothelioma, and / or lung carcinoid tumor; and / or colon or colorectal cancer is an adenocarcinoma, gastrointestinal stromal tumor (GIST), squamous cell carcinoma, carcinoid tumor, lymphoma, or sarcoma of the colon.

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

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

13. The method according to claim 12, wherein the sequence that encodes a hyaluronidase enzyme 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 thereto.

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

15. The method according to any one of claims 1-14, wherein the oncolytic adenoviral replication occurs in tumor cells with an aberrant Rb-E2F pathway and not in healthy, non-tumor, or normal cells.

16. The method according to claim 15, wherein the oncolytic adenovirus is engineered to replicate in tumor cells and not healthy, non-tumor, or normal cells by a deletion of a Rb binding domain or a A24 deletion in the sequence coding for the E1 a protein and the insertion of four binding sites to E2F-1 and one binding site to Sp1 into the endogenous promoter of E1 a to control the expression of E1 a.

17. The method according to any one of claims 1 -16, wherein the oncolytic adenovirus has the capsid modified such that the binding domain91KKTK94(SEQ ID NO: 6) of the heparan sulfates present in the adenovirus fibre has been replaced, optionally by the domain91RGDK94(SEQ ID NO: 7).

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

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

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

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

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

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

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

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

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

27. The method according to any one of claims 1-22, wherein the method results in reduced or maintained tumor size, and / or prevention or reduction of metastasis, secondary malignancy, or recurrent disease associated with colon or colorectal cancer, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

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

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

30. The method according to any one of claims 1 -22, 27 or 29, wherein the prodrug of the topoisomerase I inhibitor is a liposomal irinotecan, optionally ONIVYDE, is administered by intravenous infusion.31 . The method according to any one of claims 1 -30, wherein the method results in increased expression of the cellular transcription factor E2F1, compared to treatment with the oncolytic virus without the topoisomerase I inhibitor or a prodrug thereof.

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

33. A method for treating or preventing pancreatic cancer in a patient in need thereof, comprising coadministering to the patient (I) an oncolytic adenovirus, and (ii) 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 the NALIRIFOX (irinotecan, 5-FU, leucovorin, oxaliplatin) regimen.

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

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