Combination therapy of oncolytic adenoviruses with topoisomerase I inhibitors or prodrugs thereof

Abstract

The present disclosure relates, inter alia, to the combination therapy of an oncolytic adenovirus for the treatment of retinoblastoma, Ewing's sarcoma or neuroblastoma with a topoisomerase I inhibitor or a prodrug thereof, such as topotecan or SN-38, or a prodrug of a topoisomerase inhibitor, such as irinotecan.

Description

Technical Field

[0001] The present disclosure relates particularly to combination therapies of oncolytic adenoviruses with topotecan, irinotecan, or SN-38 for use in oncology.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 408,079, filed September 19, 2022, and U.S. Provisional Application No. 63 / 533,176, filed August 17, 2023, the entire contents of which are hereby incorporated by reference in their entirety.

[0004] Description of the XML file submitted electronically

[0005] This application contains a sequence listing, which is electronically submitted in XML file format and is hereby incorporated by reference in its entirety. The XML file was created on September 8, 2023, is named "VCN-004_Sequence_Listing.xml", and is 72,036 bytes in size. Background Art

[0006] Retinoblastoma, neuroblastoma, and Ewing sarcoma are common types of solid tumors, especially in children.

[0007] Retinoblastoma is the most common malignant intraocular tumor in children, occurring in 1 in 17,000 births. It develops in the retina and typically grows into the vitreous cavity.

[0008] Current retinoblastoma treatment is primarily for the patient's survival and secondarily for preservation of vision. Various treatment options are available, including systemic or local ocular chemotherapy, radiotherapy, cryotherapy, laser therapy, and surgery (enucleation of the affected eye). The choice of treatment depends on many factors, such as the stage of tumor development, whether the tumor is unifocal or multifocal, unilateral or bilateral, and the location and size of the tumor.

[0009] In the late 1990s, there was a shift from local, direct ocular therapies such as external beam radiation therapy (EBRT), which has always been associated with a high risk of second malignancies, to systemic chemotherapy in combination with aggressive local therapy (Rodriguez-Galindo et al., "Treatment of retinoblastoma: current status and future perspectives," Curr Treat Options Neurol. 2007 Jul;9(4):294-307). The use of systemic chemovolume reduction is effective for tumor control in eyes with less advanced tumors, avoiding the use of EBRT, but approximately 75% of retinoblastoma cases worldwide present with advanced tumors with massive vitreous or subretinal seeding and retinal detachment, in which both radiation and systemic chemotherapy are rarely eye or vision salvageable. Systemic chemovolume reduction results in reduced long-term toxicity by sparing many patients from receiving EBRT, but its acute toxicity can be fatal in up to 4%-5% of patients in less developed countries. In addition, studies have shown an additional risk of secondary malignancies when chemotherapy is combined with EBRT. Ototoxicity caused by carboplatin and cases of fatal chemotherapy-induced leukemia have also been reported in children with retinoblastoma who had not received radiation. Therefore, in the mid-2000s, physicians switched from systemic chemotherapy to advanced technologies for more selective ocular delivery to increase drug exposure in the tumor, thereby maximizing efficacy and minimizing the probability of adverse events. The introduction of ophthalmic artery chemotherapy (OAC) has been a great success in treating eyes with more advanced disease, in which systemic chemotherapy has a worse outcome. The current standard of care in high- and middle-income countries is intravitreal chemotherapy combined with OAC, which has led to improved eye preservation and eliminated the use of EBRT.

[0010] Among the antitumor agents used for retinoblastoma, topotecan is used based on its effects in preclinical models and clinical studies. Topotecan alone or in combination is active against retinoblastoma (Schaiquevich et al., "Ocular pharmacology of topotecan and its activity in retinoblastoma," Retina. 2014 Sep; 34(9): 1719-27). When administered intravenously and intraarterially, topotecan has been shown to enter the vitreous body favorably and has minimal ocular toxicity with all routes of administration. However, the clinical role, optimal dose, and route of administration of topotecan in the treatment of retinoblastoma remain to be determined. For example, a study of OAC administration of topotecan showed that the OAC route selectively delivered chemotherapy to the eye and that topotecan concentrations in the vitreous and retina of non-tumor-bearing pigs were 243- and 146-fold higher after OAC than after IV, respectively, while systemic exposure was comparable between the two routes (Schaiquevich et al., “Treatment of Retinoblastoma: What Is the Latest and What Is the Future,” Front Oncol. 2022 Apr. 1;12:822-330). The favorable distribution of topotecan in ocular tissues after OAC administration predicts successful tumor control with the OAC topotecan; however, this has not been observed clinically. Despite vitreous levels of topotecan exceeding the IC50, adequate tumor control was not achieved, which may be related to the administration schedule. Topotecan exerts its cytotoxic activity by inhibiting the nuclear enzyme topoisomerase I, which causes double-strand breaks during cell replication and thus acts on cells in the S phase. Therefore, subsequent doses are required to target quiescent tumor cells that are entering cell replication, and long-term schedules are associated with stronger antitumor effects compared to intermittent, high-dose schedules. However, daily repeated OAC delivery of topotecan is impractical, which limits its efficacy via this delivery route.

[0011] Given the above, despite advances in retinoblastoma treatment, eyes that relapse or are initially refractory to conventional therapies remain difficult to treat with currently available drug options.

[0012] Ewing's sarcoma is the second most common bone cancer in children. It most commonly develops in the long bones of the legs or arms, pelvis, chest wall, spine, and skull, but can also begin in soft tissue without bone involvement. The disease most often develops in adolescents, with nearly half of cases occurring between the ages of 10 and 20. Ewing's sarcoma is more common in males than in females.

[0013] In children and adolescents, oncologists typically prescribe a combination of five drugs given in an alternating sequence for up to a year. This combination therapy cures about 70% of young patients with localized disease. However, if the cancer is metastatic, doesn't respond to treatment, or recurs, the prognosis is often poor.

[0014] A variation of this combination therapy is also used in adults, but adults often cannot tolerate a full year of the same intensive treatment given to children. This may partly explain why cure rates for adults with Ewing sarcoma are lower than for younger patients.

[0015] Neuroblastoma accounts for 3.8% of all childhood cancer diagnoses. There are more than 650 cases in the United States each year. Neuroblastoma most often originates in the adrenal gland, located on top of each kidney. However, the tumor can begin anywhere in the body. Other common sites are the chest, neck, and pelvis. While some patients may have cancer found in only one part of the body at the time of diagnosis, in others the cancer may have metastasized from its original location to the lymph nodes, bone marrow, or bones.

[0016] Outcomes for children with neuroblastoma vary widely, ranging from cure rates of >90% for patients with low-risk disease to cure rates of <50% for patients with high-risk disease. Recent studies have revealed the biology of neuroblastoma, allowing for more accurate risk stratification and reduced treatment in many cases, but newer treatment strategies for children with high-risk and recurrent neuroblastoma are needed to improve outcomes.

[0017] Therefore, there remains a need for alternative and more effective therapies to treat retinoblastoma, Ewing's sarcoma, and neuroblastoma. In the case of retinoblastoma, such alternative therapies are needed to avoid the need for enucleation of one or both eyes. There is also a need for new treatment alternatives that allow for the elimination or reduction of a patient's risk of developing metastases, secondary malignancies, or recurrent disease. Summary of the Invention

[0018] Thus, in various aspects, the present disclosure provides methods for treating retinoblastoma, Ewing's sarcoma, or neuroblastoma in a patient in need thereof, comprising co-administering to the patient (i) an oncolytic adenovirus comprising a replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug of a topoisomerase inhibitor.

[0019] In an embodiment, the oncolytic adenovirus is administered by intraocular, intrathecal, intravitreal, intravenous, intraarterial, or intratumoral injection. In an embodiment, the topoisomerase I inhibitor or a prodrug thereof is administered intravenously, intraarterially, intravitreal, intrathecal, intracerebroventricularly, or orally.

[0020] In embodiments, oncolytic adenovirus and topoisomerase I inhibitor or its prodrug are formulated into a separate composition. In embodiments, oncolytic adenovirus and topoisomerase I inhibitor or its prodrug are formulated into a single composition. In embodiments, a separate composition is administered simultaneously or over the same period. In embodiments, an oncolytic adenovirus is first administered, and a topoisomerase I inhibitor or its prodrug is administered within about 60 minutes after the oncolytic adenovirus is administered. In embodiments, a topoisomerase I inhibitor or its prodrug is administered within about 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes, or about 1 minute after the oncolytic adenovirus is administered. In embodiments, a topoisomerase I inhibitor or its prodrug is first administered, and an oncolytic adenovirus is administered within about 60 minutes after the oncolytic adenovirus is administered. In embodiments, an oncolytic adenovirus is administered within about 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes, or about 1 minute after the oncolytic adenovirus is administered.

[0021] In embodiments, a subject is administered a single intrathecal injection of an oncolytic adenovirus, concomitantly or concurrently with systemic or intrathecal circulation of a topoisomerase I inhibitor or a prodrug thereof. In embodiments, an oncolytic adenovirus and a topoisomerase I inhibitor or a prodrug thereof are administered systemically to a subject. In embodiments, an oncolytic adenovirus and / or a topoisomerase I inhibitor or a prodrug thereof are co-administered with one or more topical and / or systemic corticosteroids. In an embodiment, the one or more topical and / or systemic corticosteroids are selected from hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, alsosterone, budesonide, fluticasone, flunisolide, ciclesonide, mometasone, beclomethasone, triamcinolone, and tixocortol. In an embodiment, the one or more topical and / or systemic corticosteroids are selected from methylprednisolone, dexamethasone, betamethasone, and triamcinolone.

[0022] 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.

[0023] In embodiments, the hyaluronidase is human hyaluronidase PH20. In embodiments, the sequence encoding the hyaluronidase is SEQ ID NO: 9 or a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto.

[0024] In an embodiment, the oncolytic adenovirus is produced by human adenovirus serotype 5.

[0025] In embodiments, oncolytic adenovirus replication occurs in tumor cells (e.g., solid tumors) with an aberrant Rb-E2F pathway, but not in healthy, non-tumor, or normal cells. In embodiments, an oncolytic adenovirus is engineered to replicate in tumor cells, but not in healthy, non-tumor, or normal cells, by deleting the Rb binding domain in the sequence encoding the E1a protein and inserting four binding sites for E2F-1 and one binding site for Sp1 into the endogenous promoter of E1a to control expression of E1a.

[0026] In embodiments, oncolytic adenovirus replication occurs in tumor cells (e.g., solid tumors) with an aberrant Rb-E2F pathway, but not in healthy, non-tumor, or normal cells. In embodiments, an oncolytic adenovirus is engineered to replicate in tumor cells, but not in healthy, non-tumor, or normal cells, by deleting Δ24 in the sequence encoding the E1a protein and inserting four binding sites for E2F-1 and one binding site for Sp1 into the endogenous promoter of E1a to control expression of E1a.

[0027] In embodiments, the capsid of an oncolytic adenovirus is modified such that the heparan sulfate binding domain present in the adenoviral fiber 91 KKTK 94 (SEQ ID NO: 8) was replaced.

[0028] In embodiments, the capsid of an oncolytic adenovirus is modified such that the heparan sulfate binding domain present in the adenoviral fiber 91 KKTK 94 (SEQ ID NO: 8) 91 RGDK 94 (SEQ ID NO: 9) was replaced.

[0029] In an embodiment, the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3) or a functional variant thereof.

[0030] In embodiments, the patient is a human patient. In embodiments, the human patient is a pediatric human patient.

[0031] In embodiments, the retinoblastoma, Ewing's sarcoma, or neuroblastoma is a retinoblastoma, Ewing's sarcoma, or neuroblastoma that is resistant to treatment with conventional chemotherapy and / or radiation therapy.

[0032] In embodiments, the method improves and / or increases and / or enhances anti-tumor efficacy compared to treatment with a topoisomerase I inhibitor or its prodrug without an oncolytic adenovirus. In embodiments, the method results in a tumor size that is reduced or maintained, and / or prevents or reduces metastasis, secondary malignancies, or trilateral retinoblastoma associated with retinoblastoma, compared to treatment with a topoisomerase I inhibitor or its prodrug without an oncolytic adenovirus. In embodiments, the method results in a tumor size that is reduced or maintained, and / or prevents or reduces metastasis, secondary malignancies, or trilateral retinoblastoma associated with retinoblastoma, compared to treatment with a topoisomerase I inhibitor or its prodrug without an oncolytic adenovirus. In embodiments, the method results in a tumor size that is reduced or maintained, and / or prevents or reduces metastasis, secondary malignancies, or recurrent disease associated with Ewing's sarcoma, compared to treatment with a topoisomerase I inhibitor or its prodrug without an oncolytic adenovirus. In embodiments, the method results in a tumor size that is reduced or maintained, and / or prevents or reduces metastasis, secondary malignancies, or recurrent disease associated with neuroblastoma, compared to treatment with a topoisomerase I inhibitor or its prodrug without an oncolytic adenovirus.

[0033] In embodiments, without wishing to be bound by theory, the administration of an oncolytic adenovirus reduces the apoptotic effect of topotecan compared to monotherapy. In embodiments, without wishing to be bound by theory, the administration of topotecan after the administration of an oncolytic adenovirus results in S-phase cell cycle arrest. In embodiments, without wishing to be bound by theory, the S-phase cell cycle arrest results in an increase in the infectivity of the oncolytic adenovirus compared to the treatment with an oncolytic adenovirus without a topoisomerase I inhibitor or its prodrug. In embodiments, without wishing to be bound by theory, the administration of topotecan after the administration of an oncolytic adenovirus results in an increase in E2F-1, p21, and / or cyclin E1 expression compared to the treatment with an oncolytic adenovirus without a topoisomerase I inhibitor or its prodrug. In embodiments, without wishing to be bound by theory, the increased E2F-1 expression results in an increase in the oncolytic activity of the oncolytic adenovirus compared to the treatment with an oncolytic adenovirus without a topoisomerase I inhibitor or its prodrug. In embodiments, without wishing to be bound by theory, the increase in the infectivity and oncolytic activity of the oncolytic adenovirus occurs when the replication of the oncolytic adenovirus is substantially not increased. In embodiments, without wishing to be bound by theory, sequential treatment wherein an oncolytic adenovirus is first administered followed by systemic administration of topotecan enhances or increases the cell infection and / or anticancer efficacy of the oncolytic adenovirus.

[0034] In various aspects, the present disclosure provides methods for treating retinoblastoma, Ewing's sarcoma, or neuroblastoma in a patient in need thereof, comprising administering to the patient (i) an oncolytic adenovirus comprising a replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug thereof.

[0035] In an embodiment, the oncolytic adenovirus is administered by intraocular, intrathecal, intravitreal, intravenous, intraarterial, or intratumoral injection. In an embodiment, the topoisomerase I inhibitor or a prodrug thereof is administered intravenously, intravitreally, intrathecally, intracerebroventricularly, or orally.

[0036] In embodiments, the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are formulated as separate compositions. In embodiments, the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are formulated as a single composition. In embodiments, the oncolytic adenovirus is administered first and the topoisomerase I inhibitor or a prodrug thereof is administered within about 12 weeks of 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 after administration of the oncolytic adenovirus.

[0037] In embodiments, an intrathecal injection of an oncolytic adenovirus is administered to a subject, and systemic or intrathecal circulation of a topoisomerase I inhibitor or its prodrug is performed concomitantly or concurrently. In embodiments, an oncolytic adenovirus and a topoisomerase I inhibitor or its prodrug are systemically administered to a subject. In embodiments, an oncolytic adenovirus and / or a topoisomerase I inhibitor or its prodrug are co-administered with one or more local and / or systemic corticosteroids. In embodiments, one or more local and / or systemic corticosteroids are selected from hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, aldosterone, budesonide, fluticasone, flunisolide, ciclesonide, mometasone, beclomethasone, triamcinolone and tixocortolone. In embodiments, one or more local and / or systemic corticosteroids are selected from methylprednisolone, dexamethasone, betamethasone and triamcinolone.

[0038] In an embodiment, the topoisomerase I inhibitor or a prodrug thereof is topotecan, SN-38, or irinotecan.

[0039] In embodiments, the hyaluronidase is human hyaluronidase PH20. In embodiments, the sequence encoding the hyaluronidase is SEQ ID NO: 9 or a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto.

[0040] In an embodiment, the oncolytic adenovirus is produced by human adenovirus serotype 5.

[0041] In embodiments, oncolytic adenovirus replication occurs in tumor cells with an aberrant Rb-E2F pathway, but not in healthy, non-tumor, or normal cells. In embodiments, an oncolytic adenovirus is engineered to replicate in tumor cells, but not in healthy, non-tumor, or normal cells, by deleting the Rb binding domain in the sequence encoding the E1a protein and inserting four binding sites for E2F-1 and one binding site for Sp1 into the endogenous promoter of E1a to control expression of E1a.

[0042] In embodiments, oncolytic adenovirus replication occurs in tumor cells with an aberrant Rb-E2F pathway, but not in healthy, non-tumor, or normal cells. In embodiments, an oncolytic adenovirus is engineered to replicate in tumor cells, but not in healthy, non-tumor, or normal cells, by deleting Δ24 in the sequence encoding the E1a protein and inserting four binding sites for E2F-1 and one binding site for Sp1 into the endogenous promoter of E1a to control expression of E1a.

[0043] In embodiments, the capsid of an oncolytic adenovirus is modified such that the heparan sulfate binding domain present in the adenoviral fiber 91 KKTK 94 (SEQ ID NO: 8) is replaced. In an embodiment, the capsid of the oncolytic adenovirus is modified so that the heparan sulfate binding domain present in the adenoviral fiber 91 KKTK 94 (SEQ ID NO: 8) 91 RGDK 94 (SEQ ID NO: 9) was replaced.

[0044] In an embodiment, the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3) or a functional variant thereof.

[0045] In embodiments, the patient is a human patient. In embodiments, the human patient is a pediatric human patient.

[0046] In embodiments, the retinoblastoma, Ewing's sarcoma, or neuroblastoma is a retinoblastoma, Ewing's sarcoma, or neuroblastoma that is resistant to treatment with conventional chemotherapy and / or radiation therapy.

[0047] In embodiments, the method improves and / or increases and / or enhances anti-tumor efficacy compared to treatment with a topoisomerase I inhibitor or its prodrug without an oncolytic adenovirus. In embodiments, the method results in a tumor size that is reduced or maintained, and / or prevents or reduces metastasis, secondary malignancies, or trilateral retinoblastoma associated with retinoblastoma, compared to treatment with a topoisomerase I inhibitor or its prodrug without an oncolytic adenovirus. In embodiments, the method results in a tumor size that is reduced or maintained, and / or prevents or reduces metastasis, secondary malignancies, or trilateral retinoblastoma associated with retinoblastoma, compared to treatment with a topoisomerase I inhibitor or its prodrug without an oncolytic adenovirus. In embodiments, the method results in a tumor size that is reduced or maintained, and / or prevents or reduces metastasis, secondary malignancies, or recurrent disease associated with Ewing's sarcoma, compared to treatment with a topoisomerase I inhibitor or its prodrug without an oncolytic adenovirus. In embodiments, the method results in a tumor size that is reduced or maintained, and / or prevents or reduces metastasis, secondary malignancies, or recurrent disease associated with neuroblastoma, compared to treatment with a topoisomerase I inhibitor or its prodrug without an oncolytic adenovirus.

[0048] In embodiments, without wishing to be bound by theory, the administration of an oncolytic adenovirus reduces the apoptotic effect of topotecan compared to monotherapy. In embodiments, without wishing to be bound by theory, the administration of topotecan after the administration of an oncolytic adenovirus results in S-phase cell cycle arrest. In embodiments, without wishing to be bound by theory, the S-phase cell cycle arrest results in an increase in the infectivity of the oncolytic adenovirus compared to the treatment with an oncolytic adenovirus without a topoisomerase I inhibitor or its prodrug. In embodiments, without wishing to be bound by theory, the administration of topotecan after the administration of an oncolytic adenovirus results in an increase in E2F-1, p21, and / or cyclin E1 expression compared to the treatment with an oncolytic adenovirus without a topoisomerase I inhibitor or its prodrug. In embodiments, without wishing to be bound by theory, the increased E2F-1 expression results in an increase in the oncolytic activity of the oncolytic adenovirus compared to the treatment with an oncolytic adenovirus without a topoisomerase I inhibitor or its prodrug. In embodiments, without wishing to be bound by theory, the increase in the infectivity and oncolytic activity of the oncolytic adenovirus occurs when the replication of the oncolytic adenovirus is substantially not increased. In embodiments, without wishing to be bound by theory, sequential treatment wherein an oncolytic adenovirus is first administered followed by systemic administration of topotecan enhances or increases the cell infection and / or anticancer efficacy of the oncolytic adenovirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1Shown is an immunoblot of E1a in retinoblastoma cells infected with VCN-01 (SEQ ID NO: 3) and treated with topotecan, carboplatin, or melphalan.

[0050] Figure 2A-2B Shows that after direct injection of VCN-01 into subcutaneously implanted Y79 tumors in nude mice and systemic treatment with topotecan, carboplatin, and hydroxyurea, the Figure 2A ) and day 15 ( Figure 2B ) Quantified VCN-01 intratumoral levels.

[0051] Figure 3 Shown are quantified intratumoral levels of VCN-01 on day 5 following direct injection of VCN-01 into subcutaneously implanted HSJD-RBT-7 tumors in nude mice and systemic treatment with topotecan, carboplatin, and hydroxyurea.

[0052] Figures 4A-4C Shown is the immunoblot of E1a in Y79 tumors implanted subcutaneously in nude mice after direct injection of VCN-01 and systemic treatment with topotecan, carboplatin, and hydroxyurea ( Figure 4A ) or histological E1a immunostaining ( Figure 4B ). Figure 4C Show Figure 4B Quantification of E1A-positive cell counts in immunoblotting.

[0053] Figure 5 Shown are the antitumor activities observed following treatment of subcutaneously implanted Y79 tumors in nude mice with topotecan + VCN-01 compared to treatment with VCN-01 alone or topotecan alone (depicted as percentage survival in Kaplan-Meier curves).

[0054] Figure 6A-Figure 6B Shown is the effect of topotecan + VCN-01 treatment on orthotopically implanted Y79 tumors in mice eyes compared to either treatment alone. Figure 6A ) or HSJD-RBT-7 orthotopic tumors ( Figure 6B ) after 48 h of nab-positive control group (depicted as survival percentage in Kaplan-Meier curves).

[0055] Figure 7 Shown are Ela immunoblots of subcutaneously grown Y79 tumors in nude mice treated with VCN-01 alone or in combination with carboplatin, melphalan, hydroxyurea, etoposide, irinotecan, and SN-38.

