Composition for treating glioma

Abstract

Provided are combinations of an oncolytic virus and temozolomide (TMZ) for the treatment of glioma, and a method of treating TMZ resistant glioma. Wherein the oncolytic virus expresses an interferon encoded by SEQ ID NO: 2, 3 or 4. Also provided are kits for the treatment of glioma comprising an oncolytic adenovirus and TMZ wherein the oncolytic adenovirus expresses a consensus interferon and the oncolytic adenovirus and TMZ are in the same or different containers.

Description

Cross Reference to Related Applications

[0001] This application claims priority to PCT application PCT / CN2023 / 122421 entitled Compositions for treating glioma, filed September 28, 2023, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of tumor therapy, in particular to the combination of oncolytic virus and temozolomide (TMZ) for treating glioma and methods of treating TMZ-resistant glioma. BACKGROUND

[0003] Glioblastoma (GBM) accounts for 55% of all primary malignant brain tumors, characterized by its highly aggressive nature, poor prognosis, and tendency to relapse. Newly diagnosed GBM patients have a median survival time of less than 20 months, with a 5-year survival rate of only 5%. The main treatment methods for GBM include surgical resection and postoperative chemotherapy-radiotherapy, while alternative methods include targeted therapy, immunotherapy, and tumor electric field therapy. Despite these efforts, the survival time of GBM patients remains unacceptably static, and the development of new drugs faces significant obstacles and high failure rates. In addition, there is a lack of proven interventions and standardized treatment for recurrent GBM. Therefore, the five-year survival rate of GBM has made little progress in the past three decades, emphasizing the urgent need for effective interventions.

[0004] Notably, the use of oncolytic viruses has shown significant efficacy in treating recurrent GBM. In particular, oncolytic virus Delytact (G47Δ), based on its impressive survival benefit observed in a phase II clinical trial, has obtained a conditional marketing approval in Japan. For recurrent glioblastoma, the median overall survival was 20.2 months after OV treatment.

[0005] OVs are naturally occurring or genetically modified viruses with the ability to replicate within tumor cells. Their anti-tumor efficacy mainly comes from their direct tumor-killing effect and their indirect anti-tumor immune effect. OVs can cause direct lysis of tumor cells by propagating within tumor cells. In addition, oncolytic virus therapy can indirectly trigger an anti-tumor immune response by recruiting immune cells to the infection site and exposing tumor antigens after tumor cell lysis, which can change the local tumor immune environment from "cold" to "hot". In clinical trials, this effect is considered to be the possible cause of long-term survival of recurrent glioblastoma multiforme (GBM) cases. However, the immune environment also plays a role in clearing OVs. Therefore, precisely regulating the tumor immune environment associated with OVs at each stage and promoting effective viral replication while inducing long-term anti-tumor immune effects have become an important direction for the next generation of OV therapy. SUMMARY

[0006] In the research, the inventors unexpectedly found that oncolytic adenovirus (such as YSCH-01 disclosed in CN111363726A) can be used for treating glioma, especially showing an unexpected therapeutic effect on TMZ-resistant glioma. In addition, the combination therapy of oncolytic adenovirus and TMZ shows an unexpected therapeutic effect on treating glioma. On this basis, the present application is completed.

[0007] The inventors developed a Cancer Targeting Gene-Viro-Therapy (CTGVT) strategy by constructing an oncolytic adenovirus expressing an interferon-like immune anticancer gene (L-IFN), named YSCH-01. Based on previous research, a new recombinant interferon called sIFN-I was optimized, which was confirmed to have better affinity to IFNAR1 and showed a prolonged half-life for anti-solid tumor in mice. Therefore, an interferon-like immune anticancer gene (L-IFN) carried by a dual-regulated replication-competent human adenovirus type 5 was developed, and YSCH-01 was constructed. The inserted L-IFN gene replicates within tumor cells together with the viral vector. Therefore, a large amount of L-IFN protein is expressed within tumor cells and secreted into the extracellular space. By direct injection, the L-IFN drug with OV is administered, and the CTGVT strategy of the present application takes advantage of the optimal time window for viral replication and expression of therapeutic genes (L-IFN). Comprehensive in vitro and in vivo studies have been conducted to verify the safety and efficacy of oncolytic adenovirus. These studies strongly support the modification strategy of the present application, which exhibits the potential for achieving effective oncolytic effect in tumor therapy.

[0008] In a first aspect, the present application provides a method for treating temozolomide (TMZ)-resistant glioma, comprising administering to a subject in need thereof a therapeutically effective amount of an oncolytic adenovirus expressing consensus interferon. The present application provides the oncolytic adenovirus expressing consensus interferon for use in treating TMZ-resistant glioma.

[0009] In some embodiments, the consensus interferon is encoded by SEQ ID NO: 2, 3 or 4. In other embodiments, the oncolytic adenovirus is deposited at China Center for Type Culture Collection with the accession number of CCTCC NO: V201871, CCTCC NO: V201957 or CCTCC NO: V201958. In some embodiments, the oncolytic adenovirus is administered by intratumoral injection, intravenous injection, intraperitoneal injection or perfusion, preferably by intratumoral injection.

[0010] In a second aspect, the present application provides a combination therapy method for treating glioma, comprising administering to a subject in need thereof a therapeutically effective amount of an oncolytic adenovirus expressing consensus interferon and TMZ.

[0011] In some embodiments, the consensus interferon is encoded by SEQ ID NO: 2, 3 or 4. In other embodiments, the oncolytic adenovirus is deposited at China Center for Type Culture Collection with the accession number of CCTCC NO: V201871, CCTCC NO: V201957 or CCTCC NO: V201958.

[0012] In some embodiments, the oncolytic adenovirus is administered by intratumoral injection, intravenous injection, intraperitoneal injection or perfusion, preferably by intratumoral injection; and TMZ is administered by oral route. In other embodiments, the oncolytic adenovirus and TMZ are administered in the same treatment cycle or in different treatment cycles.

[0013] In a third aspect, the present application provides a combination of an oncolytic adenovirus and TMZ for use in treating glioma, wherein the oncolytic adenovirus expresses consensus interferon.

[0014] In some embodiments, the consensus interferon is encoded by SEQ ID NO: 2, 3 or 4. In other embodiments, the oncolytic adenovirus is deposited at China Center for Type Culture Collection with the accession number of CCTCC NO: V201871, CCTCC NO: V201957 or CCTCC NO: V201958.

[0015] In some embodiments, the oncolytic adenovirus is administered by intratumoral injection, intravenous injection, intraperitoneal injection or perfusion, preferably by intratumoral injection; and TMZ is administered by oral route. In other embodiments, the oncolytic adenovirus and TMZ are administered in the same treatment cycle or in different treatment cycles.

[0016] In a fourth aspect, the present application provides a kit for the treatment of glioma comprising an oncolytic adenovirus and TMZ, wherein the oncolytic adenovirus expresses a consensus interferon, the oncolytic adenovirus and TMZ are in different containers. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 : Modified oncolytic adenoviruses carrying recombinant type I interferon (L-IFN) specifically target glioma cells. (A): Schematic representation of the genetic modifications of the viruses. On top OncoMul-V2, on the bottom YSCH-01. (B): Four glioma cell lines, one GBM patient-derived primary cell and the HFF-1 cell line were incubated with the viruses for 96 hours, respectively, and cell viability assays were performed to evaluate IC 50 (C): Representative images of 3D cultures of patient-derived glioma tumor tissues administered with modified oncolytic adenoviruses carrying the GFP gene. (D): Concentration of L-IFN in the supernatant of GBM patient-derived primary cells 72 