[0056] Figure 8Shown is the antitumor activity (depicted as survival percentage in Kaplan-Meier curves) observed following treatment with intrathecal VCN-01 + systemic topotecan in HSJD-RBT-7 tumors orthotopically implanted in the brain (intracerebroventricularly) of mice compared to treatment with VCN-01 alone, topotecan alone, and standard of care.

[0057] Figure 9 Shown are body weights of animals following treatment with intrathecal VCN-01 + systemic topotecan in mice bearing orthotopically implanted HSJD-RBT-7 tumors compared to treatment with VCN-01 alone.

[0058] Figure 10 Shown are the tumor burdens observed following treatment with intrathecal VCN-01 + systemic topotecan in HSJD-RBT-7 tumors orthotopically implanted in the brain (intracerebroventricularly) of mice compared to treatment with VCN-01 alone, topotecan alone, and standard of care treatment.

[0059] Figure 11 Shown are growth curves of HSJD-ES-033 Ewing's sarcoma tumors implanted subcutaneously in nude mice following treatment with the combination of intratumoral injection of VCN-01 + systemic topotecan compared to treatment with VCN-01 alone.

[0060] Figure 12 Shown are growth curves of patient-derived neuroblastoma tumors implanted subcutaneously in nude mice after treatment with intratumoral injection of VCN-01 plus systemic topotecan or irinotecan combination compared to VCN-01 alone, topotecan alone, or irinotecan alone.

[0061] Figures 13A-13H illustrate the in vitro interaction of VCN-01 with chemotherapy. Figure 13A shows the antiproliferative activity of topotecan (TPT), carboplatin (CBP), and melphalan (MEL) in Y79 cells with an MOI of 10 (VCN-01) or without (medium). Values ​​are means and standard deviations of three replicates. Lines are best-fit curves constructed using the least squares regression method in GraphPad. Cell viability is expressed as the relative percentage of the assay signal in treated cells compared to control untreated cells (which was set to 100%). Figure 13B shows immunoblots for p53, cPARP, and E1A following treatment of Y79 cells with VCN-01 (48 h; 50 MOI), TPT (24 h; 2 μM), a combination of VCN-01 and TPT (VCN-01 / TPT; 48 h; TPT added at 24 h), CBP (24 h; 12.5 μM), a combination of VCN-01 and CBP (VCN-01 / CBP; 48 h; CBP added at 24 h), MEL (24 h; 12 μM), and a combination of VCN-01 and MEL (VCN-01 / MEL; 48 h; MEL added at 24 h). Control cells were untreated. GAPDH is a loading control. Figure 13C shows representative brightfield images of Y79 cells (floating aggregates) treated with VCN-01 (48 h; 50 MOI), TPT (48 h; 2 μM), or the combination (48 h; TPT added at 24 h). Figure 13D shows the viability of Y79 cells after 6 days of sequential treatment with "TPT first" or "VCN-01 first." The treatments (TPT, 2 μM; VCN-01, 50 MOI; or medium) and sequence (where the second treatment was administered at the beginning of day 4) are detailed in the lower column. Bars are the means and standard deviations of 8 replicates. The MTS signal of control untreated cells was set to 100%. FIG13E shows immunoblots for p53, cPARP, E2F-1, and E1A following treatment of Y79 cells with VCN-01 (24 or 48 h; 50 MOI), TPT (24 or 48 h; 2 μM), or a sequential combination of "TPT first" (TPT / VCN-01; 48 h; VCN-01 added at 24 h) or "VCN-01 first" (VCN-01 / TPT; 48 h; TPT added at 24 h). β-Tubulin is a loading control. FIG13F shows quantification of E1A mRNA in Y79 cells treated with the "TPT first" or "VCN-01 first" sequence. Points are experimental replicates and bars are means and standard deviations. FIG13G shows quantification of E1B mRNA in Y79 cells treated with the "TPT first" or "VCN-01 first" sequence. FIG. 13H shows immunoblotting of E2F-1 and E1A following treatment of Y79 cells exposed to VCN-01 for 48 h (50 MOI).At 24 h, TPT (2 μM), CBP (12.5 μM), MEL (12 μM), hydroxyurea (HU; 100 μM), etoposide (ETO; 2 μM), irinotecan (IRN; 10 μM), or SN-38 (1 μM) were added. GAPDH was used as a loading control.

[0062] Figure 14 Shown are the antiproliferative activities of topotecan, carboplatin, and melphalan in combination with 10 MOI of VCN-01 or as single agents (medium) in RBT-5, RBT-7, and RBVS-10 cells. Values ​​are means and standard deviations of three replicates. Lines are best-fit curves constructed using GraphPad. Cell viability is expressed as the relative percentage of the assay signal in treated cells compared to control untreated cells (which was set to 100%).

[0063] Figures 15A-15C show the in vitro interaction of VCN-01 with chemotherapy. Figure 15A shows immunoblots of p53, cPARP, and E1A after treatment of primary retinoblastoma cells (RBT-5, RBT-7, and RBVS-10) with VCN-01 (48h; 50 MOI), topotecan (TPT; 24h; 2 μM), a combination of VCN-01 and TPT (VCN-01 / TPT; 48h; TPT added at 24h), carboplatin (CBP; 24h; 12.5 μM), a combination of VCN-01 and CBP (VCN-01 / CBP; 48h; CBP added at 24h), melphalan (MEL; 24h; 12 μM), and a combination of VCN-01 and MEL (VCN-01 / MEL; 48h; MEL added at 24h). Control cells were untreated. GAPDH is a loading control. Figure 15B shows cPARP treated with VCN-01 (48 h; 50 MOI), topotecan (24 h; 2 μM), or the combination (VCN-01 / TPT; 48 h; TPT added at 24 h) + Ratios (%) of Y79 and RBT-7 cells. Dots represent experimental replicates and bars represent mean and standard deviation. FIG15C shows representative flow cytometry histograms of cPARP expression in Y79 and RBT-7 cells.

[0064] Figure 16Shown are immunoblots for topoisomerase 1 (TOPO 1) in primary retinoblastoma cells (RBT-7 and RBVS-10) treated with VCN-01 (48 h; 50 MOI), topotecan (TPT; 24 h; 2 μM), a combination of VCN-01 and TPT (VCN-01 / TPT; 48 h; TPT added at 24 h), carboplatin (CBP; 24 h; 12.5 μM), a combination of VCN-01 and CBP (VCN-01 / CBP; 48 h; CBP added at 24 h), melphalan (MEL; 24 h; 12 μM), and a combination of VCN-01 and MEL (VCN-01 / MEL; 48 h; MEL added at 24 h). Control cells were untreated. GAPDH is a loading control.

[0065] Figures 17A-17G show the cell cycle and E2F-1 expression of retinoblastoma cells exposed to chemotherapy. Figure 17A shows the percentage of Y79 cells in the G2, S, and G1 cell cycles after treatment with culture medium (control), topotecan (TPT; 2 μM), carboplatin (CBP; 12.5 μM), or hydroxyurea (HU; 100 μM) for 24 hours. The bar graph is the mean and standard deviation of three replicates. Figure 17B shows representative images of cell cycle assessment of Y79 cells treated with TPT (2 μM) for 4 hours, 16 hours, or 24 hours. In the chart, G1 phase cells are green (left peak). S phase cells are yellow (center), and G2 phase cells are blue (right). Figure 17C shows the percentage of Y79 cells in the G2, S, and G1 cell cycles after treatment with TPT (2 μM) for up to 48 hours. The bar graph is the mean and SD of three replicates. Figure 17D shows immunoblots for E2F-1, cyclin E1, and p21 following treatment of Y79 cells with TPT (2 μM), CBP (12.5 μM), MEL (12 μM), HU (100 μM), or SN-38 (1 μM) for up to 48 hours. GAPDH is a loading control. Figure 17E shows immunoblots for E2F-1 (brown nuclear stain) in Y79 intraocular xenografts treated with TPT (0.6 mg / kg, once daily for five days) or saline. Figure 17F shows expression of cyclins and cell cycle regulatory genes induced by TPT (2 μM; 24 hours) and CBP (12.5 μM; 24 hours) in RBT-7 cells infected with VCN-01 (50 MOI; 48 hours). Values ​​are fold changes relative to expression in control infected cells exposed to medium. Figure 17G shows the expression of CDKN1A and CDK6 in RBT-7 cells infected with VCN-01 48 h after treatment with TPT (2 μM) or CBP (12.5 μM). Values ​​are fold changes relative to expression in control infected cells exposed to medium.

[0066] Figures 18A-18J show the effects of chemotherapy on adenoviral transduction and replication in retinoblastoma. Figure 18A shows a representative graph of the percentage of Y79 cells in the G2, S, and G1 cell cycles after treatment with hydroxyurea (HU; 4mM) (synchronous) or culture medium (asynchronous). The table contains quantification of the graphs. Figure 18B shows representative bright light and fluorescent images of Y79 cells pre-exposed to 4mM HU (synchronous) and treated with AdTLRGDK (50MOI) for 24h, 48h, or 72h. Culture medium was used as a control (asynchronous). Figure 18C shows a representative pseudo-color flow graph of the experiment in Figure B. Uninfected cells were used as a negative control. Figure 18D shows GFP expression in the experiment in Figure B. + Counts of cells (%). Points are experimental replicates (n=3-4), and bars are means and standard deviations. FIG18E shows GFP expression in cells pretreated with AdTLRGDK (50 MOI) for 24 h and additionally exposed to culture medium (culture medium / Medium), topotecan (TPT; 2 μM), carboplatin (CBP: 12.5 μM), melphalan (MEL; 12 μM), or HU (100 μM) for 24 h, or exposed to the reverse order of GFP expression in which cells were treated with TPT or HU 6 h before AdTLRGDK infection. + The ratio (%) of Y79 cells. Points represent experimental replicates and bars represent mean and standard deviation. Figure 18F shows the effect of TPT (2 μM) on the viral production of VCN-01 in Y79 cells, measured as the number of viral genomes per mL of cell pellet extract. Figure 18G shows the quantification of VCN-01 genomes in subcutaneous (sc) retinoblastomas (Y79 and RBT-7) 5 days after local injection of VCN-01 and subsequent treatment with saline, TPT (0.6 mg / kg, once daily for five consecutive days), CBP (40 mg / kg, a single dose on day 1), or HU (200 mg / kg, once daily for five consecutive days). Points are values ​​from individual tumors. Figure 18H shows the mRNA expression of recombinant hyaluronidase PH20 (SPAM1 gene) in subcutaneous retinoblastomas (Y79 and RBT-7) 5 days after local injection of VCN-01 and subsequent treatment with TPT or CBP. Figure 18I shows immunostaining of E1A in subcutaneous Y79 xenografts 5 days after local injection of VCN-01 and subsequent treatment with TPT, CBP, or HU. Figure 18J shows immunoblotting of E1A in subcutaneous Y79 xenografts 5 or 15 days after local injection of VCN-01 and subsequent treatment with TPT, CBP, or HU. Each sample corresponds to a separate tumor.

[0067] Figures 19A-19B show adenoviral transduction and replication in retinoblastoma cells exposed to topotecan. Figure 19A shows GFP expression in cells pretreated with AdTLRGK for 24 h (50 MOI) and additionally exposed to culture medium (culture medium / Medium), topotecan (TPT; 2 μM), carboplatin (CBP; 12.5 μM), melphalan (MEL; 12 μM), or hydroxyurea (HU; 100 μM) for 24 h. + The proportion (%) of RBT-5 and RBT-7 cells. Points represent experimental replicates and bars represent means and standard deviations. Figure 19B shows quantification of the VCN-01 genome in subcutaneous retinoblastomas (Y79 and RBT-7) 15 days after local injection of VCN-01 and subsequent treatment with saline, TPT (0.6 mg / kg, once daily for five consecutive days), or CBP (40 mg / kg, a single dose on day 1). Points are values ​​from individual tumors.

[0068] Figures 20A-20I show the activity of topical VCN-01 and systemic topotecan in retinoblastoma xenografts. Figure 20A shows the eye survival rate of eyes with Y79 xenografts treated with intravitreal VCN-01, standard of care chemotherapy (SoC), topotecan (TPT), or a combination. Figure 20B shows the eye survival rate of eyes with Y79 xenografts treated with topotecan, VCN-01, or a combination in the order of topotecan first (TPT+VCN-01) or virus first (VCN-01+TPT). Figure 20C shows the eye survival rate of eyes with RBT-7 xenografts treated with topotecan, VCN-01, or a combination. Figure 20D shows representative images of mice with bilateral RBT-7 intraocular xenografts, the left eye of which was treated with systemic topotecan and a dose of VCN-01, and the right eye was treated with saline. The images were obtained 30 days after the start of treatment. Figure 20E shows representative H&E staining of surviving eyes (day 80) bearing RBT-7 xenografts treated with VCN-01 and topotecan. Figure 20F shows E1A immunostaining in RBT-7 intraocular xenografts treated with VCN-01 and topotecan and enucleated at endpoint (65 days after VCN-01 injection). Figure 20G shows ocular survival of eyes bearing RBT-2 xenografts treated with a maximal dosing schedule including VCN-01 (two injections), topotecan (6 cycles), and the combination. Figure 20H shows the survival of mice bearing Y79 or RBT-7 subcutaneous xenografts treated with a single intratumoral injection of VCN-01, systemic topotecan, or the combination. Figure 20I shows the antitumor activity of a single intratumoral injection of VCN-01, systemic topotecan, or the combination in subcutaneous retinoblastoma Y79 and RBT-7. The graph shows the growth of individual tumors.

[0069] Figure 21 Shown is the intratumoral accumulation of lactone topotecan (TPT) and total topotecan at steady state (i.e., constant concentration in plasma) in orthotopic retinoblastoma xenografts (RBT-2) pretreated with local intraocular injection of adenovirus (VCN-01) or vehicle solution (Vehicle). Points are values ​​obtained from individual tumors, and lines are means and standard deviations.

[0070] Figures 22A-22J show the activity of topical VCN-01 and systemic topotecan in CNS disseminated retinoblastoma. Figure 22A shows the transplantation of RBT-7 cells in the mouse brain at endpoint. The entire brain was stained with hematoxylin and eosin (H&E). Cancer cells in high-magnification images were stained brown with anti-human nuclear antibodies. Figure 22B shows the transplantation of RBT-7 cells in the meninges surrounding the spinal cord of the mouse at endpoint (H&E and anti-human nuclear). Figure 22C shows the survival rate of mice bearing RBT-7 brain tumors treated with standard of care chemotherapy (SoC), topotecan (TPT) alone, VCN-01 alone, or a combination of VCN-01 and topotecan. Figure 22D shows images of mice that received VCN-01 alone or VCN-01 with topotecan 25 days after tumor inoculation (i.e., 17 days after VCN-01 injection). The numbers on the mice are individual weights (g). Figure 22E shows immunostaining of human cells (anti-human nuclei, brown) in mice sacrificed 26 days after tumor inoculation. Figure 22F shows tumor burden (CRX gene expression) in brain homogenates at the end of treatment (5 days after intraventricular injection of VCN-01; i.e., day 12 after tumor inoculation). The dots are data from individual brains and the bars are the mean and standard deviation. Figure 22G shows the expression of CDKN1A and E2F1 in brain homogenates at the end of treatment. The dots are individual brains, with the highest value identified by the mouse number. Figure 22H shows the quantification of the VCN-01 genome in brain homogenates at the end of treatment. Figure 22I shows the quantification of the mRNA of the human hyaluronidase gene in brain homogenates at the end of treatment. Figure 22J shows an immunoblot of adenovirus hexon in brain homogenates obtained at the end of treatment. GAPDH is a loading control. Samples are numbered according to the identity of the mouse.

[0071] Figures 23A-23C show toxicity and infection of intraventricular VCN-01 treatment in mice with CNS disseminated retinoblastoma (RBT-7). Figure 23A shows individual body weights of mice bearing RBT-7 brain tumors treated with a single intraventricular injection of VCN-01, standard of care chemotherapy (SoC), topotecan (TPT) alone, VCN-01 alone, or a combination of VCN-01 and TPT. Control mice were treated with a single intraventricular injection of vehicle. Figure 23B shows immunostaining for the leukocyte marker CD45 in the brain of a mouse bearing an RBT-7 brain xenograft, treated with a combination of intraventricular VCN-01 and SoC, and sacrificed 9 days after VCN-01 inoculation due to acute weight loss. High-magnification images correspond to CD45. +Figure 23C shows immunostaining for E1A in the brains of mice bearing RBT-7 xenografts treated with a single intracerebroventricular dose of VCN-01 alone (VCN-01) or in combination with TPT (VCN-01+TPT). Mice were sacrificed and samples obtained at the endpoint of treatment. High-magnification images correspond to tumor areas.

[0072] Figures 24A-24G show the activity of topical VCN-01 and systemic topotecan in Ewing's sarcoma and neuroblastoma PDXs. Figure 24A shows the gene expression of E2F1, CXADR, and ITGA5 in retinoblastoma, neuroblastoma, Ewing's sarcoma, and their control tissues, fetal retina, pediatric brain, and muscle, respectively. Each tumor was compared to its control (Mann-Whitney test). Figure 24B shows immunostaining of E2F-1 in patient biopsies of retinoblastoma (positive control), Ewing's sarcoma, and neuroblastoma, corresponding to the HSJD patients from whom PDXs ES-033 and NB-005 were established. Figure 24C shows immunoblots of E2F-1 and cyclin E1 in NB-005 and ES-003 cells treated with topotecan for up to 48 hours. GAPDH is a loading control. Figure 24D shows the survival of mice bearing neuroblastoma treated with topical VCN-01, topotecan (TPT), irinotecan (IRN), or a combination. Figure 24E shows the survival of PDX ES-033 treated with topical VCN-01, TPT, or a combination thereof. Figure 24F shows the tumor volume (individual values) of neuroblastoma PDXs treated with topical VCN-01, TPT, IRN, or a combination. Figure 24G shows the tumor volume (individual values) of Ewing's sarcoma PDXs treated with topical VCN-01, TPT, or a combination.

[0073] Figure 25 Figure 2 shows the antiproliferative activity of VCN-01 (6-day treatment) against PDX-derived primary cells in culture. Values ​​are the means and standard deviations of six replicates. Lines are the best-fit curves constructed using the least-squares regression method in GraphPad. Cell viability is the relative percentage of the assay signal in treated cells compared to control untreated cells, which was set to 100%. DETAILED DESCRIPTION

[0074] Overview

[0075] The present disclosure is based, inter alia, on the discovery that a genetically modified oncolytic adenovirus can be used in combination with a topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38, or irinotecan) to treat retinoblastoma, Ewing's sarcoma, or neuroblastoma. This discovery is surprising for a number of reasons, including but not limited to the fact that topotecan and irinotecan (a prodrug of the topoisomerase I inhibitor SN-38), but not other chemotherapy agents such as carboplatin or melphalan, enhance the viral activity of the modified oncolytic adenovirus in retinoblastoma cells.

[0076] The inventors of the present disclosure have surprisingly discovered that the oncolytic adenoviruses described herein, i.e., oncolytic adenoviruses comprising a sequence encoding hyaluronidase inserted into their genome and a replication machinery specific for tumor cells, can be used in combination with a topoisomerase I inhibitor or a prodrug thereof (e.g., topotecan or a prodrug of a topoisomerase I inhibitor (e.g., irinotecan, which is converted to SN-38 in vivo) to effectively treat retinoblastoma, Ewing's sarcoma, or neuroblastoma. Without wishing to be bound by theory, it is believed that administration of topotecan, SN-38, or irinotecan can sensitize retinoblastoma, Ewing's sarcoma, or neuroblastoma to the oncolytic adenovirus and enhance the replication of the oncolytic virus in these tumors.

[0077] Treatment of retinoblastoma

[0078] In various aspects, the present disclosure provides methods of treating retinoblastoma in a patient in need thereof, comprising administering to the patient (i) an oncolytic adenovirus comprising a replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug thereof, e.g., topotecan, SN-38, or irinotecan.

[0079] In various aspects, the present disclosure provides methods for preventing, eliminating, or reducing retinoblastoma-associated metastases, secondary malignancies, and or trilateral retinoblastomas in a patient in need thereof, the methods comprising administering to the patient (i) an oncolytic adenovirus comprising a replication machinery specific for tumor cells, and optionally, a polynucleotide sequence encoding a hyaluronidase inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug thereof, e.g., topotecan, SN-38, or irinotecan.

[0080] Retinoblastoma is the most common type of eye cancer in children. It is usually found in children younger than two years old. As the eyes develop, they develop progenitor cells called retinoblastomas. These cells divide into new cells and populate the part of the eye that will become the retina.

[0081] Non-hereditary retinoblastoma, also known as sporadic retinoblastoma, occurs sporadically. Approximately 60% of children with retinoblastoma have non-hereditary retinoblastoma. Children are born with two normal copies of the RB1 gene. A mutation in both copies of the RB1 gene in the retinoblastoma cells causes retinoblastoma tumors to form in the eye. Children with non-hereditary retinoblastoma develop a tumor in only one eye, called unilateral retinoblastoma. They typically do not pass the RB1 mutation on to their future children.

[0082] Hereditary retinoblastoma is passed down from parents to children. Approximately 40% of children with retinoblastoma have hereditary retinoblastoma. Hereditary retinoblastoma can be familial or sporadic. In familial hereditary retinoblastoma (also called familial retinoblastoma), a parent or other family member has retinoblastoma. In sporadic hereditary retinoblastoma, no one else in the family has a history of retinoblastoma. The RB1 gene mutation occurs as a germline mutation in the egg or sperm before conception and is passed down to the child. In all cases of hereditary retinoblastoma, a child is born with one copy of the RB1 mutation in all cells of the body. A second copy of the RB1 gene then mutates in a retinoblastoma, leading to the development of retinoblastoma. Children with hereditary retinoblastoma may have more than one tumor, and tumors may occur in one or both eyes, a condition called bilateral retinoblastoma. Children with hereditary retinoblastoma can pass the RB1 mutation on to their future children. They are also at increased risk of developing other cancers.

[0083] In embodiments, the retinoblastoma is non-hereditary retinoblastoma. In embodiments, the retinoblastoma is hereditary retinoblastoma.

[0084] Retinoblastoma is classified using several staging systems based on how far the cancer has spread. The prognosis for children with retinoblastoma depends partly on the stage of the cancer. Staging is also an important factor in choosing treatment.

[0085] Retinoblastoma is typically staged based on the results of an eye exam, imaging studies, and any other relevant tests. When determining treatment options, retinoblastoma is typically divided into two major categories: (i) intraocular retinoblastoma, in which the cancer is still inside the eye; and (ii) extraocular retinoblastoma, in which the cancer has spread outside the eye. Extraocular cancer can be further divided into orbital retinoblastoma (which has spread only to the orbit of the eye) and metastatic retinoblastoma (which has spread to distant parts of the body, such as the brain or bone marrow).

[0086] In embodiments, the retinoblastoma is intraocular retinoblastoma. In embodiments, the retinoblastoma is extraocular retinoblastoma. In embodiments, the retinoblastoma is orbital retinoblastoma. In embodiments, the retinoblastoma is metastatic retinoblastoma.

[0087] In embodiments, the method prevents progression of intraocular retinoblastoma to extraocular retinoblastoma. In embodiments, the method prevents progression of intraocular retinoblastoma to orbital retinoblastoma. In embodiments, the method prevents progression of intraocular retinoblastoma to metastatic retinoblastoma. In embodiments, the method prevents progression of orbital retinoblastoma to metastatic retinoblastoma.

[0088] In the United States, most retinoblastomas are diagnosed before they spread outside the eye, so staging systems that apply only to intraocular retinoblastomas are most commonly used. There are two commonly used staging systems for intraocular retinoblastoma: the International Classification for Intraocular Retinoblastoma and the Reese-Ellsworth Staging System.

[0089] The International Classification of Intraocular Retinoblastoma divides intraocular retinoblastoma into five groups, A through E, based on the extent of the cancer and based on the chance that the eye can be saved using current treatment options.

[0090] Group A: Small tumors (no larger than 3 mm in diameter) located only within the retina and not near important structures such as the optic disc (where the optic nerve enters the retina) or the fovea (the center of vision).

[0091] Group B: All other tumors (greater than 3 mm or close to the optic disc or fovea), still located only in the retina.

[0092] Group C: Tumors with well-defined margins and minimal subretinal extension, either subretinal seeding or vitreous seeding.

[0093] Group D: Tumors are large or poorly circumscribed, with extensive vitreous or subretinal infiltration. The retina may have detached from the back of the eye.

[0094] Group E: The tumor is very large, extends near the front of the eye, bleeds or causes glaucoma (high pressure inside the eye), or has other characteristics that mean there is little chance of saving the eye.

[0095] In embodiments, retinoblastoma is classified using the International Classification of Intraocular Retinoblastoma and is classified as Group A. In embodiments, retinoblastoma is classified using the International Classification of Intraocular Retinoblastoma and is classified as Group B. In embodiments, retinoblastoma is classified using the International Classification of Intraocular Retinoblastoma and is classified as Group C. In embodiments, retinoblastoma is classified using the International Classification of Intraocular Retinoblastoma and is classified as Group D. In embodiments, retinoblastoma is classified using the International Classification of Intraocular Retinoblastoma and is classified as Group E.

[0096] In embodiments, the method prevents progression of Group A retinoblastoma to Group B retinoblastoma, Group C retinoblastoma, Group D retinoblastoma, or Group E retinoblastoma. In embodiments, the method prevents progression of Group B retinoblastoma to Group C retinoblastoma, Group D retinoblastoma, or Group E retinoblastoma. In embodiments, the method prevents progression of Group C retinoblastoma to Group D retinoblastoma or Group E retinoblastoma. In embodiments, the method prevents progression of Group D retinoblastoma to Group E retinoblastoma.

[0097] The Reese-Ellsworth staging system divides intraocular retinoblastoma into five groups. The higher the group number (from 1 to 5), the lower the chance of controlling the retinoblastoma or saving the eye or any useful vision.

[0098] Group 1 (very favorable for salvaging or eye preservation) - 1A: One tumor, smaller than 4 disc diameters (DD), located at or behind the equator; 1B: Multiple tumors smaller than 4 DD, all located at or behind the equator.

[0099] • Group 2 (favorable for salvage or eye preservation) - 2A: one tumor, 4 to 10 DD, located at or behind the equator; 2B: multiple tumors, at least one of which is 4 to 10 DD, and all located at or behind the equator.

[0100] • Group 3 (doubtful to salvage or preserve the eye) - 3A: any tumor anterior to the equator; 3B: one tumor greater than 10 DD posterior to the equator.

[0101] • Group 4 (Not Favorable for Salvage (or Saving) of the Eye) - 4A: Multiple tumors, some larger than 10 DD; 4B: Any tumor extending to the front of the eye to the ora serrata (front edge of the retina).

[0102] • Group 5 (very unfavorable for salvaging (or preserving) the eye) - 5A: tumor involving more than half of the retina; 5B: vitreous implants.

[0103] In embodiments, retinoblastoma is classified using the Reese-Ellsworth system and is classified into Group 1A. In embodiments, retinoblastoma is classified using the Reese-Ellsworth system and is classified into Group 1B. In embodiments, retinoblastoma is classified using the Reese-Ellsworth system and is classified into Group 2A. In embodiments, retinoblastoma is classified using the Reese-Ellsworth system and is classified into Group 2B. In embodiments, retinoblastoma is classified using the Reese-Ellsworth system and is classified into Group 3A. In embodiments, retinoblastoma is classified using the Reese-Ellsworth system and is classified into Group 3B. In embodiments, retinoblastoma is classified using the Reese-Ellsworth system and is classified into Group 4A. In embodiments, retinoblastoma is classified using the Reese-Ellsworth system and is classified into Group 4B. In embodiments, retinoblastoma is classified using the Reese-Ellsworth system and is classified into Group 5A. In an embodiment, retinoblastoma is classified using the Reese-Ellsworth system and is classified as Group 5B.

[0104] In embodiments, the method prevents progression of Group 1A retinoblastoma to Group 1B retinoblastoma, Group 2A retinoblastoma, Group 2B retinoblastoma, Group 3A retinoblastoma, Group 3B retinoblastoma, Group 4A retinoblastoma, Group 4B retinoblastoma, Group 5A retinoblastoma, or Group 5B retinoblastoma. In embodiments, the method prevents progression of Group 1B retinoblastoma to Group 2A retinoblastoma, Group 2B retinoblastoma, Group 3A retinoblastoma, Group 3B retinoblastoma, Group 4A retinoblastoma, Group 4B retinoblastoma, Group 5A retinoblastoma, or Group 5B retinoblastoma. In embodiments, the method prevents progression of Group 2A retinoblastoma to Group 2B retinoblastoma, Group 3A retinoblastoma, Group 3B retinoblastoma, Group 4A retinoblastoma, Group 4B retinoblastoma, Group 5A retinoblastoma, or Group 5B retinoblastoma. In embodiments, the method prevents the progression of Group 2B retinoblastoma to Group 3A retinoblastoma, Group 3B retinoblastoma, Group 4A retinoblastoma, Group 4B retinoblastoma, Group 5A retinoblastoma, or Group 5B retinoblastoma. In embodiments, the method prevents the progression of Group 3A retinoblastoma to Group 3B retinoblastoma, Group 4A retinoblastoma, Group 4B retinoblastoma, Group 5A retinoblastoma, or Group 5B retinoblastoma. In embodiments, the method prevents the progression of Group 3B retinoblastoma to Group 4A retinoblastoma, Group 4B retinoblastoma, Group 5A retinoblastoma, or Group 5B retinoblastoma. In embodiments, the method prevents the progression of Group 4A retinoblastoma to Group 4B retinoblastoma, Group 5A retinoblastoma, or Group 5B retinoblastoma. In embodiments, the method prevents the progression of Group 4B retinoblastoma to Group 5A retinoblastoma or Group 5B retinoblastoma. In embodiments, the method prevents progression of Group 5A retinoblastoma to Group 5B retinoblastoma.

[0105] Some doctors may use other staging systems that include both intraocular retinoblastomas and those that have spread outside the eye (extraocular retinoblastomas). These staging systems may be particularly useful in countries where these cancers are likely to have spread by the time they are discovered. For example, the American Joint Committee on Cancer (AJCC) staging system considers four key pieces of information:

[0106] T: The size of the primary tumor and its extent of growth inside and outside the eye

[0107] N: Whether the cancer has spread to nearby lymph nodes in the head or neck

[0108] M: Whether the cancer has spread to distant parts of the body, such as the bone marrow, brain, skull, or long bones

[0109] H: Whether the child has a heritable form of retinoblastoma

[0110] This system is used to detail the extent of retinoblastoma, particularly those that have spread outside the eye.

[0111] In an embodiment, retinoblastoma is classified using the AJCC staging system.

[0112] In embodiments, the methods described herein eliminate the need for a patient to undergo an enucleation procedure.

[0113] Treatment of Ewing's sarcoma

[0114] In various aspects, the present disclosure provides methods of treating Ewing sarcoma in a patient in need thereof, comprising administering to the patient (i) an oncolytic adenovirus comprising a replication machinery specific for tumor cells, and optionally, inserted into its genome, a polynucleotide sequence encoding a hyaluronidase and a replication machinery specific for tumor cells, and (ii) a topoisomerase I inhibitor or a prodrug thereof, e.g., topotecan, SN-38, or irinotecan.

[0115] In various aspects, the present disclosure provides methods of treating Ewing sarcoma in a patient in need thereof, the methods comprising administering to the patient (i) an oncolytic adenovirus comprising a replication machinery specific for tumor cells and a polynucleotide sequence encoding a hyaluronidase inserted into its genome and a replication machinery specific for tumor cells, and (ii) a topoisomerase I inhibitor or a prodrug thereof, e.g., topotecan, SN-38, or irinotecan.

[0116] Ewing tumors (also called Ewing sarcomas) are a group of cancers that begin in bones or nearby soft tissue and share some common characteristics. These tumors can develop in people of any age but are most common in older children and adolescents.

[0117] The main types of Ewing tumors are Ewing sarcoma of bone, extraosseous Ewing tumor (EOE), and peripheral primitive neuroectodermal tumor (PPNET). Ewing sarcoma of bone is the most common and is the type of Ewing sarcoma that begins in the bone. EOE tumors begin in the soft tissue surrounding the bones, but they look and behave very similarly to Ewing sarcomas in the bones. They are also called extraskeletal Ewing sarcomas. PPNET is a rare childhood cancer that also begins in the bones or soft tissues and shares many features with Ewing sarcoma of bone and EOE. Peripheral PNETs that begin in the chest wall are called Askin tumors.

[0118] Most Ewing tumors develop in bones. The most common sites are the pelvis (hip bones), chest wall (such as the ribs or shoulder blades), and legs (primarily in the middle of the long bones). Extraskeletal Ewing tumors can develop almost anywhere.

[0119] In embodiments, the Ewing's sarcoma is Ewing's sarcoma of bone. In embodiments, the Ewing's sarcoma is EOE. In embodiments, the Ewing's sarcoma is PPNET.

[0120] Treatment of Ewing sarcoma is based primarily on its location in the body and how far it has spread when it is first discovered, and it is generally classified as localized, metastatic, or recurrent Ewing sarcoma.

[0121] A localized Ewing's tumor is one that appears to remain confined to the area where it started (and may also be confined to nearby tissues, such as muscle or tendon), based on imaging tests and biopsy results. However, even people with a localized Ewing's tumor often have small areas of cancer elsewhere in the body that can't be seen with imaging tests but can grow. Therefore, chemotherapy is an important part of treatment to prevent localized Ewing's tumors from spreading.

[0122] Once Ewing's tumor is diagnosed and staged, the first treatment is chemotherapy. It is called neoadjuvant chemotherapy because it is given before any surgery or radiation therapy. In the United States, patients are given a chemotherapy regimen called VDC / IE (or VAC / IE), which is a combination of vincristine, doxorubicin (Adriamycin), and cyclophosphamide, alternating with ifosfamide and etoposide, but other combinations of the same drugs are also effective.

[0123] After at least nine weeks of chemotherapy, imaging tests (such as CT, MRI, PET, or bone scans) are done to see if the tumor has shrunk or at least has not grown and can be removed surgically. If so, surgery is done at this time, and a pathologist analyzes the surgical specimen. If cancer cells are found at or near the edges of the surgical specimen, meaning there may be some cancer cells left, radiation therapy and chemotherapy are continued for several months. If there are no cancer cells at or near the edges of the surgical specimen, chemotherapy may be used instead of radiation therapy.

[0124] If surgery isn't an option after initial chemotherapy, but the tumor hasn't grown, radiation therapy and chemotherapy are usually given as the next step. In some cases, this may shrink the tumor enough to allow surgery. This is then followed by more chemotherapy and possibly more radiation. In other cases, radiation therapy and chemotherapy are the main treatments.

[0125] If the Ewing tumor continues to grow despite initial chemotherapy, a second type of chemotherapy may be tried. Surgery or radiation therapy may also be tried to help keep the tumor under control. More chemotherapy may also follow.

[0126] Patients who have metastatic disease when first diagnosed are more difficult to treat than those with localized disease. The outlook is often better when the cancer has spread only to the lungs than when it has spread to other bones or the bone marrow.

[0127] Treating metastatic disease is similar in many ways to treating localized disease. Chemotherapy is the first treatment, but it usually requires a stronger regimen than used when the cancer is localized. After a few months, tests (such as CT or MRI scans, bone scans or PET scans, and / or a bone marrow biopsy) are done to see how the cancer responded to treatment.

[0128] If cancer remains in only a few small areas after chemotherapy, surgery may be used to remove the primary tumor and all known metastatic areas. Other options include surgery plus radiation therapy (before and / or after surgery) or radiation therapy alone to all known metastatic sites. During and after these treatments, chemotherapy is also given for several months.

[0129] Another treatment being studied is high-intensity chemotherapy followed by a stem cell transplant to try to improve outcomes for these patients.

[0130] Ewing tumors are less likely to come back after treatment than they used to be, but they can still happen. If the tumor comes back, treatment depends on many factors, including: the size and location of the tumor, whether it has spread to different parts of the body, what types of treatments were used previously, and how long it has been since treatment.

[0131] Depending on the condition, recurrent tumors may be treated with chemotherapy, surgery, radiation therapy, or some combination of these.

[0132] Other treatments being studied include high-dose chemotherapy followed by a stem cell transplant and targeted drugs and immunotherapy.

[0133] In embodiments, the Ewing sarcoma is a localized Ewing tumor. In embodiments, the Ewing sarcoma is a metastatic Ewing tumor. In embodiments, the Ewing sarcoma is a recurrent Ewing tumor.

[0134] In embodiments, the method prevents progression of a localized Ewing tumor to a metastatic Ewing tumor or a recurrent Ewing tumor. In embodiments, the method prevents progression of a metastatic Ewing tumor to a recurrent Ewing tumor.

[0135] The American Joint Committee on Cancer (AJCC) uses a system to describe all bone cancers, including Ewing tumors that begin in the bones. The AJCC stages Ewing's tumors, which do not begin in the bones like soft tissue sarcomas. The AJCC staging system for bone cancer and soft tissue sarcomas is based on four key pieces of information:

[0136] T describes the size of the primary tumor and whether it occurs in different areas of the bone.

[0137] N describes the extent of spread to regional lymph nodes. Bone tumors rarely spread to lymph nodes.

[0138] M indicates whether the cancer has spread to other organs in the body. (The most common sites of spread are the lungs or other bones.)

[0139] G stands for the grade of the tumor, which describes the appearance of the cells in the biopsy sample. Low-grade tumor cells look more like normal cells and are less likely to grow and spread quickly, while high-grade tumor cells look more abnormal.

[0140] Once the T, N, M, and G categories are determined, the information is combined and presented as an overall stage. The process of assigning stage numbers is called stage grouping. The stages are described with Roman numerals from I to IV and are sometimes further divided.

[0141] Stage I is not used for Ewing sarcoma. This is because all Ewing sarcomas are high grade (G2 or G3). Stage I is used for other types of bone cancer.

[0142] Stage II is divided into 2 groups:

[0143] Stage IIA. The tumor is no larger than 8 centimeters (cm) in diameter and is high-grade. The cancer has not spread to nearby lymph nodes or organs elsewhere in the body.

[0144] Stage IIB. The tumor is larger than 8 centimeters (cm) in diameter and is high-grade. The cancer has not spread to nearby lymph nodes or organs elsewhere in the body.

[0145] Stage III means the tumor is in more than one spot in the same bone and is high grade. The cancer has not spread to nearby lymph nodes or organs in other parts of the body.

[0146] Stage IV is divided into 2 groups:

[0147] Stage IVA. Cancer has spread to the lungs but not to the lymph nodes or organs elsewhere in the body. It can be any size or grade.

[0148] Stage IVB is any of these:

[0149] The cancer has spread to nearby lymph nodes. It may or may not have spread to organs elsewhere in the body. It can be any size or grade.

[0150] ○ Cancer has spread to organs elsewhere in the body, but not to the lungs. It can be any size or grade.

[0151] In embodiments, Ewing's sarcoma is stage II. In embodiments, Ewing's sarcoma is stage III. In embodiments, Ewing's sarcoma is stage IV. In embodiments, Ewing's sarcoma is stage IIA. In embodiments, Ewing's sarcoma is stage IIB. In embodiments, Ewing's sarcoma is stage IIIA. In embodiments, Ewing's sarcoma is stage IIIB. In embodiments, Ewing's sarcoma is stage IVA. In embodiments, Ewing's sarcoma is stage IVB.

[0152] In embodiments, the method prevents the progression of stage II Ewing's sarcoma to stage III or stage IV Ewing's sarcoma. In embodiments, the method prevents the progression of stage III Ewing's sarcoma to stage IV Ewing's sarcoma. In embodiments, the method prevents the progression of stage IIA Ewing's sarcoma to stage IIB, stage IIIA, stage IIIB, stage IVA, or stage IVB Ewing's sarcoma. In embodiments, the method prevents the progression of stage IIB Ewing's sarcoma to stage IIIA, stage IIIB, stage IVA, or stage IVB Ewing's sarcoma. In embodiments, the method prevents the progression of stage IIIA Ewing's sarcoma to stage IIIB, stage IVA, or stage IVB Ewing's sarcoma. In embodiments, the method prevents the progression of stage IIIB Ewing's sarcoma to stage IVA or stage IVB Ewing's sarcoma. In embodiments, the method prevents the progression of stage IVA Ewing's sarcoma to stage IVB Ewing's sarcoma.

[0153] Treatment of neuroblastoma

[0154] In various aspects, the present disclosure provides methods of treating neuroblastoma in a patient in need thereof, comprising administering to the patient (i) an oncolytic adenovirus comprising a replication machinery specific for tumor cells, and optionally, a polynucleotide sequence encoding a hyaluronidase and a replication machinery specific for tumor cells inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug thereof, e.g., topotecan, SN-38, or irinotecan.

[0155] In various aspects, the present disclosure provides methods of treating neuroblastoma in a patient in need thereof, comprising administering to the patient (i) an oncolytic adenovirus comprising a replication machinery specific for tumor cells and a polynucleotide sequence encoding a hyaluronidase inserted into its genome and a replication machinery specific for tumor cells, and (ii) a topoisomerase I inhibitor or a prodrug thereof, e.g., topotecan, SN-38, or irinotecan.

[0156] Neuroblastoma is a cancer that begins in cells called neuroblastomas, which are cells in the sympathetic nervous system. This type of cancer most often develops in infants and young children.

[0157] Most neuroblastomas begin in the abdomen, in the adrenal glands or sympathetic ganglia. Most others begin in the sympathetic ganglia near the spine in the chest or neck or in the pelvis. Rarely, by the time a neuroblastoma is discovered, it has spread so extensively that doctors cannot pinpoint its origin. Some neuroblastomas grow and spread rapidly, while others grow slowly. Sometimes, in very young children, the cancer cells die for no apparent reason, and the tumor disappears on its own. In other cases, the cells mature into normal ganglion cells and stop dividing, making the tumor a benign ganglioneuroma.

[0158] There are two systems used to stage neuroblastoma. The main difference between them is whether the staging system can be used to help determine a child's risk group before treatment begins. The International Neuroblastoma Risk Group Staging System (INRGSS) uses the results of imaging tests (such as CT or MRI and MIBG scans) to help determine the stage. The INRGSS stage can be determined before treatment begins. The International Neuroblastoma Staging System (INSS) is based on the results of surgery to remove the child's tumor, not on imaging tests.

[0159] The INRGSS was developed to help determine a child's stage and risk group before treatment begins. The INRGSS uses imaging tests (usually CT or MRI scans and MIBG scans) along with examinations and biopsies to help define the stage. The stage can then be used to help predict how removable the tumor will be. The INRGSS uses imaging-defined risk factors (IDRFs), which are factors seen on imaging tests that may mean the tumor will be harder to remove. This includes things like the tumor growing into nearby vital organs or growing around important blood vessels. The INRGSS divides neuroblastoma into 4 stages:

[0160] L1: The tumor has not spread from its initial location and has not grown into vital structures as defined by the IDRF list. It is confined to one area of ​​the body, such as the neck, chest, or abdomen.

[0161] L2: The tumor has not spread far from where it started (for example, it may have grown from the left side of the abdomen to the left side of the chest), but it has at least one IDRF.

[0162] M: The tumor has metastasized to distant parts of the body (except in patients with MS).

[0163] MS: Metastatic disease in children younger than 18 months, with cancer that has spread only to the skin, liver, and / or bone marrow.

[0164] In embodiments, the neuroblastoma is a L1 neuroblastoma. In embodiments, the neuroblastoma is a L2 neuroblastoma. In embodiments, the neuroblastoma is a M neuroblastoma. In embodiments, the neuroblastoma is a MS neuroblastoma.

[0165] In embodiments, the method prevents progression of L1 neuroblastoma to L2, M, or MS neuroblastoma. In embodiments, the method prevents progression of L2 neuroblastoma to M or MS neuroblastoma.

[0166] The INSS takes into account the results of surgery to remove the tumor. It does not help doctors determine the stage before any treatment begins, so it is less effective for children who do not need or cannot have surgery. The stages are:

[0167] Stage 1: The cancer is still in the area where it started. It is on one side of the body (right or left). All visible tumor has been completely removed by surgery (although the edges of the tumor may show some cancer cells when viewed under a microscope after surgery). Lymph nodes near the tumor do not have cancer (although nodes contained within the tumor may contain neuroblastoma cells).

[0168] Stage 2A: The cancer is still in the area where it started and is on one side of the body, but surgery may not be able to remove all visible tumor. Lymph nodes near the tumor are free of cancer (although nodes contained within the tumor may contain neuroblastoma cells).

[0169] Stage 2B: The cancer is on one side of the body and may or may not have been completely removed by surgery. Nearby lymph nodes outside the tumor contain neuroblastoma cells, but the cancer has not spread to lymph nodes on the other side of the body or to other locations.

[0170] Stage 3: The cancer has not spread to distant parts of the body, but one of the following is true:

[0171] The cancer cannot be completely removed with surgery, and it has crossed the midline (defined as the spine) to the other side of the body. It may or may not have spread to nearby lymph nodes.

[0172] The cancer is still in the area where it started and is on one side of the body. It has spread to relatively close lymph nodes that are on the other side of the body.

[0173] The cancer is in the middle of the body and has grown toward the sides (either directly or by spreading to nearby lymph nodes).

[0174] Stage 4: The cancer has spread to distant parts of the body, such as distant lymph nodes, bones, liver, skin, bone marrow, or other organs (but children do not meet the criteria for Stage 4S).

[0175] Stage 4S (also called "special" neuroblastoma): Children are younger than 1 year old. The cancer is located on one side of the body. It may have spread to lymph nodes on the same side of the body but not to the lymph nodes on the other side. The neuroblastoma has spread to the liver, skin, and / or bone marrow. However, no more than 10% of the bone marrow cells are cancerous, and imaging tests (such as MIBG scans) do not show cancer in the bone marrow.

[0176] Recurrence: Although not officially part of the staging system, this term is used to describe cancer that has come back after treatment. Cancer can come back in the area where it originally started or in other parts of the body.

[0177] In embodiments, the neuroblastoma is a stage 1 neuroblastoma. In embodiments, the neuroblastoma is a stage 2A neuroblastoma. In embodiments, the neuroblastoma is a stage 2B neuroblastoma. In embodiments, the neuroblastoma is a stage 3 neuroblastoma. In embodiments, the neuroblastoma is a stage 4 neuroblastoma. In embodiments, the neuroblastoma is a stage 4S neuroblastoma. In embodiments, the neuroblastoma is a recurrent neuroblastoma.

[0178] In embodiments, the method treats stage 1 neuroblastoma. In embodiments, the method treats stage 2A neuroblastoma. In embodiments, the method treats stage 2B neuroblastoma. In embodiments, the method treats stage 3 neuroblastoma. In embodiments, the method treats stage 4 neuroblastoma. In embodiments, the method treats stage 4S neuroblastoma. In embodiments, the method treats recurrent neuroblastoma.

[0179] Prognostic markers are characteristics that help predict whether a child's prognosis will be better or worse than predicted by staging alone. Many of these prognostic markers are used along with a child's staging to assign them a risk group:

[0180] Age: Younger children (under 12-18 months) are more likely to have better outcomes than older children.

[0181] Tumor histology: Tumors that contain more normal-looking cells and tissues tend to have a better prognosis and are said to have favorable histology. Tumors whose cells and tissues appear more abnormal under a microscope tend to have a worse prognosis and are said to have unfavorable histology.

[0182] DNA ploidy: Neuroblastoma cells with approximately the same amount of DNA as normal cells (DNA index of 1) are classified as diploid. Cells with increased DNA amount (DNA index above 1) are called hyperdiploid. Neuroblastoma cells with more DNA are associated with a better prognosis, especially in children under 2 years old. DNA ploidy is less useful for understanding prognosis in older children.

[0183] MYCN gene amplification: MYCN is a gene that normally helps regulate cell growth. Changes in the MYCN gene can cause it to become an oncogene. Neuroblastomas with amplified MYCN oncogenes tend to grow quickly and may be more difficult to treat.

[0184] Chromosomal changes: Tumor cells missing parts of chromosomes 1 or 11 (called 1p deletion or 11q deletion) predict a less favorable prognosis. Having an extra part of chromosome 17 (17q gain) is also associated with a worse prognosis.

[0185] Neurotrophin (nerve growth factor) receptors: These are substances on the surface of normal nerve cells and some neuroblastoma cells. They normally allow cells to recognize neurotrophins, which are hormone-like chemicals that help nerve cells mature. Neuroblastomas that have more of certain neurotrophin receptors, particularly the nerve growth factor receptor TrkA, may have a better prognosis.

[0186] Serum levels of certain substances can also be used to help predict prognosis. Ferritin is a chemical that is an important part of the body's normal iron metabolism, and patients with high ferritin levels tend to have a worse prognosis. Elevated levels of lactate dehydrogenase (LDH) in the blood are also associated with a worse outlook for children with neuroblastoma.

[0187] Co-administration of oncolytic adenoviruses and topoisomerase inhibitors

[0188] In one aspect, the present disclosure provides a method for improving and / or increasing and / or enhancing anti-tumor efficacy in a patient in need thereof, comprising administering to the patient (i) an oncolytic adenovirus comprising a replication machinery specific for tumor cells, and optionally, a polynucleotide sequence encoding a hyaluronidase inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug thereof, e.g., topotecan, SN-38, or irinotecan, and (b) observing improved anti-tumor efficacy compared to treatment with a topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0189] In one aspect, the present disclosure provides a method for improving and / or increasing and / or enhancing anti-tumor efficacy in a patient in need thereof, the method comprising administering to the patient (i) an oncolytic adenovirus comprising a replication machinery specific for tumor cells and a polynucleotide sequence encoding a hyaluronidase inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug thereof, e.g., topotecan, SN-38, or irinotecan, and (b) observing improved anti-tumor efficacy compared to treatment with a topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0190] In an embodiment, an oncolytic adenovirus of the present disclosure, a topoisomerase I inhibitor, or a prodrug thereof (e.g., topotecan, SN-38, or irinotecan) is co-administered. In an embodiment, co-administration can occur simultaneously or sequentially.

[0191] In embodiments, oncolytic adenovirus and topoisomerase I inhibitor or its prodrug (for example, topotecan, SN-38 or irinotecan) are applied to subject simultaneously.As used herein, term " simultaneously " means oncolytic adenovirus and topoisomerase I inhibitor or its prodrug (for example, topotecan, SN-38 or irinotecan) with 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 time interval administration.Oncolytic adenovirus and topoisomerase I inhibitor or its prodrug (for example, topotecan, SN-38 or irinotecan) administration can be by simultaneously administering single preparation (for example, comprising oncolytic adenovirus and topoisomerase I inhibitor or its prodrug (for example, topotecan, SN-38 or irinotecan) preparation) or separate preparation (for example, comprising oncolytic adenovirus first preparation and comprising topoisomerase I inhibitor or its prodrug (for example, topotecan, SN-38 or irinotecan) second preparation) carry out.

[0192] In embodiments, the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38, or irinotecan) are administered to the subject simultaneously, but the release of the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38, or irinotecan) from their respective dosage forms (or a single unit dosage form if co-formulated) can occur sequentially.

[0193] If, for example, the administration time of an oncolytic adenovirus and a topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38 or irinotecan) is such that the pharmacological activity of the oncolytic adenovirus and topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38 or irinotecan) overlaps in time, then co-administration does not require them to be administered simultaneously. For example, an oncolytic adenovirus and a topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38 or irinotecan) can be administered sequentially or over the same period. As used herein, the terms "sequentially" and "over the same period" mean that an oncolytic adenovirus and a topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38 or irinotecan) are administered at a time interval of more than about 60 minutes. For example, the time interval between the sequential administration of an oncolytic adenovirus and a topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38 or irinotecan) may be greater than about 60 minutes, greater than about 2 hours, greater than about 5 hours, greater than about 10 hours, greater than about 1 day, greater than about 2 days, greater than about 3 days or greater than about 1 week. The administration time will depend on the metabolic rate, excretion rate and / or pharmacodynamic activity of the oncolytic adenovirus and topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38 or irinotecan) administered. An oncolytic adenovirus or a topoisomerase I inhibitor or its prodrug, e.g., topotecan or SN-38, may be administered first.

[0194] In embodiments, the oncolytic adenovirus is administered first and the topoisomerase I inhibitor or a prodrug thereof, e.g., topotecan, SN-38, or irinotecan, 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.

[0195] In embodiments, co-administration also does not require that the oncolytic adenovirus and the topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38, or irinotecan) be administered to the subject by the same route of administration. Instead, in embodiments, each therapeutic agent can be administered by any appropriate route, e.g., intravenous, intravitreal, intrathecal, intracerebroventricular, intraocular, intratumoral, intraperitoneal, oral, non-vitreal, non-intrathecal, non-cerebroventricular, non-intraocular, non-intratumoral, non-intraperitoneal, or non-oral.

[0196] In embodiments, the administration of the present compositions and preparations comprising oncolytic adenovirus and / or topoisomerase I inhibitors or their prodrugs (e.g., topotecan, SN-38 or irinotecan) can 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 conventional additional therapies for treating retinoblastoma, Ewing's sarcoma or neuroblastoma. In embodiments, additional agents are one or more of the following: vincristine, carboplatin, etoposide, melphalan, doxorubicin, cyclophosphamide, ifosfamide, cisplatin (cisplatin), paclitaxel, docetaxel (docetaxel), oxaliplatin (oxaliplatin) and temozolamide (temozolamide). In embodiments, the co-administration of additional agents and the present compositions / preparations can be simultaneous or sequential. In embodiments, additional agents may be included in the present compositions and preparations comprising oncolytic adenovirus and / or topoisomerase I inhibitors or their prodrugs (e.g., topotecan, SN-38 or irinotecan). In embodiments, additional agents can be administered as a composition separated from the present composition and preparation, and the present composition and preparation include oncolytic adenovirus and / or topoisomerase I inhibitor or its prodrug, for example, topotecan, SN-38 or irinotecan. In embodiments, additional agents are administered simultaneously with the present composition and preparation comprising oncolytic adenovirus and / or topoisomerase I inhibitor or its prodrug (for example, topotecan, SN-38 or irinotecan). In embodiments, additional agents are administered before the present composition and preparation comprising oncolytic adenovirus and / or topoisomerase I inhibitor or its prodrug (for example, topotecan, SN-38 or irinotecan). In embodiments, additional agents are administered after the present composition and preparation comprising oncolytic adenovirus and / or topoisomerase I inhibitor or its prodrug (for example, topotecan, SN-38 or irinotecan). In embodiments, the present compositions and formulations comprising an oncolytic adenovirus and / or a topoisomerase I inhibitor or a prodrug thereof (e.g., topotecan, SN-38, or irinotecan) can be used alone in a treatment regimen for treating retinoblastoma, Ewing's sarcoma, or neuroblastoma, i.e., without the use of other anti-tumor agents and / or other conventional therapies.

[0197] In embodiments, the present compositions comprising oncolytic adenovirus and / or topoisomerase I inhibitors or their prodrugs (e.g., topotecan, SN-38 or irinotecan) and the administration of the preparation can be combined with additional agents (such as one or more local and / or systemic corticosteroids). In embodiments, one or more local and / or systemic corticosteroids are selected from hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, aldosterone, budesonide, fluticasone, flunisolide, ciclesonide, mometasone, beclomethasone, triamcinolone and triamcinolone. In embodiments, one or more local and / or systemic corticosteroids are selected from methylprednisolone, dexamethasone, betamethasone and triamcinolone.

[0198] In addition, the present composition / preparation may include additional agents (e.g., via co-formulation). For example, additional agents and oncolytic adenovirus and / or topoisomerase I inhibitors or their prodrugs (e.g., topotecan, SN-38, or irinotecan) may be combined into a single formulation. Alternatively, additional agents and oncolytic adenovirus and / or topoisomerase I inhibitors or their prodrugs, e.g., topotecan, SN-38, or irinotecan, may be formulated separately.

[0199] Oncolytic adenovirus

[0200] The present disclosure relates in part to pharmaceutical compositions, formulations, and uses of one or more oncolytic adenoviruses.

[0201] As used in this document, "oncolytic adenovirus" and its plural form refer to an adenovirus that is capable of self-replication or replication-competent in tumor cells. Oncolytic adenoviruses are distinguished from non-replicating adenoviruses because the latter cannot self-replicate in target cells.

[0202] Adenovirus

[0203] In embodiments, the oncolytic adenovirus used in the present disclosure is an oncolytic adenovirus with a replication mechanism and a capsid that allows infection and replication in human cancer cells. In embodiments, the oncolytic adenovirus is generated by an adenovirus that infects people. Examples of adenoviruses that infect people include, but are not limited to, human adenovirus serotypes 1 to 51, such as serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 adenovirus or a combination thereof, such as a hybrid recombinant of two or more human adenoviruses of different serotypes.

[0204] The 51 identified human adenovirus serotypes are divided into six groups from A to F. Human adenovirus serotype 5 (Ad5) belonging to group C is a virus formed by an icosahedral protein capsid containing 36 kilobases of linear deoxyribonucleic acid (DNA). In adults, Ad5 infection is usually asymptomatic, and causes the common cold and conjunctivitis in children. Ad5 usually infects epithelial cells, cells of the bronchial epithelium during natural infection. It enters the cell by means of a fiber (a viral protein extending from the twelve vertices of the capsid like an antenna) and a cell protein (called Coxsackie-adenovirus receptor (CAR)) that participates in cell-to-cell adhesion. When viral DNA arrives inside the nucleus, it begins to systematically transcribe the early genes (E1 to E4) of the virus. The first viral gene to be expressed corresponds to early region gene 1A (E1A). E1A binds to the cell protein of retinoblastoma to release E2F, and therefore activates the transcription of other viral genes (such as E2, E3, E4 and cell genes that activate the cell cycle). For its part, E1B binds to the p53 protein to activate the cell cycle and prevent apoptosis in infected cells. E2 encodes viral replication proteins; E3 encodes proteins that suppress antiviral immune responses; and E4 encodes proteins that transport viral RNA. Expression of early genes leads to the replication of viral DNA, and once this is replicated, the major late promoter is activated, leading to the expression of messenger RNA (RNA) transcripts, which, through cleavage and differential splicing, generate all the RNA encoding the structural proteins that form the capsid.

[0205] In embodiments, the oncolytic adenovirus used in the present disclosure is generated from human adenovirus serotype 5.

[0206] Hyaluronidase

[0207] In an embodiment, the oncolytic adenovirus used in the present disclosure comprises a sequence encoding a hyaluronidase inserted into its genome. In an embodiment, the oncolytic adenovirus used in the present disclosure does not comprise a sequence encoding a hyaluronidase inserted into its genome.

[0208] Hyaluronidase is a class of enzymes responsible for degrading hyaluronic acid. In humans, six genes encoding hyaluronidases have been identified to date, each with different properties and locations. Isoforms Hyal1 and Hyal2 are present in most tissues, with Hyal1 being the predominant form in human plasma. Hyal3 is located in the bone marrow and testes, but its function remains unclear. Hyaluronidase PH20 is highly expressed in the testes and is involved in the fertilization of the oocyte by sperm. Hyaluronidase PH20 is anchored to the cytoplasmic membrane and inner acrosomal membrane of sperm, enabling sperm to penetrate the extracellular matrix of cumulus cells (which is rich in hyaluronic acid) and reach the zona pellucida of the oocyte. During the acrosome reaction, some hyaluronidases anchored to the sperm membrane are enzymatically processed to produce a soluble form of the protein, which is released from the acrosomal membrane. Membrane protein PH20 is the only enzyme in the mammalian hyaluronidase family that is active at neutral pH.

[0209] Without wishing to be bound by theory, it is believed that the hyaluronidase expression of oncolytic adenoviruses can help degrade the dense physical and immunosuppressive matrix barrier surrounding solid tumors, thereby ensuring that the oncolytic adenovirus and co-administered therapies better penetrate the tumor. Without wishing to be bound by theory, this ensures that the adenovirus can reach and infect a large number of tumor cells. Degrading the tumor matrix can also expose tumor neoantigens, thereby stimulating an anti-tumor immune response by the patient's immune system.

[0210] In embodiments, the hyaluronidase is a mammalian testicular hyaluronidase, optionally a human hyaluronidase (e.g., GenBank Gene ID: 6677), also known as SPAM1 or sperm adhesion molecule 1 or PH20. In embodiments, the sequence of the hyaluronidase has a sequence corresponding to an enzyme from which the membrane-binding carboxyl-terminal domain is deleted, rendering the enzyme soluble. When the carboxyl-terminal domain is deleted, the resulting enzyme is secreted into the extracellular environment.

[0211] In an embodiment, the hyaluronidase encoding sequence inserted into the oncolytic adenoviral genome is SEQ ID NO: 1, wherein nucleotides 1471 to 1527 corresponding to the carboxyl terminal domain are deleted.

[0212] In addition, in embodiments, the hyaluronidase is a hyaluronidase variant. Compared to the parent wild-type sequence, the hyaluronidase variant has at least one or more amino acid modifications, typically amino acid substitutions. In embodiments, the hyaluronidase of the present disclosure comprises a hyaluronidase that has at least about 60% (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77% or about 78% of any sequence disclosed herein. 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%). In addition, in embodiments, the hyaluronidase variants retain most or all of their biochemical activity as measured by any suitable method known in the art.

[0213] In embodiments, the hyaluronidase of the present disclosure comprises a hyaluronidase that has at least about 60% (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79% or about 80% of the amino acid sequence encoded by SEQ ID NO: 1. or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%).

[0214] In embodiments, the hyaluronidase of the present disclosure comprises a hyaluronidase that has 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%) similarity to the amino acids encoded by SEQ ID NO: 1 (wherein nucleotides 1471 to 1527 corresponding to the carboxyl terminal domain are deleted). , 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.

[0215] In embodiments, the hyaluronidase of the present disclosure comprises a hyaluronidase that has at least about 60% (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79% or about 80% of the amino acid sequence encoded by SEQ ID NO: 9. or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%).

[0216] In embodiments, the hyaluronidase of the present disclosure comprises a hyaluronidase that has at least about 60% (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79% or about 80% of the hyaluronidase sequence of SEQ ID NO: 8. or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) amino sequences with sequence identity.

[0217] In embodiments, the hyaluronidase of the present disclosure comprises a hyaluronidase that has at least about 60% (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79% or about 80% of the hyaluronidase sequence of SEQ ID NO: 10. or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) amino sequences with sequence identity.

[0218] In embodiments, the oncolytic adenovirus used in the present disclosure comprises one or more regulatory elements, modifications or variants that provide for tumor-specific enzyme expression or viral replication.

[0219] In embodiments, the expression of hyaluronidase is controlled by a promoter that operates in tumor cells. In embodiments, the expression of hyaluronidase is controlled by a promoter that operates in retinoblastoma, Ewing's sarcoma, or neuroblastoma cells to be treated. Optionally, in embodiments, the expression of the enzyme is controlled by a promoter that operates in animal cells. Optionally, in embodiments, the promoter is selected from a cytomegalovirus promoter, adenovirus major late promoter, SV40 promoter, herpes simplex virus thymidine kinase promoter, RSV promoter, EF1-α promoter, β-actin promoter, human IL-2 promoter, human IL-4 promoter, IFN promoter, E2F promoter, human GM-CSF promoter, or a combination thereof.

[0220] In an embodiment, the promoter that regulates the expression of the enzyme may be naturally present in the adenovirus, as is the case with the adenovirus major late promoter. In an embodiment, the promoter may also be inserted together with the sequence encoding the enzyme. In an embodiment, the promoter is an adenovirus major late promoter, and it is already located in the oncolytic adenovirus genome. In an embodiment, it is not necessary to introduce the promoter together with the hyaluronidase sequence, but rather to introduce the latter into the oncolytic adenovirus genome so that it is still under the control of the promoter.

[0221] In embodiments, the oncolytic adenovirus used in the present disclosure includes additional sequences that allow the protein translation of the sequence encoding hyaluronidase to be promoted or optimized. In embodiments, the sequence is located inside or outside the hyaluronidase gene. For example, in embodiments, additional sequences are selected from cutting and splicing sequences, IRES (internal ribosome entry site) sequences, picornavirus sequence 2A, or combinations thereof that allow RNA to be processed.

[0222] Replication mechanism specific to tumor cells

[0223] In embodiments, the tumor cell-specific replication mechanism included in the oncolytic virus used in the present disclosure is a mechanism that causes an oncolytic adenovirus to replicate in a specific form in tumor cells rather than in healthy, non-tumor or normal cells. In embodiments, if the mechanism provides the ability for an oncolytic adenovirus to replicate only in or mainly in tumor cells (e.g., retinoblastoma, Ewing's sarcoma or neuroblastoma cells), the mechanism may take different forms. In other words, in embodiments, adenoviruses have selective replication, wherein their replication mechanism requires the virus to enter cancer or tumor cells in order to make the virus self-replicate. In embodiments, the oncolytic adenovirus used may have a modification in its genomic sequence that gives it selective replication in tumor cells.

[0224] In embodiments, this is achieved by incorporating a tissue-specific promoter or a tumor-specific promoter, wherein the promoter controls the expression of one or more genes in the E1a, E1b, E2, and E4 groups. In embodiments, the promoter is selected from the group consisting of the E2F promoter, the telomerase hTERT promoter, the tyrosinase promoter, the prostate-specific antigen (PSA) promoter, the alpha-fetoprotein promoter, the COX-2 promoter, and artificial promoters formed by binding sites for various transcription factors, such as the binding sites for hypoxia-inducible factor (HIF-1), ETS transcription factors, tumor cytotoxicity factor (TCF), E2F transcription factors, or Sp1 transcription factors. In embodiments, the promoter controls the expression of E1a.

[0225] In embodiments, treatment is the treatment of tumors with abnormal Rb-E2F pathways. In healthy, non-tumor or normal cells, the E1A protein expressed by 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 abnormal Rb-E2F mechanisms, free E2F allows cells to continue dividing (and viral replication) without being initiated by viral E1A. Therefore, genetic modifications that inhibit the expression of normal viral E1A prevent the virus from replicating in healthy, non-tumor or normal cells, while allowing the virus to replicate in tumor cells. Almost all solid tumors have abnormal Rb-E2F pathways, including but not limited to retinoblastoma, Ewing's sarcoma and neuroblastoma. In embodiments, the oncolytic adenovirus used in the present disclosure is characterized by the deletion of the Rb binding domain. In an embodiment, the oncolytic adenovirus used in the present disclosure is characterized by a Δ24 deletion, which affects the interaction of E1a with the retinoblastoma protein, and the insertion of four E2F-1 binding sites and one Sp1 binding site into the endogenous E1a promoter to control the expression of E1a. The DNA sequence corresponds to SEQ ID NO: 2.

[0226] In addition, in embodiments, it is envisioned that the oncolytic adenovirus used in the present disclosure comprises one or more modifications in the capsid, thereby allowing for improved biodistribution of the oncolytic adenovirus and reduced clearance of the oncolytic adenovirus from the body. In embodiments, the capsid of the oncolytic adenovirus used in the present disclosure is modified to reduce its sequestration and destruction in the liver so that the heparan sulfate binding domain KKTK present in the adenoviral fiber has been replaced, for example, by the domain RGDK. The modifications relate to positions 91 to 94 of the adenoviral fiber, with the standard sequence of adenovirus serotype 5 fiber as a reference. Sequence SEQ ID NO: 4 shows the complete sequence of adenovirus type 5 fiber protein, whose heparan sulfate binding domain has a modified version (modified RGDK).

[0227] Thus, in embodiments, the oncolytic adenovirus used in the present disclosure is generated from human adenovirus serotype 5 and comprises:

[0228] a sequence encoding a hyaluronidase inserted into its genome, optionally the human testicular hyaluronidase sequence (PH20, SEQ ID NO: 1), in which the sequence corresponding to the membrane-binding carboxyl-terminal domain has been deleted to render the enzyme soluble, optionally encoding a hyaluronidase having the amino acid sequence of SEQ ID NO: 10;

[0229] The oncolytic adenovirus replicates in tumor cells with an aberrant Rb-E2F pathway but not in healthy, non-tumor or normal cells; optionally, the oncolytic adenovirus contains a deletion of the Rb binding domain or a Δ24 deletion, which affects the interaction of E1a with the Rb protein, and four binding sites for E2F-1 and one binding site for Sp1 are inserted into the endogenous E1a promoter to control the expression of E1a; and / or

[0230] - Modification of the adenoviral capsid to improve the biodistribution of the oncolytic adenovirus and to reduce the clearance of the oncolytic adenovirus from the body; optionally, the heparan sulfate binding domain KKTK present in the adenoviral fiber has been replaced, for example, by the domain RGDK.

[0231] In embodiments, the oncolytic adenovirus of the present disclosure comprises an oncolytic adenovirus 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% of the sequence of SEQ ID NO: 3. In some embodiments, the oncolytic adenovirus has a nucleotide sequence with at least one nucleotide sequence identity to the oncolytic adenovirus of SEQ ID NO: 3. In some embodiments, the oncolytic adenovirus has a nucleotide sequence with at least one nucleotide sequence identity to the oncolytic adenovirus of SEQ ID NO: 3.

[0232] In some embodiments, the oncolytic adenoviruses of the present disclosure comprise an oncolytic adenovirus 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%) sequence identity to VCN-01. 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%, of which the sequence identity is nucleotide sequence. In embodiments, the oncolytic adenovirus has the nucleotide sequence of VCN-01.

[0233] Methods for preparing oncolytic adenoviruses of the present disclosure

[0234] In an embodiment, in order to construct the oncolytic adenovirus to be used in the present disclosure, any method of constructing a genetically modified adenovirus known in the field of gene therapy and virotherapy using adenovirus is used. The most commonly used method is based on first constructing the desired genetic modification in a plasmid containing the adenovirus region to be modified, and then performing homologous recombination with a plasmid containing the rest of the viral genome in bacteria. It will be understood by those skilled in the art that due to the degeneracy of the genetic code, the protein sequences depicted herein can be encoded by any number of possible nucleic acid sequences.

[0235] In embodiments, nucleic acids encoding components of the present disclosure can be incorporated into oncolytic adenoviruses as known in the art and, depending on the host cell, used to produce hyaluronidase of the present disclosure. Typically, nucleic acids are operably linked to any number of regulatory elements (promoters, origins of replication, selectable markers, ribosome binding sites, inducers, etc.).

[0236] In embodiments, the oncolytic adenovirus used in the present disclosure is propagated and amplified in cell lines commonly used in the field of gene therapy and viral therapy, such as cell lines HEK-293 (reference number: ATCC CRL-1573) and A549 (reference number: ATCC CCL185). In embodiments, the adenovirus propagation method is by infecting a cell line that allows adenovirus replication. The lung adenocarcinoma cell line A549 is an example of a cell line with such characteristics. In embodiments, for example, propagation is performed as follows: A549 cells are grown on plastic cell culture plates and infected with 100 virus particles / cell. After two days, a cytopathic effect reflecting the production of the virus as cell aggregation is observed. In embodiments, cells are collected and stored in tubes. In embodiments, after centrifugation at about 1000g 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 1000g for about 5 minutes, and the supernatant containing the virus is loaded onto a cesium chloride gradient and centrifuged at about 35,000g for about 1 hour. In embodiments, the viral band obtained from the gradient is reloaded onto another cesium chloride gradient and centrifuged at about 35,000 g for about 16 hours. In embodiments, the viral band is collected and dialyzed against PBS-10% glycerol. In embodiments, the viral dialysate is aliquoted and stored at about -80°C. The number of plaque forming units and particles can be quantified according to standard protocols known in the art. Phosphate buffered saline (PBS) containing 5% glycerol is a standard formulation for storing adenovirus and is used in embodiments. However, new formulations that improve viral stability have been described and are used in embodiments.

[0237] Topoisomerase I inhibitors

[0238] Topoisomerase inhibitors can inhibit cell proliferation by, for example, preventing DNA replication, stimulating DNA damage, and inducing cell cycle arrest.

[0239] Topotecan is a semisynthetic derivative of the cytotoxic alkaloid camptothecin. SN-38 is the active topoisomerase I-inhibiting metabolite prodrug of irinotecan (CPT-11). Typically, topoisomerase I inhibitors cause cell cycle arrest in S phase by stabilizing the complex between topoisomerase I and DNA, thereby inhibiting the religation of topoisomerase I-mediated single-strand DNA breaks and generating potentially lethal double-strand DNA breaks.

[0240] In embodiments, a topoisomerase I inhibitor or a prodrug thereof (e.g., topotecan, SN-38, or irinotecan) is administered in a variety of ways to treat retinoblastoma, Ewing's sarcoma, or neuroblastoma, including systemic, intra-arterial, and intravitreal administration.

[0241] In embodiments, for systemic administration, a topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38 or irinotecan) is administered intravenously or orally. Topoisomerase I inhibitors or their prodrugs (e.g., topotecan, SN-38 or irinotecan) then enter the bloodstream and are distributed throughout the body. Irinotecan is converted into active topoisomerase I inhibitor SN-38 by liver and tumor carboxylesterases. In embodiments, systemic chemotherapy is used to treat children with bilateral retinoblastoma, or for treating children who cannot have unilateral retinoblastoma treated with centralized intervention (such as cryotherapy or transpupillary thermotherapy) or who cannot use OAC. In embodiments, systemic chemotherapy is also used to treat primary and metastatic Ewing's sarcoma or neuroblastoma. For OAC, chemotherapy drugs are injected directly into the ophthalmic artery, the main artery that supplies blood to the eye. In many cases, this method of administration allows doctors to save eyes that would otherwise need to be removed. The chemotherapy doses used in this method are much lower. For intravitreal chemotherapy, chemotherapy drugs are injected directly into the vitreous humor. This approach is sometimes used (along with systemic or intra-arterial chemotherapy) to treat tumors that have spread widely within the eye and have not helped with other treatments.

[0242] In an embodiment, the topoisomerase I inhibitor or a prodrug thereof (eg, topotecan, SN-38, or irinotecan) is administered intravenously, intraarterially, intravitreally, intrathecally, intracerebroventricularly, or orally.

[0243] In an embodiment, the methods of the present disclosure improve and / or increase and / or enhance anti-tumor efficacy compared to treatment with a topoisomerase I inhibitor or a prodrug thereof without an oncolytic adenovirus. In an embodiment, the methods make the patient eligible for treatment with a combination therapy of more than one cancer therapy.

[0244] In embodiments, without wishing to be bound by theory, the administration of an oncolytic adenovirus reduces the apoptotic effect of topotecan compared to monotherapy. In embodiments, without wishing to be bound by theory, the administration of topotecan after the administration of an oncolytic adenovirus results in S-phase cell cycle arrest. In embodiments, without wishing to be bound by theory, the S-phase cell cycle arrest results in an increase in the infectivity of the oncolytic adenovirus compared to the treatment with an oncolytic adenovirus without a topoisomerase I inhibitor or its prodrug. In embodiments, without wishing to be bound by theory, the administration of topotecan after the administration of an oncolytic adenovirus results in an increase in E2F-1, p21, and / or cyclin E1 expression compared to the treatment with an oncolytic adenovirus without a topoisomerase I inhibitor or its prodrug. In embodiments, without wishing to be bound by theory, the increased E2F-1 expression results in an increase in the oncolytic activity of the oncolytic adenovirus compared to the treatment with an oncolytic adenovirus without a topoisomerase I inhibitor or its prodrug. In embodiments, without wishing to be bound by theory, the increase in the infectivity and oncolytic activity of the oncolytic adenovirus occurs when the replication of the oncolytic adenovirus is substantially not increased.

[0245] In embodiments, without wishing to be bound by theory, sequential treatment wherein an oncolytic adenovirus is first administered followed by systemic administration of topotecan enhances or increases the cell infection and / or anti-cancer efficacy of the oncolytic adenovirus, i.e., addresses one of the major limitations of oncolytic adenoviruses, namely, limited penetration and distribution in tumors.

[0246] preparation

[0247] The present disclosure provides the oncolytic adenovirus and / or topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38 or irinotecan) composition in various formulations. Any oncolytic adenovirus and / or topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38 or irinotecan) described herein can be in the form of a solution, emulsion, suspension, delayed release formulation, sustained release formulation, controlled release formulation, or any other form suitable for parenteral use.

[0248] It should be understood that the compositions used in the present disclosure are used in a pharmaceutically acceptable form. This means that any oncolytic adenovirus and / or topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38 or irinotecan) described herein can be administered to a subject as a component of a composition comprising a pharmaceutically acceptable carrier or vehicle. Such compositions may optionally include a suitable amount of a pharmaceutically acceptable excipient to provide a form for appropriate administration.

[0249] Pharmaceutical excipients can be liquids, such as water and oil, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil etc. Pharmaceutical excipients can be, for example, saline, gum arabic, gelatin, starch paste, talc, keratin, silica gel, urea etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can be used. In embodiments, when applied to a subject, a pharmaceutically acceptable excipient is sterile. When any agent described herein is administered intravenously, water is a useful excipient. Saline solutions and dextrose aqueous solutions and glycerol solutions can also be used as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, cellulose, hypromellose, lactose, sucrose, trehalose, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, polyvidone, cross-linked polyvinylpyrrolidone, water, ethanol etc. If desired, any of the agents described herein may also contain 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 ed., 19th ed. 1995), which is incorporated herein by reference.

[0250] Administration and dosage

[0251] It will be understood that the actual dosage of the oncolytic adenovirus and / or topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38 or irinotecan) to be administered according to the present disclosure will vary according to different parameters, such as the volume of the vitreous cavity, the size and stage of the retinoblastoma, Ewing's sarcoma or neuroblastoma to be treated, the age and weight of the patient to be treated, the specific dosage form and the mode of administration. It will be understood that the dosage of the oncolytic adenovirus and the co-administered topoisomerase I inhibitor or its prodrug (e.g., topotecan, SN-38 or irinotecan) must be sufficient to ensure that the combination of the methods produces a positive therapeutic effect on retinoblastoma, Ewing's sarcoma or neuroblastoma. In embodiments, a positive therapeutic effect on retinoblastoma, Ewing's sarcoma or neuroblastoma refers to cessation of tumor growth, or reduction in tumor volume, or prevention of tumor infiltration into surrounding tissues, or prevention of metastasis.

[0252] Those skilled in the art may consider many factors (e.g., body weight, sex, diet, administration time, administration route, excretion rate, subject's condition, drug combination, genetic predisposition and reaction sensitivity) that may alter the effects of oncolytic adenovirus and / or topoisomerase I inhibitors or their prodrugs (e.g., topotecan, SN-38 or irinotecan). Administration may be continuous or in one or more discrete doses up to the maximum tolerated dose. Those skilled in the art may use conventional assessments of dose administration and dose tolerance to determine the optimal administration rate under a given set of conditions.

[0253] The composition of the present invention (for example, a composition comprising an oncolytic adenovirus disclosed herein) is administered by any route ensuring that the necessary amount of oncolytic adenovirus reaches the tumor site. In the case of retinoblastoma, the composition of the present invention is injected by any route ensuring that the necessary amount of oncolytic adenovirus reaches the inside of the eyeball, for example, a composition comprising an oncolytic adenovirus disclosed herein. In the case of Ewing's sarcoma or neuroblastoma, the composition of the present invention is injected by any route ensuring that the necessary amount of oncolytic adenovirus reaches the tumor site, for example, a composition comprising an oncolytic adenovirus disclosed herein. Optional routes of administration include but are not limited to intravenous injection, intraarterial injection, intratumoral injection, intraocular injection, intraperitoneal injection, intrathecal injection, and intravitreal injection. The advantage of intraocular or intravitreal injection in retinoblastoma patients is that the injection is performed in the organ eye, which provides an immune privileged environment, thereby facilitating a reduction in the immune response to the oncolytic adenovirus contained in the composition or an absence of response to the oncolytic adenovirus contained in the composition. This makes it possible to ensure that the effect of the oncolytic adenovirus will be confined to the eye to a large extent, because once the oncolytic adenovirus leaves the eye, it may be neutralized by the immune system. Thus, in embodiments, preferred compositions comprising the oncolytic adenovirus disclosed herein are in a suitable form for intravitreal, intraarterial, intrathecal or intraocular administration or injection.Another possible route of administration for the compositions provided herein is intratumoral injection.

[0254] In embodiments, the oncolytic adenovirus and / or topoisomerase I inhibitor or a prodrug thereof (e.g., topotecan, SN-38, or irinotecan) can be administered, for example, more than once daily (e.g., about twice, about three times, about four times, about five times, about six times, about seven times, about eight times, nine times, or about ten times per day), about once a day, about once every other day, about once every three days, about once a week, about once every two weeks, about once a month, about once every two months, about once every three months, about once every six months, or about once a year.

[0255] In an embodiment, a subject is administered a single intrathecal injection of VCN-01 concurrently with systemic or intrathecal circulation of topotecan. In an embodiment, the subject has neuroblastoma or retinoblastoma, optionally with leptomeningeal metastatic retinoblastoma or CNS metastatic retinoblastoma. In an embodiment, the subject is administered VCN-01 and topotecan or irinotecan systemically. In an embodiment, the subject has extracranial disseminated disease, optionally Ewing's sarcoma. In an embodiment, the subject has an aggressive and / or chemorefractory pediatric solid tumor that expresses E2F-1. In an embodiment, VCN-01 and / or topotecan are co-administered with one or more local and / or systemic corticosteroids. In an embodiment, the one or more topical and / or systemic corticosteroids are selected from hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, aldosterone, budesonide, fluticasone, flunisolide, ciclesonide, mometasone, beclomethasone, triamcinolone, and tixocortolone. In an embodiment, the one or more topical and / or systemic corticosteroids are selected from methylprednisolone, dexamethasone, betamethasone, and triamcinolone.

[0256] definition

[0257] As used herein, "a," "an," or "the" may mean one or more than one.

[0258] Additionally, when used in conjunction with a reference number, the term "about" means the reference number plus or minus up to 10% of the reference number. For example, the language "about 50%" encompasses a range of 45% to 55%.

[0259] When used in connection with medical uses, an "effective amount" is an amount effective to provide a measurable treatment, prevention, or reduction in the incidence of the target condition.

[0260] As used herein, something is "reduced" if, in the presence of an agent or stimulus, the activity and / or effect readout 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%) relative to the absence of such modulation. As will be understood by one of ordinary skill in the art, in embodiments, activity is reduced and some downstream readouts will be reduced but other readouts may increase.

[0261] In contrast, an activity is "increased" if the readout of activity and / or effect is increased by a significant amount in the presence of an agent or stimulus relative to the absence of such agent or stimulus, e.g., 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.

[0262] As mentioned herein, unless otherwise specified, all composition percentages are by weight of the total composition. As used herein, the word "include" and its variants are intended to be non-restrictive, so that the detailed description of the items in the list does not exclude other similar items that may also be applicable to the compositions and methods of this technology. Similarly, the terms "can" and "may" and their variants are intended to be non-restrictive, so that the detailed description of an embodiment that may or may include certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.

[0263] Although the open-ended term "comprising," as a synonym for terms such as including, containing, or having, is used herein to describe and claim the present disclosure, the disclosure or embodiments thereof may alternatively be described using alternative terminology such as "consisting of" or "consisting essentially of.

[0264] As used herein, the words "preferred" and "preferably" refer to embodiments of the present technology that offer 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 mean that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the present technology.

[0265] 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 a non-human primate, such as a monkey, chimpanzee, or baboon. In embodiments, the subject and / or animal is a non-mammal, such as, for example, a zebrafish.

[0266] In embodiments, the methods of the present invention can be used to treat human subjects. In embodiments, the human is a human child. In embodiments, the human is an adult. In embodiments, the human is an elderly person. 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.

[0267] In embodiments, the human is within the age range of about 1 to about 18 months, about 18 to about 36 months, about 1 to about 5 years, about 5 to about 10 years, about 10 to about 15 years, about 15 to about 20 years, about 20 to about 25 years, about 25 to about 30 years, about 30 to about 35 years, about 35 to about 40 years, about 40 to about 45 years, about 45 to about 50 years, about 50 to about 55 years, about 55 to about 60 years, about 60 to about 65 years, about 65 to about 70 years, about 70 to about 75 years, about 75 to about 80 years, about 80 to about 85 years, about 85 to about 90 years, about 90 to about 95 years, or about 95 to about 100 years.

[0268] The amount of each component in the composition required for achieving therapeutic effect as described herein can be determined empirically according to conventional procedures for a specific purpose. Typically, therapeutic agents (e.g., oncolytic adenovirus and / or topoisomerase I inhibitors or their prodrugs, e.g., topotecan, SN-38 or irinotecan, compositions as described herein) are administered for therapeutic purposes, and the therapeutic agent is given in a pharmacologically effective dose. "Pharmacologically effective amount," "pharmacologically effective dose," "therapeutically effective amount" or "effective amount" refers to an amount sufficient to produce a desired physiological effect or to achieve the desired result (particularly the amount of the treatment disorder or disease). As used herein, an effective amount includes an amount sufficient to, for example, delay the development of the symptoms of a disorder or disease, change the course of the symptoms of a disorder or disease (e.g., slowing down the progress of disease symptoms), reduce or eliminate one or more symptoms or manifestations of a disorder or disease and reverse the amount of the symptoms of a disorder or disease. Therapeutic benefit also includes stopping or slowing down the progress of a potential disease or disorder, whether or not improvement is achieved.

[0269] The effective amount, toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures, tissue samples, tissue homogenates or experimental animals, for example, to determine 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 on the dosage form adopted and the route of administration utilized. The dose ratio between toxicity and therapeutic effect is the therapeutic index and can be expressed as the ratio LD50 / ED50. In embodiments, compositions and methods that exhibit a large therapeutic index are preferred. The therapeutically effective dose can be initially assessed by in vitro assays (including, for example, cell culture assays or measurements or methane production in fecal samples). In addition, the dosage can be formulated in an animal model to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture or in an appropriate animal model. The level of the composition in plasma can be measured, for example, by high performance liquid chromatography. The effect of any particular dose can be monitored by suitable bioassays. The dosage can be determined by a physician and, if necessary, adjusted to suit the observed therapeutic effect.

[0270] 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 greater. A therapeutic benefit also includes halting or slowing the progression of the underlying disease or condition, regardless of whether improvement is achieved.

[0271] As used herein, "method of treatment" also applies to the use of a composition for treating a disease or condition described herein and / or in the manufacture of a medicament for treating a disease or condition described herein.

[0272] Example

[0273] Example 1: Treatment of retinoblastoma with oncolytic adenovirus VCN-01 and topotecan

[0274] Two million HSJD-RBT-5, HSJD-RBT-7, HSJD-RBVS-10, and Y79 retinoblastoma cells were infected with 50 MOI of VCN-01 having the nucleotide sequence of SEQ ID NO: 3. After 24 h, cells were treated with topotecan (2 μM), carboplatin (12.5 μM), or melphalan (10 μM). After 48 h, total cell lysates were extracted and immunoblotted for E1a, using GAPDH as a loading control. E1A is the first protein expressed by the adenovirus and drives the expression of the remaining viral proteins. Results Figure 1, and demonstrated that topotecan, but not carboplatin or melphalan, enhanced VCN-01 protein expression in retinoblastoma cells (including 1 cell line and 3 patient-derived models).

[0275] Y79 cells were inoculated into the abdominal cavity of athymic nude mice. 3 ), intratumoral injection of VCN-01 (3x 10E 9 vp / tumor; single dose, day 1). Mice then received daily topotecan (0.6 mg / kg / day) or hydroxyurea (200 mg / kg / day) from day 1 to day 5, or a single dose of carboplatin (40 mg / kg) on ​​day 1. Mice were sacrificed at two different times: 5 days after inoculation with VCN-01 or 15 days after inoculation with VCN-01, and viral genomes were quantified. Results are in Figure 2A-2B In the present study, we demonstrated that systemic administration of topotecan, but not carboplatin, following intratumoral injection of VCN-01 increased the genomic content of VCN-01 in an in vivo retinoblastoma tumor model. Of note, hydroxyurea is a model drug that arrests retinoblastoma cells in S phase. Although it can be hypothesized that the enhanced activity of topotecan against VCN-01 is due to the arrest of retinoblastoma cells in S phase (a property shared with hydroxyurea), the enhancement of VCN-01 replication in vivo appears to be specific to topotecan.

[0276] HSJD-RBT-7 cells were inoculated into the abdominal cavity of athymic nude mice. 3 ), intratumoral injection of VCN-01 (3x 10E 9 vp / tumor; single dose, day 1). Mice then received daily topotecan (0.6 mg / kg / day) or hydroxyurea (200 mg / kg / day) from day 1 to day 5, or a single dose of carboplatin (40 mg / kg) on ​​day 1. Mice were sacrificed 5 days after VCN-01 inoculation, and viral genomes were quantified. Figure 3 Shown in , it is demonstrated that systemic administration of topotecan, but not carboplatin, followed by intratumoral injection of VCN-01, increased VCN-01 genomic content in an in vivo retinoblastoma tumor model.

[0277] Y79 cells were inoculated into the abdominal cavity of athymic nude mice. 3 ), intratumoral injection of VCN-01 (3x 10E 9vp / tumor; single dose, day 1). Mice then received daily topotecan (0.6 mg / kg / day) or hydroxyurea (200 mg / kg / day) from day 1 to day 5, or a single dose of carboplatin (40 mg / kg) on ​​day 1. Mice were sacrificed at two different times: 5 days after inoculation with VCN-01 or 15 days after inoculation with VCN-01. Figures 4A-4C Figure 3 demonstrates that systemic administration of topotecan, but not carboplatin, after intratumoral injection of VCN-01 enhances VCN-01 activity in an in vivo retinoblastoma tumor model. Immunoblots show E1A and loading control GAPDH ( Figure 4A Immunostaining shows representative images of E1A staining in subcutaneous tumors obtained 5 days after VCN-01 injection and topotecan or carboplatin treatment ( Figure 4B ). Figure 4C The graph in shows the E1A-positive cell counts in 7 different fields of view for each sample, where each dot represents one field of view.

[0278] Effect of topotecan on the antitumor activity of VCN-01 in the Y79 subcutaneous model Figure 5 The doses were as follows: VCN-01: intratumoral, 3 x 10 9 vp / tumor, day 1 (single dose); and topotecan: systemically, 0.6 mg / kg / day, days 1, 2, 3, 4, and 5.

[0279] The effect of topotecan on the antitumor activity of VCN-01 in Y79 and HSJD-RBT-7 orthotopic (intraocular retinoblastoma) models was Figure 6A-Figure 6B The doses were as follows: VCN-01: intratumoral, 3 x 10 9 vp / tumor, day 1 (single dose); and topotecan: systemically, 0.6 mg / kg / day, days 1, 2, 3, 4, and 5. The orthotopic xenograft model recapitulates intraocular tumor growth and recapitulates molecular markers of human disease and dissemination.

[0280] Example 2: In vitro evaluation of VCN-01 in combination with a topoisomerase I inhibitor

[0281] Two million Y79 retinoblastoma cells were infected with 50 MOI of VCN-01. 24 hours later, cells were treated with topotecan (2 μM), carboplatin (12.5 μM), melphalan (10 μM), hydroxyurea (100 μM), etoposide (2 μM), irinotecan (10 μM), or SN-38 (1 μM). Total cell lysates were extracted 48 hours later and immunoblotted for E1a, using GAPDH as a loading control. The results were shown in Figure 5. Figure 7The results show that SN-38, another topoisomerase 1 inhibitor from the camptothecin family, increases VCN-01 protein expression in retinoblastoma cells (Y79 model) similarly to topotecan. The SN-38 prodrug, irinotecan, is ineffective in vitro due to a lack of conversion to SN-38 in the absence of carboxylesterase. Interestingly, etoposide, another topoisomerase inhibitor, does not induce the same effect. Etoposide is a topoisomerase 2 inhibitor.

[0282] Example 3: Treatment of brain retinoblastoma with systemic topotecan and intrathecal VCN-01

[0283] One million retinoblastoma HSJD-RBT-7 cells in 25 μL of Matrigel were inoculated intrathecally exclusively into the fourth ventricle of athymic nude mice on day 0. Combination treatment was initiated on day 6 after tumor cell inoculation, with a single dose of 3 x 10 retinoblastoma cells in 25 μL administered by stereotactic intrathecal injection (in the fourth ventricle) on the morning of day 6. 9 vp / tumor, and topotecan at a concentration of 0.6 mg / kg / day systemically (intraperitoneally) on the afternoons of days 6, 7, 8, 9, and 10. This combination, consisting of carboplatin 40 mg / kg systemically (intraperitoneally) on day 6 and etoposide 6 mg / kg intraperitoneally on days 7 and 8, was compared to both individual treatments and standard of care treatment. Figure 8 and demonstrated that the combination therapy was more effective than monotherapy and standard of care treatment. Additional results are presented in Figure 9 , and demonstrated that 26 days after the start of treatment, VCN-01 + topotecan mice experienced less weight loss than mice treated with VCN-01 alone. At that time, all control mice had died of disease. Additional results are in Figure 10 , and demonstrates that 26 days after the start of treatment, the tumor burden in the intraventricular thecal space (measured as the amount of human cells stained with anti-human nuclear antibody; darkly stained areas within the brain) was less in VCN-01 + topotecan mice than in mice treated with each of the other treatments.

[0284] Example 4: Treatment of Ewing's sarcoma with topotecan and VCN-01

[0285] Patient-derived xenografts (PDX) of Ewing's sarcoma (a pediatric solid tumor) were implanted subcutaneously in athymic nude mice. The tumor model was HSJD-ES-033. After implantation (tumor volume ranged from 100-500 mm 3), in which topotecan was administered systemically (intraperitoneally) at a concentration of 0.6 mg / kg / day on days 1, 2, 3, 4, and 5, and a single dose of 3 x 10 9 The combination treatment of VCN-01 with vp / tumor was compared with either treatment alone. Figure 11 Shown in , this graph shows the mean tumor size from 2-4 tumors per group and demonstrates that the combination treatment was more effective than either treatment alone.

[0286] Example 5: Treatment of neuroblastoma with a combination of topotecan or irinotecan and VCN-01

[0287] Neuroblastoma (a pediatric solid tumor) PDX was subcutaneously implanted into athymic nude mice. The tumor model was HSJD-NB-005. After implantation (tumor volume ranged from 100-500 mm 3 ) and treatment was started. The activity of topotecan in combination with irinotecan was evaluated compared with that of VCN-01. The following groups of mice were included in the study, with n = 6 tumors per group:

[0288] Group 1: No treatment

[0289] Group 2: Topotecan 0.6 mg / kg intraperitoneally on days 1-5

[0290] Group 3: irinotecan 10 mg / kg intraperitoneally on days 1-5

[0291] Group 4: VCN-01 administered intratumorally at 3E9 vp / tumor, day 1

[0292] Group 5: VCN-01 at 3E9 vp / tumor intratumorally on day 1 (morning) + topotecan at 0.6 mg / kg intraperitoneally on days 1-5

[0293] Group 6: VCN-01 at 3E9 vp / tumor intratumorally on day 1 (morning) + irinotecan at 10 mg / kg intraperitoneally on days 1-5

[0294] The results are Figure 12 It is shown in and demonstrated that the combination treatment of topotecan and VCN-01 is more effective than either monotherapy. The combination treatment of irinotecan and VCN-01 is significantly more effective than VCN-01 and slightly more effective than irinotecan.

[0295] Example 6: Treatment of childhood cancer with a combination of topotecan and VCN-01

[0296] This example describes, among other things, studies of the interaction of VCN-01 with the chemotherapeutic agents topotecan, carboplatin, and melphalan, all of which are standard of care for retinoblastoma and other childhood cancers. Topotecan is a topoisomerase 1 inhibitor of the camptothecin family. These drugs produce double-strand breaks in DNA (Pommier, "Topoisomerase I inhibitors: camptothecins and beyond," Nature Reviews Cancer 2006; 6(10): 789-802) and S-phase cell cycle arrest (Ohneseit et al., "Cell cycle effects of topotecan alone and in combination with irradiation," Radiother Oncol 2005; 75(2): 237-45). Carboplatin and melphalan are alkylating agents. Here, we demonstrate that topotecan enhances the infectivity of VCN-01 in cancer cells and provides significant therapeutic benefit in several E2F-1-expressing childhood cancer xenografts, including intraocular retinoblastoma, central nervous system (CNS) disseminated retinoblastoma, and subcutaneous (sc) patient-derived xenografts (PDXs) of Ewing sarcoma and neuroblastoma.

[0297] Cell culture, tumor models, viral constructs, and chemotherapeutic agents. Primary retinoblastoma cell cultures established from enucleated eyes of four human patients were used (Pascual-Pasto et al., "Preclinical platform of retinoblastoma xenografts recapitulating human disease and molecular markers of dissemination," Cancer Letters 2016; 380(1): 10-19). The Y79 cell line was obtained from ATCC (Manassas, VA, USA). Clinical details of the cell lines are shown in Table 1.

[0298] Table 1. Clinical details of retinoblastoma cell models.

[0299]

[0300] 1 RB1 mut : Mutations in the gene RB1; MYCN A : Amplification of the MYCN gene.

[0301] 2Five tandem doses of topotecan and melphalan were administered intra-ophthalmic arterially.

[0302] 3 Six cycles of systemic carboplatin, etoposide, and vincristine, three tandem doses of topotecan and melphalan delivered intra-ophthalmic arterially, and ruthenium brachytherapy.

[0303] 4 Eight cycles of systemic carboplatin, etoposide, and vincristine, ten cycles of systemic cyclophosphamide and etoposide, one dose of intravitreal melphalan, and three tandem doses of topotecan and melphalan administered intravitreally.

[0304] Primary tumor cells were cultured in neural stem cell medium (serum-free medium supplemented with growth factors), and Y79 cells were cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% FBS as described (Pascual-Pasto et al., 2016). The short tandem repeat (STR) profiles of primary cells have been reported elsewhere (Cuadrado-Vilanova et al., "Identification of immunosuppressive factors in retinoblastoma cell secretomes and aqueous humor from patients," The Journal of Pathology 2022; 257(3): 327-39). Ewing's sarcoma and neuroblastoma PDXs were obtained from the HSJD repository with available clinical annotation data (Pascual-Pasto et al., "LowBcl-2 is a robust biomarker of sensitivity to nab-paclitaxel in Ewingsarcoma," Biochem Pharmacol 2023;208:115408; Aschero et al., "Prognostic value of xenograft engraftment in patients with metastatic high-risk neuroblastoma," Pediatric blood & cancer 2023:e30318). Cell cultures and PDXs are identified with the institutional prefix HSJD (Table 1), which has been omitted in the text and figures for clarity.

[0305] Cell proliferation. Cancer cells were incubated with VCN-01 and chemotherapeutic agents. First, the antiproliferative activity of topotecan (ranging from 10 μM to 0.0000256 μM), carboplatin (200 μM to 0.78 μM), or melphalan (10 μM to 0.00015 μM) was evaluated in retinoblastoma cells (2 × 104 cells per well in a 96-well plate) infected with VCN-01 at a multiplicity of infection (MOI; i.e., viral transduction units per cell) of 10 for the first three days. This concentration of virus was subtoxic after six days of incubation in these cells (Pascual-Pasto et al., "Therapeutic targeting of the RB1 pathway inretinoblastoma with the oncolytic adenovirus VCN-01," Science Translational Medicine 2019; 11(476): eaat9321). Three days after drug addition, cell viability was measured using the compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H]tetrazole (MTS assay; Promega, Fitchburg, WI, USA).

[0306] To address the effect of treatment sequence on cell viability and apoptosis markers, topotecan (2 μM) and VCN-01 at 50 MOI (a concentration sufficient to achieve viral protein transduction at early time points) were incubated in the order of "topotecan first" (three days before VCN-01) or "VCN-01 first" (three days before topotecan). Control experiments were performed by adding culture medium instead of the second treatment.

[0307] Similar experiments were performed to address the anti-proliferative activity of VCN-01 (100-0.4 MOI) in primary cultures of Ewing's sarcoma and neuroblastoma PDXs.

[0308] Genomic Analysis: The expression of viral genes E1A, E1B, and SPAM1 (recombinant PH20 hyaluronidase), as well as cell cycle genes such as CDKN1A and CDK6, was assessed in retinoblastoma cells treated with chemotherapeutic agents, VCN-01, and combinations in different orders.

[0309] In the presence of 2×10 6Expression of the viral genes E1A and E1B was studied in 6-well plates of retinoblastoma cells. Cells were exposed to topotecan (2 μM) and VCN-01 (50 MOI) and incubated in either a "topotecan first" (24 hours before VCN-01) or "VCN-01 first" (24 hours before topotecan) order. Control experiments were performed by adding culture medium instead of topotecan. 24 hours after the second treatment, cell pellets were collected and mRNA was isolated using the TRIzol method (Thermo Fisher Scientific, Waltham, MA, USA). Gene expression was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR) using SYBR technology (Thermo Fisher Scientific). Primers were viral E1A (forward 5'-ATC GAAGAG GTA CTG GCT GA-3' (SEQ ID NO: 11), reverse 5'-CCT CCG GTG ATA ATG ACA AG-3' (SEQ ID NO: 12)) and E1B (forward 5'-GAG GGT AAC TCC AGG GTG CG-3' (SEQ ID NO: 13), reverse 5'-TTTCAC TAG CAT GAA GCA ACC ACA-3' (SEQ ID NO: 14)).

[0310] To evaluate the expression of genes involved in the cell cycle, a 2 × 10 6 6-well culture plates of retinoblastoma cells were plated. VCN-01 was added at 50 MOI for 48 h, followed by topotecan (2 μM), carboplatin (12.5 μM), or culture medium. Cell pellets were collected 24 h later. The samples were placed in Human Cyclin and Cell Cycle Regulation Array (Thermo Fisher Scientific). This array contains measurements of 92 cell cycle-related genes as well as measurements of GAPDH, 18S, HPRT1, and GUSB as endogenous control genes. A cycle threshold (Ct) value ≥ 35 was defined as absent expression. The average expression value of the control was used to normalize gene expression values. The thresholds for upregulation and downregulation were 2 -ΔΔCt ≥2.0 and 2 -ΔΔCt ≤0.5 (Livak and Schmittgen, "Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method," Methods 2001; 25(4): 402-8).

[0311] RT-qPCR was performed to validate the results for the top genes that were overexpressed or downregulated in the array. SYBR primers were CDKN1A (forward 5'-GGA CAG GAG AGG AAG ACC ATG T-3' (SEQ ID NO: 15), reverse 5'-TGG AGTGGT AGA ATT CTG TCA TGC-3' (SEQ ID NO: 16)) and CDK6 (forward 5'-CCA GGC AGG CTT TTCATT CA-3' (SEQ ID NO: 17), reverse 5'-AGG TCC TGG AAG TAT GGG TG-3' (SEQ ID NO: 18)).

[0312] To quantify the recombinant PH20 hyaluronidase gene (SPAM1) in culture and mouse tissues, SYBR primers were used: forward 5′-TAC ACA CTC CTT GCT CCT GG-3′ (SEQ ID NO: 19), reverse: 5′-CTT AGT CTCACA GAG GCC AC-3′ (SEQ ID NO: 20) (Bazan-Peregrino et al., “VCN-01 disrupts spancreatic cancer stroma and exerts antitumor effects,” Journal for ImmunoTherapy of Cancer 2021;9(11):e003254).

[0313] To quantify the burden of retinoblastoma cells in mouse tissues (i.e., to assess tumor burden), SYBR primers for the gene CRY (forward: 5′-AGG TGG CTC TGA AGA TCA ATC TG-3′ (SEQ ID NO: 21), reverse: 5′-TTA GCC CTC CGG TTC TTG AA-3′ (SEQ ID NO: 22)) were used (Pascual-Pasto, 2016).

[0314] To evaluate the expression of E2F1, CXADR, and ITGA5 in pediatric cancers, available datasets for fetal retina (GSE12621; n = 12), pediatric brain (GSE44971 and GSE13564; n = 28), muscle tissue (GSE17679; n = 18), retinoblastoma (GSE29683; n = 55), neuroblastoma (GSE16237; n = 51), and Ewing's sarcoma (GSE34620; n = 44) were used. Data from CEL files were normalized and processed using the robust multichip average (RMA) algorithm using R statistical software and the Affymetrix library available through Bioconductor (Gautier et al., "affy--analysis of Affymetrix GeneChip data at the probe level," Bioinformatics 2004; 20(3): 307-15).

[0315] To quantify genomic copies of VCN-01 in cell culture or mouse tissues, total DNA was extracted and real-time quantitative polymerase chain reaction (RT-qPCR) was performed as described (Cascallo et al., "Systemic toxicity-efficacy profile of ICOVIR-5, a potent and selective oncolytic adenovirus based on the pRB pathway," Mol Ther 2007; 15(9): 1607-15).

[0316] Protein Expression. Proteins associated with adenoviral infection, cell cycle, and apoptosis in retinoblastoma cells exposed to chemotherapeutic agents and VCN-01 were investigated.

[0317] In all experiments, 2 × 10 6 6-well culture plates with 10 cells. First, cells were infected with VCN-01 (50 MOI, 24 hours), exposed to topotecan (2 μM), carboplatin (12.5 μM) or melphalan (12 μM), and precipitates were collected after 24 hours to analyze apoptosis, cell cycle and infection markers. The experiment was controlled by adding culture medium instead of the second treatment. To address whether the treatment order affects their interaction, the order of "topotecan first" (24 hours before VCN-01) or "VCN-01 first" (24 hours before topotecan) was applied. The analysis was extended to other chemotherapeutic agents, including hydroxyurea (100 μM), etoposide (2 μM), irinotecan (10 μM) or SN-38 (1 μM).

[0318] In a time course experiment, cell cycle proteins were analyzed after cells were exposed to chemotherapeutic agents. Retinoblastoma cells were treated with topotecan (2 μM), carboplatin (12.5 μM), melphalan (12 μM), hydroxyurea (100 μM), or SN-38 (1 μM), and cell pellets were collected at 1 h, 2 h, 4 h, 8 h, 16 h, 24 h, and 48 h.

[0319] Cells were lysed with RIPA buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton-X 100, 0.1% SDS, 1% sodium deoxycholate) to obtain total protein extracts. Protein was quantified using a BCA assay (Thermo Fisher Scientific) and immunoblotting was performed as previously described (Mandigo et al., "Relevance of pRB Loss in Human Malignancies," Clin Cancer Res 2022; 28(2): 255-64).

[0320] For immunoblotting assays, the primary antibodies were p53 (2527S, 1:1000, Cell Signaling, Danvers, MA, USA), E1A (adenovirus type 5 infection marker; ab33183, 1 μg / mL, Abcam, Cambridge, MA, USA), cleaved poly (ADP-ribose) polymerase (cPARP; apoptosis marker; 9541S, 1:1000, Cell Signaling), p21 (2947, 1:1000, Cell Signaling), E2F-1 (VCN-01 promoter; 3742, 1:1000, Cell Signaling), cyclin E1 (sc-377100, 1:200, Santa Cruz Biotechnology, Santa Cruz), and cyclin B1 (sc-177100, 1:200, Santa Cruz Biotechnology, Santa Cruz). Cruz, CA, US), topoisomerase 1 (ab85038, 1:1000, Abcam), adenovirus type 5 (hexon; ab6982, 1:1000, Abcam), β-tubulin (MAB374, 1:10,000, Millipore, Darmstadt, Germany), and GAPDH (1:10,000, Millipore). Detection was performed using near-infrared-labeled secondary antibodies (Odyssey CLx, LI-COR Inc. Bad Homburg, Germany).

[0321] To study proteins induced by adenovirus, AdTLRGDK was used, which expresses green fluorescent protein (GFP) in a genetic background similar to VCN-01 (Rodríguez-García et al., "Safety and Efficacy of VCN-01, an Oncolytic Adenovirus Combining Fiber HSG-Binding Domain Replacement with RGD and Hyaluronidase Expression," Clinical Cancer Research 2015; 21(6): 1406-18). Green fluorescent protein (GFP) transfected by AdTLRGDK was studied (Shiozawa et al., "Immunohistochemical analysis of the expression of cdk4 and p16INK4 in humanendometrioid-type endometrial carcinoma," Cancer 1997; 80(12): 2250-6). In a first experiment, Y79 cells were arrested in S phase with hydroxyurea (4 mM). After 24 hours, cells were washed with phosphate-buffered saline (PBS) and exposed to 50 MOI of AdTLRGDK. Cell fluorescence images were acquired at 24, 48, and 72 hours post-infection, and pellets were collected for GFP analysis by flow cytometry (NovoCyte Flow Cytometry System, ACEA Biosciences, San Diego, CA, USA). Infected cell signals were gated using autofluorescence from untreated cells. Data were analyzed using NovoExpress software (ACEA Biosciences). In a second cytometry experiment, Y79, RBT-5, and RBT-7 cells were infected with 50 MOI of AdTLRGDK. After 24 hours, topotecan (2 μM), carboplatin (12.5 μM), melphalan (10 μM), hydroxyurea (100 μM), or fresh medium were added. GFP expression was quantified 24 hours later. To address the effect of treatment order on GFP expression, cells were exposed to topotecan (2 μM) or hydroxyurea (100 μM) 6 h before VCN-01 infection at an MOI of 50 and harvested 48 h later.

[0322] Virus production assay. Y79 cells were infected at a multiplicity of infection (MOI; i.e., viral transduction units per cell) of 500, a concentration sufficient to achieve 80% to 100% infectivity. After 4 h, cells were washed three times with PBS, fresh medium was added, and cells were incubated with or without topotecan (2 μM). At 30 h, 48 h, and 72 h post-infection, cell pellets were collected and exposed to three cycles of freeze-thaw lysis. Virus titers in the pellets were determined in triplicate following an anti-hexon staining method in HEK293 cells (Cascallo et al., 2007).

[0323] Cell cycle. To address the effect of chemotherapy on the retinoblastoma cell cycle, we used hydroxyurea (100 μM) as a positive control for S-phase cell cycle arrest (Alvino et al., "Replication in hydroxyurea: it's a matter of time," Mol Cell Biol 2007; 27(18): 6396-406).

[0324] Put 10 6 Retinoblastoma cells were treated with topotecan (2 μM), carboplatin (12.5 μM), melphalan (10 μM), or hydroxyurea (100 μM) for 24 h. Cells were then collected and fixed in 70% ethanol for 2 h. Propidium iodide (PI)-based assay FxCycle was used before quantification by flow cytometry (NovoCyte). TM PI / RNase (ThermoFisher Scientific) was used as the staining solution.

[0325] In order to synchronize the cells in the S phase of the cycle, 10 6 Cells were exposed to a high concentration of hydroxyurea (4 mM). After 24 h, cells were washed with PBS, fresh medium was added, and cells were incubated for 2 h, 4 h, 8 h, and 24 h until processed for flow cytometry.

[0326] Apoptosis. The apoptosis marker cPARP was assessed in retinoblastoma cells exposed to topotecan, VCN-01, or the combination.

[0327] Put 10 6Retinoblastoma cells were seeded in each well of a 6-well plate. After 24 hours, VCN-01 was added at a 50 MOI. The next day, topotecan (2 μM) was added. After 24 hours, cells were harvested, washed twice with 1% bovine serum albumin in PBS, and fixed with 70% ethanol for 2 hours at 4°C. Cells were labeled with a primary antibody against cPARP (5625S, 1:1000, Cell Signaling) and a secondary antibody (ab150077, 1:2000, Abcam) prior to flow cytometry (NovoCyte).

[0328] Accumulation of topotecan in retinoblastoma cells infected with VCN-01. Animal experiments were approved by the Ethical Committee for Animal Experimentation of the University of Barcelona (animal protocol number 482 / 18). Retinoblastoma cells (Envigo, Barcelona, ​​Spain) were cultured in the eyes of athymic nude mice as described previously (Pascual-Pasto et al., 2016). After 14 days, VCN-01 (3 × 10 9 vp / eye). After another 14 days, a subcutaneous osmotic pump loaded with topotecan was implanted to achieve constant topotecan in the plasma, as previously described (Pascual-Pasto et al., "Increased delivery of chemotherapy to the vitreous by inhibition of the blood-retinal barrier," J Control Release 2017; 264: 34-44), and tumor samples were collected for topotecan analysis.

[0329] Animal experiments were performed in accordance with institutional and European guidelines (EU Directive 2010 / 63 / EU) and ARRIVE guidelines (van den Heuvel and Dyson, "Conserved functions of the pRB and E2F families," Nature Reviews Molecular Cell Biology 2008; 9(9): 713-24). Retinoblastoma cells (2 x 10 5 ) were inoculated into the posterior segment of the eye of six-week-old athymic nude mice as described previously (Giani et al., 1994). Fourteen days later, a single dose of VCN-01 (3×10 9vp / eye). After another 14 days, Alzet osmotic pumps (2001D, Durect, Palo Alto, CA, USA) loaded with 1 mg / mL topotecan were implanted. These pumps released topotecan at a dose of 25 μg / h, achieving a constant level of approximately 100 ng / mL in plasma. Tumors and plasma were collected at steady state (6 h). Samples were analyzed by high-performance liquid chromatography (HPLC) as previously described (Nevins, "The Rb / E2F pathway and cancer," Human Molecular Genetics 2001; 10(7): 699-703).

[0330] Intratumoral infection with VCN-01. Subcutaneous tumors were established in athymic nude mice. Following xenograft implantation, mice received a single intratumoral injection of VCN-01 alone or in combination with a cycle of topotecan, carboplatin, or hydroxyurea. Five and 15 days after VCN-01 inoculation, genomic copies of VCN-01 in tumors were quantified and immunostained for E1A and E2F-1.

[0331] To establish subcutaneous tumors, 10 μL of 1% WT mice suspended in 25 μL of matrigel (Corning, Glendale, AZ, USA) were placed in a 4% flask. 6 Retinoblastoma cells were injected into athymic nude mice. After xenograft implantation (tumor volume ranged from 100-200 mm 3 ), treatment was started. Mice received VCN-01 alone (3×10 9vp, a single intratumoral injection on day 1) or VCN-01 in combination with a cycle of topotecan (0.6 mg / kg, intraperitoneally -ip-, once daily on days 1-5), carboplatin (40 mg / kg, intraperitoneally, a single dose on day 1), or hydroxyurea (200 mg / kg, intraperitoneally, once daily on days 1-5). One group of mice received a single intratumoral injection of VCN-01 in a vehicle (20 mM pH 8.0 tris buffer, 25 mM NaCl, and 2.5% glycerol). Mice were sacrificed 5 and 15 days after VCN-01 inoculation, tumors were dissected, and tumor homogenates were prepared by adding 10 μL of water per mg of tissue and homogenizing with a Bullet Blender turbine Storm 24 (Quasar instruments, Colorado Springs, CO, USA). Genomic copies of VCN-01 were quantified in the tumor homogenates. Some tumors were treated with 4% paraformaldehyde and embedded in paraffin for immunohistochemical analysis. 4 μm paraffin sections were stained with hematoxylin and eosin (H&E), and human nuclei (MAB4383, 1:200, Merck Millipore) were immunostained for viral proteins E1A (ab33183, 1:200, Abcam) and E2F-1 (sc-251, 1:50, Santa Cruz Biotechnology).

[0332] Antitumor activity in retinoblastoma. For orthotopic intraocular implantation, 2 x 10 cells / eye were inoculated in 2 μL of matrigel. 5 To obtain subcutaneous xenografts, 10 retinoblastoma cells in 25 μL of matrigel were injected. 6 For ocular tumors, treatment begins on day 8 after inoculation (when tumors can be found by fundus examination). For subcutaneous tumors, when the volume reaches 100-300mm 3 Treatment was initiated on day 1. After treatment, survivors were followed up until endpoint, and median survival was calculated for each group as previously described (Mandigo et al., 2022; Rodríguez-García et al., 2015).

[0333] In the first study, one dose of VCN-01 (3 × 10 8vp, intravitreal, day 15), topotecan, or standard of care (SoC) retinoblastoma chemotherapy, or a combination thereof. For topotecan, mice received 0.6 mg / kg intraperitoneal injections on days 8-12 and 29-33. For SoC, mice received 40 mg / kg intraperitoneal carboplatin on days 8 and 29, and 6 mg / kg etoposide on days 8-10 and 29-31.

[0334] In the second experiment, the order of administration of a single VCN-01 injection and a 5-day cycle of topotecan was addressed. Mice bearing intraocular tumors (Y79 or RBT-7) received intraperitoneal topotecan 0.6 mg / kg on days 8-12 and a single VCN-01 (3 × 10 8 vp) or received a combination of topotecan and VCN-01. Y79-bearing mice also underwent a switched treatment sequence, receiving VCN-01 on day 12 after the last dose of topotecan.

[0335] In the third study, the number of topotecan cycles was maximized (injections on days 8-12, 15-19, 29-33, 36-40, 50-54, and 57-61), and VCN-01 (3 × 10 7 vp / eye) by intraocular injection (Pascual-Pasto et al., 2019).

[0336] In a fourth study, to abrogate the effect of the blood-retinal barrier on drug delivery (Pascual-Pasto et al., “Therapeutic targeting of the RB1 pathway in retinoblastoma with theoncolytic adenovirus VCN-01,” Science Translational Medicine 2019;11(476):eaat9321), mice were inoculated subcutaneously bilaterally with Y79 or RBT-7 cells. These animals were treated with intraperitoneal topotecan 0.6 mg / kg, VCN-01 (3 × 10 in 20 μL vehicle) on days 1–5. 9 vp, intratumoral, day 1) or a combination of the same order.

[0337] Antitumor activity in CNS-disseminated retinoblastoma. A model of retinoblastoma dissemination in the leptomeninges of the CNS was established. Anesthetized mice were fixed in a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA). Using a mouse brain atlas (Paxinos and Franklin, "The mouse brain in stereotaxic coordinates," San Diego: Academic Press; 2001), the coordinates of the fourth ventricle were located and a hole was drilled on the skull surface. Then, 10 μL of 10 μL of matrigel was added to the cerebral canal. 6 Millions of cells (RBT-7) were injected with a blunt 22G needle attached to a 50 μL syringe (Hamilton, Bonaduz, Switzerland). One week later, VCN-01 (3×10 9 vp, intracerebroventricular, day 8), topotecan (0.6 mg / kg, intraperitoneal, days 8-12), SoC chemotherapy (carboplatin, 40 mg / kg, intraperitoneal, day 8, and etoposide, 6 mg / kg, intraperitoneal, days 8-10), or VCN-01 in combination with chemotherapy was initiated. The control group received the vehicle of VCN-01 (intracerebroventricular). Animals reached the experimental endpoint when the disease worsened or the body weight decreased by 20%. Brains collected at the end point were embedded in paraffin and stained with hematoxylin and eosin (H&E), human nuclei (MAB4383, 1:200, Millipore), viral protein E1A (ab33183, 1:200, Abcam), and CD45 (leukocyte common antigen; 70257, 1:200, Cell Signaling).

[0338] In the second experiment, 12 mice were injected with 10 6 One week later, mice were treated with vehicle, VCN-01 (3×10 9 Treatment was with topical injection of 1 mg of topotecan (vp), systemic topotecan, or a combination. Mice were sacrificed 2 hours after the last dose of topotecan. Brain homogenates were prepared by adding 10 μL of water per mg of tissue. DNA, protein, and mRNA were extracted as previously described for subcutaneous tumors. Tumor sections were processed for immunohistochemical analysis.

[0339] Antitumor activity in extracranial pediatric solid tumors. 5-10 mm Ewing sarcoma or neuroblastoma PDX 3 Fresh fragments were implanted into the bilateral abdominal cavity of mice. 3 ), mice received topotecan (0.6 mg / kg, intraperitoneally, days 1-5), VCN-01 (3 × 109 In two additional groups, we used the alternative topoisomerase 1 inhibitor irinotecan (10 mg / kg, intraperitoneally, days 1-5) instead of topotecan. After treatment completion, tumor volume was followed until endpoint (1500 mm 3 ) or until the 80th day.

[0340] Statistical analyses and graphical presentation of the data were performed using GraphPad Prism 8 software (La Jolla, CA, USA) and the R Core Team (R Foundation for Statistical Computing, Vienna, Austria).

[0341] At least three replicates were performed for each in vitro condition. In order to compare the mean values ​​of two different groups, a t-test or Mann-Whitney test was used. For more than two groups, an ANOVA test or a Kruskal-Wallis test with Dunn correction was used. Median survival was calculated using the Kaplan-Meier method, and the log-rank test with Bonferroni correction was used to compare for multiple comparisons. In most studies, all groups were compared with a control group (vehicle group). In the study of CNS disseminated retinoblastoma, all groups were compared with a group receiving a combination of VCN-01 and topotecan. For all analyses, P < 0.05 was defined as statistically significant.

[0342] Interactions between VCN-01 and chemotherapy in retinoblastoma: apoptosis and infection. Infection of retinoblastoma cells with VCN-01 reduced the sensitivity of the cells to topotecan, carboplatin, and melphalan (Figures 13A and Figure 14 ) and increased the drug concentration required to reduce cell proliferation by 50% (Table 2).

[0343] Table 2. In vitro susceptibility of retinoblastoma cells to VCN-01, chemotherapeutic agents, or combinations.

[0344]

[0345] 1 The activity of VCN-01 against retinoblastoma models was previously published [Pascual-Pastó et al., 2019].

[0346] 2 Values ​​are expressed as half maximal inhibitory concentration (IC50; MOI), ie, the concentration of anticancer agent required to cause a 50% reduction in cell viability. Values ​​in brackets represent 95% confidence intervals.

[0347] 3Values ​​are IC50 (μM) with 95% confidence intervals.

[0348] np, not performed

[0349] Viral infection hindered the expected increase in proteins triggered by DNA damage induced by cytotoxic agents such as cPARP and p53 (Figures 13B and 15A-15C). Unexpectedly, the early viral protein E1A was highly overexpressed in infected cells treated with topotecan (Figures 13B and 15A). Even in the presence of topotecan, infected cells maintained their shape and integrity during the early stages of infection (48h) (Figure 13C). Cells pre-exposed to topotecan before VCN-01 infection were sensitive to the antiproliferative activity of the drug, regardless of whether the virus was present in the later stages (Figure 13D). In contrast, the previous infection partially eliminated the activity of the drug (Figure 13D). Topotecan pretreatment inhibited subsequent infection of cells with VCN-01, resulting in low expression of E1A (protein and gene), accumulation of E2F-1, and increased expression of pro-apoptotic proteins p53 and cPARP (Figures 13E, 13F). In contrast, post-viral addition of topotecan boosted the expression of E1A (Figures 13E and 13F) and E1B (Figure 13G) by two orders of magnitude. Another camptothecin, SN-38, increased the expression of E2F-1 and E1A similarly to topotecan in cells pre-exposed to VCN-01, in contrast to the alkylating drug, the antimetabolite hydroxyurea, and the topoisomerase II inhibitor etoposide, which did not alter the levels of these proteins (Figure 13H). Topoisomerase 1, the molecular target of camptothecin, increased after VCN-01 infection in one primary culture but remained unchanged in another (Figure 13E and Figure 13F). Figure 16 Overall, the results indicate that adenovirus infection blocks the apoptotic effect of topotecan, while topotecan favors early viral protein expression in cells pre-exposed to VCN-01. Without wishing to be bound by theory, it is believed that this occurs by increasing expression of the viral promoter E2F-1 in the cells.

[0350] Interaction of VCN-01 with Chemotherapy: Cell Cycle and E2F-1 Expression. Because S-phase cells are more amenable to adenoviral infection, cell cycle alterations were addressed in retinoblastoma cells exposed to standard-of-care chemotherapy for this disease. Topotecan alone increased the percentage of S-phase cells, similar to the positive control, hydroxyurea (Alvino et al., 2007) ( FIG. 17A ). The effect of topotecan was evident early and persisted for at least 48 hours ( FIG. 17B , FIG. 17C ). Topotecan, but not carboplatin, melphalan, or hydroxyurea, promoted cellular events consistent with G1 / S cell cycle progression, such as increased protein expression of E2F-1, p21, and cyclin E1, which were evident 1 hour after exposure and persisted for at least 48 hours ( FIG. 17D ). SN-38 produced similar effects, but they occurred slightly later than topotecan ( FIG. 17D ). In mice bearing intraocular Y79 tumors, E2F-1 expression in cancer cells from animals treated with topotecan was higher compared to that found in untreated animals ( FIG. 17E ).

[0351] In primary retinoblastoma cells pre-infected with VCN-01, gene expression arrays revealed that topotecan upregulated genes that promote S-phase arrest during the cell cycle, with a 10-fold increase in expression of the gene CDKN1A (encoding the p21 protein) (Figure 17F). Conversely, it downregulated genes that promote the G1 / S transition, with a 15-fold decrease in CDK6 expression (Figure 17F). qPCR confirmed the array findings (Figure 17G).

[0352] Interaction of VCN-01 with chemotherapy: gene transduction and viral replication. To determine whether S-phase cell cycle arrest favors viral gene expression, Y79 cells were synchronized in S phase using high concentrations of hydroxyurea (Figure 18A). After a wash step, treatment of synchronized cells with adenovirus AdTLRGDK significantly increased transgene expression (GFP) (Figures 18B-18D). After 72 hours, 44% of asynchronous cells were infected, compared to 90% of synchronized cells (Figure 18D). In Y79 cells and primary retinoblastoma cultures pre-infected with adenovirus AdTLRGDK, topotecan and hydroxyurea increased transgene expression compared to carboplatin and melphalan (Figures 18E and 19A). The opposite treatment sequence (i.e., drug exposure before viral infection) did not enhance viral transduction (Figure 18E). Topotecan did not increase viral production in Y79 cells pre-infected with a high viral load of 500 MOI (Figure 18F). After a single intratumoral injection of VCN-01 in subcutaneous xenografts, subsequent treatment with systemic topotecan resulted in higher viral genome counts after 5 days ( FIG. 18G ) and higher expression of recombinant hyaluronidase ( FIG. 18H ) compared to carboplatin treatment. No difference in viral genome counts was observed after 15 days ( FIG. 19B ). Five days after topotecan treatment, tumors pretreated with VCN-01 had the greatest number of E1A-positive cells ( FIG. 18I ) and the highest level of E1A protein expression ( FIG. 18J ) compared to tumors treated with saline, carboplatin, or hydroxyurea.

[0353] Activity of VCN-01 and topotecan in retinoblastoma xenografts. Because long-term exposure to clinically relevant concentrations of VCN-01 and chemotherapy in vitro is not feasible, in vivo experiments were performed to address the therapeutic effect of the treatment combination. Administering a single intraocular dose of VCN-01 between two cycles of topotecan extended the median ocular survival of Y79 xenografts by more than 80 days, a significant therapeutic benefit compared to untreated control eyes (37 days; P = 0.0005). Treatment with topotecan or standard of care (carboplatin and etoposide) resulted in a moderate improvement in median survival (58 days and 55 days, respectively), but was not significant compared to controls (P = 0.062 and P = 0.674, respectively) (Figure 20A). As a single agent, the selected VCN-01 dose was subtherapeutic (40 days, P = 1) (Figure 20A). The order of administration that produced better-than-additive efficacy was one in which VCN-01 was administered first, followed by topotecan ( FIG. 20B ). This treatment sequence resulted in a median ocular survival of 62 days, significantly longer than the control (46 days, P = 0.0436). The reverse order (topotecan followed by VCN-01) did not result in a therapeutic benefit (52 days, P = 1) ( FIG. 20B ). The synergistic effect of VCN-01 and topotecan was demonstrated in a primary retinoblastoma xenograft, which achieved a median survival of 67 days, longer than the control (35 days, P < 0.0001), whereas VCN-01 and topotecan were less effective as single agents (33 and 39 days, respectively) ( FIG. 20C - FIG. 20E ). In tumors treated with the combination, active VCN-01-infected area was detected at the endpoint (i.e., time of extirpation due to tumor progression) even on day 65 after a single VCN-01 injection ( FIG. 20F ). Maximizing the number of topotecan cycles and VCN-01 injections resulted in a significant prolongation of median ocular survival ( FIG. 20G ; P < 0.0001). The biodistribution of topotecan in intraocular tumors was not altered following concurrent treatment with VCN-01 ( Figure 21 The excellent therapeutic activity of the combination therapy was confirmed in two subcutaneous retinoblastoma xenografts, with the median survival of Y79 and RBT-7 reaching 67 days and over 80 days, respectively, which were significantly longer than those of the control group (35 days, P = 0.0009 and 35 days, P = 0.0021, respectively) ( Figure 20H , Figure 20I ).

[0354] The activity of VCN-01 and topotecan in CNS disseminated retinoblastoma. The mouse model of CNS disseminated retinoblastoma occupied and expanded the entire pia mater space, including the meninges around the spinal cord (Figure 22A, Figure 22B). To a lesser extent, tumor cells invaded the brain parenchyma (Figure 22A). In the absence of treatment, the median survival was 22 days (Figure 22C). Mice treated with intraventricular VCN-01 and systemic topotecan achieved a median survival of 43 days, which was significantly longer (Figure 22C) compared with controls (P=0.0004), topotecan (28 days, P=0.0072), standard care (26 days, P=0.0040) and VCN-01 (28 days, P=0.0372). Under selected doses of intraventricular VCN-01, relevant toxicity (acute weight loss) was observed (Figure 23A). Due to toxicity, 50% of the animals in the VCN-01 group, 30% of the animals in the combination of VCN-01 and topotecan, and 70% of the animals in the combination of VCN-01 and standard of care died on days 7-9 after treatment, possibly due to brain inflammation. Areas of lymphocyte infiltration in the brain were observed at autopsy (Figure 23B). For survival analysis, animals that died due to toxicity and the entire group in which more than 50% of the animals had lethal toxicity were excluded. To alleviate the brain inflammation observed during the first few days of the experiment, dexamethasone (5 mg / kg, intraperitoneally, once a day for 5 consecutive days) was empirically administered to all mice starting on day 15. The survivors recovered from the treatment and gained weight (Figure 23A). Animals treated with VCN-01 and topotecan experienced less acute toxicity and recovered completely (Figure 22D). Of the animals sacrificed on day 26 to study tumor burden, one animal treated with VCN-01 and topotecan presented a smaller tumor burden than the rest of the animals (Figure 22E).

[0355] In mice sacrificed on the last day of treatment, all mice had similar brain retinoblastoma cell loads (Figure 22F). Brain homogenates from mice treated with systemic topotecan (alone or after intraventricular VCN-01) had the highest expression of CDKN1A (Figure 22G). E2F1 expression levels did not vary between groups (Figure 22G). Compared to mice treated with intraventricular injection of VCN-01 alone, the number of E1A-positive cells in mice treated with the combination was higher (Figure 23C). Brain homogenates from the group treated with VCN-01 in combination with topotecan showed the highest VCN-01 genome counts (Figure 22H), the highest expression of human hyaluronidase (Figure 22I), and the highest viral hexon positivity (Figure 22J).

[0356] Activity of VCN-01 and topotecan in Ewing's sarcoma and neuroblastoma. First, the gene expression databases GSE16237 (Ohtaki et al., "A robust method for estimating gene expression states using Affymetrix microarray probe level data," BMC Bioinformatics 2010; 11: 183-83) and GSE34620 (Postel-Vinay et al., "Common variants near TARDBP and EGR2 are associated with susceptibility to Ewing sarcoma," Nature Genetics 2012; 44(3): 323-27) were used to verify that Ewing's sarcoma and neuroblastoma express E2F1 and the adenovirus receptors CXADR and ITGA5 (Figure 24A). E2F-1 protein expression was positive in biopsies and corresponding PDXs from one patient each of Ewing's sarcoma and neuroblastoma (Figure 24B). In vitro, overexpression of E2F-1 and cyclin E1 was observed after topotecan exposure (Figure 24C). VCN-01 inhibited the proliferation of PDX-derived primary cells ( Figure 25 ). Both subcutaneous xenografts were highly resistant to monotherapy with topotecan, irinotecan, or VCN-01 (Figures 24D-24G). In neuroblastoma PDXs, the combination of VCN-01 with topotecan or irinotecan provided significant survival benefits (P = 0.0050 and P = 0.0002, respectively) and tumor growth inhibition compared to controls (Figures 24D, 24F). In Ewing's sarcoma PDXs, the combination of VCN-01 with topotecan achieved super-additive therapeutic activity in terms of animal survival (P = 0.0303 compared to controls) and tumor growth (Figures 24E, 24G).

[0357] discuss:

[0358] In summary, this example demonstrates, inter alia, the synergistic antitumor activity of VCN-01 and camptothecin in highly aggressive and chemorefractory pediatric solid tumors expressing E2F-1. Without wishing to be bound by theory, it is believed that molecular data support that the accumulation and stabilization of E2F-1 following DNA damage produced by topotecan leads to increased selectivity and infectivity of VCN-01. This work has broad applications in improving the oncolytic activity of viruses containing the E2F-1 promoter and supports the development of clinical trials.

[0359] Equivalent solutions

[0360] Although the present disclosure has been disclosed in conjunction with specific embodiments thereof, it should be understood that the present disclosure is capable of further modifications, and this application is intended to cover any variations, uses or changes that generally follow the principles of the present disclosure, include known technologies or commonly used technical means in the field to which the present disclosure belongs, although they do not fall within the scope of the present disclosure, and can be applied to the essential features set forth above and listed in the scope of the appended claims.

[0361] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed by the following claims.

[0362] Incorporated by reference

[0363] All patents and publications mentioned herein are hereby incorporated by reference in their entirety.

[0364] 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.

[0365] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The contents of any individual section may apply equally to all sections.

[0366] Implementation Plan

[0367] The embodiments listed below provide various additional embodiments of the present disclosure, which embodiments can be combined in any number and in any combination.

[0368] Embodiment 1. A method for treating retinoblastoma, Ewing's sarcoma, or neuroblastoma in a patient in need thereof, comprising co-administering to the patient (i) an oncolytic adenovirus comprising a replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug thereof.

[0369] Embodiment 2. The method of embodiment 1, wherein the oncolytic adenovirus is administered by intraocular, intravenous, intraarterial, intrathecal, intravitreal, or intratumoral injection.

[0370] Embodiment 3. The method of embodiment 1 or 2, wherein the topoisomerase I inhibitor or a prodrug thereof is administered intravenously, intraarterially, intravitreally, intrathecally, intracerebroventricularly, or orally.

[0371] Embodiment 4. The method according to any one of Embodiments 1 to 3, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are formulated into separate compositions.

[0372] Embodiment 5. The method of any one of Embodiments 1 to 3, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are formulated into a single composition.

[0373] Embodiment 6. The method according to any one of embodiments 1 to 5, wherein the separate compositions are administered simultaneously or contemporaneously.

[0374] Embodiment 7. The method according to any one of embodiments 1 to 6, wherein the oncolytic adenovirus is administered first and the topoisomerase I inhibitor or a prodrug thereof is administered within about 60 minutes after the administration of the oncolytic adenovirus.

[0375] Embodiment 8. The method of 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 after the administration of the oncolytic adenovirus.

[0376] Embodiment 9. The method of any one of Embodiments 1 to 8, wherein the subject is administered a single intrathecal injection of the oncolytic adenovirus concurrently with systemic or intrathecal circulation of the topoisomerase I inhibitor or a prodrug thereof.

[0377] Embodiment 10. The method of any one of Embodiments 1 to 9, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are administered systemically to the subject.

[0378] Embodiment 11. The method of any one of Embodiments 1 to 10, wherein the oncolytic adenovirus and / or topoisomerase I inhibitor or a prodrug thereof is co-administered with one or more topical and / or systemic corticosteroids, optionally wherein the one or more topical and / or systemic corticosteroids are selected from hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, aldosterone, budesonide, fluticasone, flunisolide, ciclesonide, mometasone, beclomethasone, triamcinolone and tixocortolone.

[0379] Embodiment 12. The method of any one of claims 1 to 11, wherein the topoisomerase I inhibitor or a prodrug thereof is topotecan, SN-38, or irinotecan.

[0380] Embodiment 13. The method of any one of embodiments 1 to 12, wherein the hyaluronidase is human hyaluronidase PH20.

[0381] Embodiment 14. The method of embodiment 13, wherein the sequence encoding the hyaluronidase is SEQ ID NO: 9 or a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% or at least about 99% sequence identity thereto.

[0382] Embodiment 15. The method of any one of Embodiments 1 to 14, wherein the oncolytic adenovirus is produced by human adenovirus serotype 5.

[0383] Embodiment 16. The method of any one of Embodiments 1 to 15, wherein the treatment is treatment of retinoblastoma and oncolytic adenovirus replication occurs in tumor cells with an aberrant Rb-E2F pathway but not in healthy, non-tumor or normal cells.

[0384] Embodiment 17. The method of embodiment 16, wherein the oncolytic adenovirus is engineered to replicate in tumor cells, but not in healthy, non-tumor or normal cells, by deleting Δ24 in the sequence encoding the E1a protein and inserting four binding sites for E2F-1 and one binding site for Sp1 into the endogenous promoter of E1a to control the expression of E1a.

[0385] Embodiment 18. The method according to any one of claims 1 to 17, wherein the capsid of the oncolytic adenovirus is modified so that the heparan sulfate binding domain present in the adenoviral fiber 91 KKTK 94 (SEQ ID NO: 8) optionally by a domain 91 RGDK 94 (SEQ ID NO: 9) was replaced.

[0386] Embodiment 19. The method of any one of Embodiments 1 to 18, wherein the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3).

[0387] Embodiment 20. The method of any one of Embodiments 1 to 19, wherein the patient is a human patient.

[0388] Embodiment 21. The method of embodiment 20, wherein the human patient is a pediatric human patient.

[0389] Embodiment 22. The method of any one of Embodiments 1 to 21, wherein the retinoblastoma, Ewing's sarcoma, or neuroblastoma is a retinoblastoma, Ewing's sarcoma, or neuroblastoma that is resistant to treatment with conventional chemotherapy and / or radiotherapy.

[0390] Embodiment 23. The method of any one of Embodiments 1 to 22, wherein the method improves and / or increases and / or enhances anti-tumor efficacy compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0391] Embodiment 24. The method of any one of Embodiments 1 to 23, wherein the method results in reduced or maintained tumor size and / or prevention or reduction of retinoblastoma-associated metastases, secondary malignancies, or trilateral retinoblastomas compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0392] Embodiment 25. The method of any one of Embodiments 1 to 23, wherein the method results in reduced or maintained tumor size and / or prevention or reduction of metastasis, secondary malignancies, or recurrent disease associated with Ewing sarcoma compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0393] Embodiment 26. The method of any one of Embodiments 1 to 23, wherein the method results in reduced or maintained tumor size and / or prevention or reduction of metastasis, secondary malignancies, or recurrent disease associated with neuroblastoma compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0394] Embodiment 27. The method of any one of Embodiments 1 to 26, wherein administration of the oncolytic adenovirus reduces the apoptotic effect of topotecan compared to monotherapy.

[0395] Embodiment 28. The method of any one of Embodiments 1 to 27, wherein administration of topotecan after administration of the oncolytic adenovirus results in S phase cell cycle arrest.

[0396] Embodiment 29. The method of any one of Embodiments 1 to 28, wherein the S phase cell cycle arrest results in increased infectivity of the oncolytic adenovirus compared to treatment with the oncolytic adenovirus without the topoisomerase I inhibitor or a prodrug thereof.

[0397] Embodiment 30. The method of any one of embodiments 1 to 29, wherein administration of topotecan following administration of the oncolytic adenovirus results in increased expression of E2F-1, p21, and / or cyclin E1 compared to treatment with the oncolytic adenovirus without the topoisomerase I inhibitor or a prodrug thereof.

[0398] Embodiment 31. The method of embodiment 30, wherein the increased E2F-1 expression results in increased oncolytic activity of the oncolytic adenovirus compared to treatment with the oncolytic adenovirus without the topoisomerase I inhibitor or a prodrug thereof.

[0399] Embodiment 32. The method of any one of Embodiments 1 to 31, wherein the increased infectivity and oncolytic activity of the oncolytic adenovirus occurs without substantially increasing the replication of the oncolytic adenovirus.

[0400] Embodiment 33. The method according to any one of embodiments 1 to 32, wherein sequential treatment of first administering the oncolytic adenovirus followed by systemic administration of topotecan enhances or increases the cell infection and / or anti-cancer efficacy of the oncolytic adenovirus.

[0401] Embodiment 34. A method for treating retinoblastoma, Ewing's sarcoma, or neuroblastoma in a patient in need thereof, comprising administering to the patient (i) an oncolytic adenovirus comprising a replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug thereof.

[0402] Embodiment 35. The method of embodiment 34, wherein the oncolytic adenovirus is administered by intraocular, intravenous, intraarterial, intrathecal, intravitreal, or intratumoral injection.

[0403] Embodiment 36. The method of embodiment 34 or 35, wherein the topoisomerase I inhibitor or a prodrug thereof is administered intravenously, intravitreally, intraarterially, intrathecally, intracerebroventricularly, or orally.

[0404] Embodiment 37. The method of any one of Embodiments 34 to 36, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are formulated into separate compositions.

[0405] Embodiment 38. The method of any one of Embodiments 34 to 37, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are formulated into a single composition.

[0406] Embodiment 39. The method of any one of Embodiments 34 to 38, wherein the oncolytic adenovirus is administered first and the topoisomerase I inhibitor or a prodrug thereof is administered within about 12 weeks after the administration of the oncolytic adenovirus.

[0407] Embodiment 40. The method of embodiment 39, 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 after the administration of the oncolytic adenovirus.

[0408] Embodiment 41. The method of any one of Embodiments 1 to 32, wherein the subject is administered a single intrathecal injection of the oncolytic adenovirus concurrently with systemic or intrathecal circulation of the topoisomerase I inhibitor or a prodrug thereof.

[0409] Embodiment 42. The method of any one of Embodiments 1 to 32, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are administered systemically to the subject.

[0410] Embodiment 43. A method according to any one of embodiments 1 to 32, wherein the oncolytic adenovirus and / or topoisomerase I inhibitor or a prodrug thereof is co-administered with one or more topical and / or systemic corticosteroids, optionally wherein the one or more topical and / or systemic corticosteroids are selected from hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, aldosterone, budesonide, fluticasone, flunisolide, ciclesonide, mometasone, beclomethasone, triamcinolone and tixocortolone.

[0411] Embodiment 44. The method of any one of Embodiments 34 to 43, wherein the topoisomerase I inhibitor is topotecan or SN-38 and the topoisomerase I inhibitor prodrug is irinotecan.

[0412] Embodiment 45. The method of any one of embodiments 34 to 44, wherein the hyaluronidase is human hyaluronidase PH20.

[0413] Embodiment 46. The method of embodiment 45, wherein the sequence encoding the hyaluronidase is SEQ ID NO: 9 or a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% or at least about 99% sequence identity thereto.

[0414] Embodiment 47. The method of any one of Embodiments 34 to 46, wherein the oncolytic adenovirus is produced by human adenovirus serotype 5.

[0415] Embodiment 48. The method of any one of Embodiments 34 to 47, wherein oncolytic adenovirus replication occurs in tumor cells with an aberrant Rb-E2F pathway but not in healthy, non-tumor or normal cells.

[0416] Embodiment 49. The method of embodiment 48, wherein the oncolytic adenovirus is engineered to replicate in tumor cells, but not in healthy, non-tumor or normal cells, by deleting the Rb binding domain or Δ24 deletion in the sequence encoding the E1a protein and inserting four binding sites for E2F-1 and one binding site for Sp1 into the endogenous promoter of E1a to control the expression of E1a.

[0417] Embodiment 50. The method of any one of claims 34 to 49, wherein the capsid of the oncolytic adenovirus is modified such that the heparan sulfate binding domain present in the adenoviral fiber 91 KKTK 94 (SEQ ID NO: 8) optionally by a domain 91 RGDK 94 (SEQ ID NO: 9) was replaced.

[0418] Embodiment 51. The method of any one of Embodiments 34 to 50, wherein the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3).

[0419] Embodiment 52. The method of any one of Embodiments 34 to 51, wherein the patient is a human patient.

[0420] Embodiment 53. The method of embodiment 52, wherein the human patient is a pediatric human patient.

[0421] Embodiment 54. The method of any one of Embodiments 34 to 53, wherein the retinoblastoma, Ewing's sarcoma, or neuroblastoma is a retinoblastoma, Ewing's sarcoma, or neuroblastoma that is resistant to treatment with conventional chemotherapy and / or radiotherapy.

[0422] Embodiment 55. The method of any one of Embodiments 34 to 54, wherein the method improves and / or increases and / or enhances anti-tumor efficacy compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0423] Embodiment 56. The method of any one of Embodiments 34 to 55, wherein the method results in reduced or maintained tumor size and / or prevention or reduction of retinoblastoma-associated metastases, secondary malignancies, or trilateral retinoblastomas compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0424] Embodiment 57. The method of any one of Embodiments 34 to 55, wherein the method results in reduced or maintained tumor size and / or prevention or reduction of metastasis, secondary malignancies, or recurrent disease associated with Ewing sarcoma compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0425] Embodiment 58. The method of any one of Embodiments 34 to 55, wherein the method results in reduced or maintained tumor size and / or prevention or reduction of metastasis, secondary malignancies, or recurrent disease associated with neuroblastoma compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

[0426] Embodiment 59. The method of any one of Embodiments 34 to 58, wherein administration of the oncolytic adenovirus reduces the apoptotic effect of topotecan compared to monotherapy.

[0427] Embodiment 60. The method of any one of embodiments 34 to 59, wherein administration of topotecan after administration of the oncolytic adenovirus results in S phase cell cycle arrest.

[0428] Embodiment 61. The method of embodiment 60, wherein the S phase cell cycle arrest results in increased infectivity of the oncolytic adenovirus compared to treatment with the oncolytic adenovirus without the topoisomerase I inhibitor or a prodrug thereof.

[0429] Embodiment 62. The method of any one of Embodiments 34 to 61, wherein administration of topotecan following administration of the oncolytic adenovirus results in increased expression of E2F-1, p21, and / or cyclin E1 compared to treatment with the oncolytic adenovirus without the topoisomerase I inhibitor or a prodrug thereof.

[0430] Embodiment 63. The method of embodiment 62, wherein the increased E2F-1 expression results in increased oncolytic activity of the oncolytic adenovirus compared to treatment with the oncolytic adenovirus without the topoisomerase I inhibitor or a prodrug thereof.

[0431] Embodiment 64. The method of any one of Embodiments 34 to 63, wherein the increased infectivity and oncolytic activity of the oncolytic adenovirus occurs without substantially increasing the replication of the oncolytic adenovirus.

[0432] Embodiment 65. The method according to any one of embodiments 34 to 64, wherein sequential treatment of first administering the oncolytic adenovirus followed by systemic administration of topotecan enhances or increases the cell infection and / or anti-cancer efficacy of the oncolytic adenovirus.

[0433] sequence

[0434] SEQ ID NO: 1

[0435] The complete PH20 cDNA sequence including the carboxyl-terminal domain. (From ATG to the stop codon, both inclusive, with nt 1471-1527 underlined and in bold)

[0436]

[0437]

[0438] SEQ ID NO: 2

[0439] Modified endogenous promoter of E1a in VCN-01 + sequence encoding the E1a-δ24 region (including four binding sites for E2F-1, a binding site for Sp1, and the coding region of E1a-δ24)

[0440]

[0441]

[0442] SEQ ID NO: 3

[0443] Complete sequence of VCN-01

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463] SEQ ID NO: 4

[0464] Amino acid sequence of a modified version of the fiber of an adenovirus serotype, in which the RGDK modification is introduced

[0465]

[0466] SEQ ID NO: 5

[0467] RGD-C peptide

[0468]

[0469] SEQ ID NO: 6

[0470] Binding domain

[0471]

[0472] SEQ ID NO: 7

[0473] Binding domain

[0474]

[0475] SEQ ID NO: 8

[0476] Translation of SEQ ID NO: 1 (PH20 with carboxyl terminal domain present)

[0477]

[0478] SEQ ID NO: 9

[0479] cDNA encoding PH20 with a carboxyl-terminal domain deletion

[0480]

[0481] SEQ ID NO: 10

[0482] Translation of SEQ ID NO: 9 (PH20 with a carboxyl terminal domain deletion)

[0483]

[0484]

Claims

1. A method for treating retinoblastoma, Ewing's sarcoma, or neuroblastoma in a patient in need thereof, comprising co-administering to the patient (i) an oncolytic adenovirus comprising a replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug thereof.

2. The method of claim 1, wherein the oncolytic adenovirus is administered by intraocular, intravenous, intraarterial, intrathecal, intravitreal, or intratumoral injection.

3. The method of claim 1 or 2, wherein the topoisomerase I inhibitor or a prodrug thereof is administered intravenously, intraarterially, intravitreally, intrathecally, intracerebroventricularly or orally.

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

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

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

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

9. The method according to any one of claims 1 to 8, wherein the subject is administered a single intrathecal injection of the oncolytic adenovirus concurrently with systemic or intrathecal circulation of the topoisomerase I inhibitor or a prodrug thereof.

10. The method of any one of claims 1 to 8, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are administered systemically to the subject.

11. The method of any one of claims 1 to 10, wherein the oncolytic adenovirus and / or topoisomerase I inhibitor or a prodrug thereof is co-administered with one or more topical and / or systemic corticosteroids, optionally wherein the one or more topical and / or systemic corticosteroids are selected from hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, aldosterone, budesonide, fluticasone, flunisolide, ciclesonide, mometasone, beclomethasone, triamcinolone and tixocortolone.

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

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

14. The method of claim 13, wherein the sequence encoding the hyaluronidase is SEQ ID NO: 9 or a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto.

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

16. The method of any one of claims 1 to 15, wherein the treatment is treatment of retinoblastoma and oncolytic adenovirus replication occurs in tumor cells with an aberrant Rb-E2F pathway but not in healthy, non-tumor or normal cells.

17. The method of claim 16, wherein the oncolytic adenovirus is engineered to replicate in tumor cells, but not in healthy, non-tumor or normal cells, by deleting the Rb binding domain or Δ24 deletion in the sequence encoding the E1a protein and inserting four binding sites for E2F-1 and one binding site for Sp1 into the endogenous promoter of E1a to control the expression of E1a.

18. The method according to any one of claims 1 to 17, wherein the capsid of the oncolytic adenovirus is modified so that the heparan sulfate binding domain present in the adenoviral fiber 91 KKTK 94 (SEQ ID NO: 8) optionally by a domain 91 RGDK 94 (SEQ ID NO: 9) was replaced.

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

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

21. The method of claim 20, wherein the human patient is a pediatric human patient.

22. The method of any one of claims 1 to 21, wherein the retinoblastoma, Ewing's sarcoma, or neuroblastoma is a retinoblastoma, Ewing's sarcoma, or neuroblastoma that is resistant to treatment with conventional chemotherapy and / or radiotherapy.

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

24. The method of any one of claims 1 to 23, wherein the method results in reduced or maintained tumor size and / or prevention or reduction of retinoblastoma-associated metastases, secondary malignancies, or trilateral retinoblastomas compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

25. The method of any one of claims 1 to 23, wherein the method results in reduced or maintained tumor size and / or prevention or reduction of metastasis, secondary malignancies, or recurrent disease associated with Ewing's sarcoma compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

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

27. The method of any one of claims 1 to 26, wherein administration of the oncolytic adenovirus reduces the apoptotic effect of topotecan compared to monotherapy.

28. The method of any one of claims 1 to 27, wherein administration of topotecan after administration of the oncolytic adenovirus results in S phase cell cycle arrest.

29. The method of claim 28, wherein the S phase cell cycle arrest results in increased infectivity of the oncolytic adenovirus compared to treatment with the oncolytic adenovirus without the topoisomerase I inhibitor or a prodrug thereof.

30. The method of any one of claims 1 to 29, wherein administration of topotecan following administration of the oncolytic adenovirus results in increased expression of E2F-1, p21, and / or cyclin E1 compared to treatment with the oncolytic adenovirus without the topoisomerase I inhibitor or a prodrug thereof.

31. The method of claim 30, wherein the increased E2F-1 expression results in increased oncolytic activity of the oncolytic adenovirus compared to treatment with the oncolytic adenovirus without the topoisomerase I inhibitor or a prodrug thereof.

32. The method of any one of claims 1 to 31, wherein the increased infectivity and oncolytic activity of the oncolytic adenovirus occurs without substantially increasing replication of the oncolytic adenovirus.

33. The method according to any one of claims 1 to 32, wherein sequential treatment of first administering the oncolytic adenovirus followed by systemic administration of topotecan enhances or increases the cell infection and / or anti-cancer efficacy of the oncolytic adenovirus.

34. A method for treating retinoblastoma, Ewing's sarcoma or neuroblastoma in a patient in need thereof, the method comprising administering to the patient (i) an oncolytic adenovirus comprising a replication machinery specific for tumor cells and, optionally, a polynucleotide sequence encoding a hyaluronidase inserted into its genome, and (ii) a topoisomerase I inhibitor or a prodrug thereof.

35. The method of claim 34, wherein the oncolytic adenovirus is administered by intraocular, intravenous, intraarterial, intrathecal, intravitreal, or intratumoral injection.

36. The method of claim 34 or 35, wherein the topoisomerase I inhibitor or a prodrug thereof is administered intravenously, intraarterially, intravitreally, intrathecally, intracerebroventricularly, or orally.

37. The method of any one of claims 34 to 36, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are formulated into separate compositions.

38. The method of any one of claims 34 to 37, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are formulated into a single composition.

39. The method of any one of claims 34 to 38, wherein the oncolytic adenovirus is administered first and the topoisomerase I inhibitor or a prodrug thereof is administered within about 12 weeks after the administration of the oncolytic adenovirus.

40. The method of claim 39, 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 after the administration of the oncolytic adenovirus.

41. The method of any one of claims 34 to 40, wherein the subject is administered a single intrathecal injection of the oncolytic adenovirus concurrently with systemic or intrathecal circulation of the topoisomerase I inhibitor or a prodrug thereof.

42. The method of any one of claims 34 to 40, wherein the oncolytic adenovirus and the topoisomerase I inhibitor or a prodrug thereof are administered systemically to the subject.

43. The method of any one of claims 34 to 42, wherein the oncolytic adenovirus and / or topoisomerase I inhibitor or a prodrug thereof is co-administered with one or more topical and / or systemic corticosteroids, optionally wherein the one or more topical and / or systemic corticosteroids are selected from hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, aldosterone, budesonide, fluticasone, flunisolide, ciclesonide, mometasone, beclomethasone, triamcinolone and tixocortolone.

44. The method of any one of claims 34 to 43, wherein the topoisomerase I inhibitor is topotecan or SN-38 and / or the prodrug of the topoisomerase I inhibitor is irinotecan.

45. The method of any one of claims 34 to 44, wherein the hyaluronidase is human hyaluronidase PH20.

46. ​​The method of claim 45, wherein the sequence encoding the hyaluronidase is SEQ ID NO: 9 or a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity thereto.

47. The method of any one of claims 34 to 46, wherein the oncolytic adenovirus is produced by human adenovirus serotype 5.

48. The method of any one of claims 34 to 47, wherein oncolytic adenovirus replication occurs in tumor cells with an aberrant Rb-E2F pathway but not in healthy, non-tumor or normal cells.

49. The method of claim 48, wherein the oncolytic adenovirus is engineered to replicate in tumor cells but not in healthy, non-tumor or normal cells by deleting Δ24 in the sequence encoding the E1a protein and inserting four binding sites for E2F-1 and one binding site for Sp1 into the endogenous promoter of E1a to control the expression of E1a.

50. The method of any one of claims 34 to 49, wherein the capsid of the oncolytic adenovirus is modified such that the heparan sulfate binding domain present in the adenoviral fiber 91 KKTK 94 (SEQ ID NO: 8) optionally by a domain 91 RGDK 94 (SEQ ID NO: 9) was replaced.

51. The method of any one of claims 34 to 50, wherein the oncolytic adenovirus is VCN-01 (SEQ ID NO: 3).

52. The method of any one of claims 34 to 51, wherein the patient is a human patient.

53. The method of claim 52, wherein the human patient is a pediatric human patient.

54. The method of any one of claims 34 to 53, wherein the retinoblastoma, Ewing's sarcoma, or neuroblastoma is a retinoblastoma, Ewing's sarcoma, or neuroblastoma that is resistant to treatment with conventional chemotherapy and / or radiotherapy.

55. The method of any one of claims 34 to 54, wherein the method improves and / or increases and / or enhances anti-tumor efficacy compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

56. The method of any one of claims 34 to 55, wherein the method results in reduced or maintained tumor size and / or prevention or reduction of retinoblastoma-associated metastases, secondary malignancies, or trilateral retinoblastomas compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

57. The method of any one of claims 34 to 55, wherein the method results in reduced or maintained tumor size and / or prevention or reduction of metastasis, secondary malignancies, or recurrent disease associated with Ewing's sarcoma, compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

58. The method of any one of claims 34 to 55, wherein the method results in reduced or maintained tumor size and / or prevention or reduction of metastasis, secondary malignancies, or recurrent disease associated with neuroblastoma compared to treatment with the topoisomerase I inhibitor or a prodrug thereof without the oncolytic adenovirus.

59. The method of any one of claims 34 to 58, wherein administration of the oncolytic adenovirus reduces the apoptotic effect of topotecan compared to monotherapy.

60. The method of any one of claims 34 to 59, wherein administration of topotecan after administration of the oncolytic adenovirus results in S phase cell cycle arrest.

61. The method of claim 60, wherein the S phase cell cycle arrest results in increased infectivity of the oncolytic adenovirus compared to treatment with the oncolytic adenovirus without the topoisomerase I inhibitor or a prodrug thereof.

62. The method of any one of claims 34 to 61, wherein administration of topotecan following administration of the oncolytic adenovirus results in increased expression of E2F-1, p21, and / or cyclin E1 compared to treatment with the oncolytic adenovirus without the topoisomerase I inhibitor or a prodrug thereof.

63. The method of claim 62, wherein the increased E2F-1 expression results in increased oncolytic activity of the oncolytic adenovirus compared to treatment with the oncolytic adenovirus without the topoisomerase I inhibitor or a prodrug thereof.

64. The method of any one of claims 34 to 63, wherein the increased infectivity and oncolytic activity of the oncolytic adenovirus occurs without substantially increasing replication of the oncolytic adenovirus.

65. The method of any one of claims 34 to 64, wherein sequential treatment of first administering the oncolytic adenovirus followed by systemic administration of topotecan enhances or increases the cell infection and / or anti-cancer efficacy of the oncolytic adenovirus.

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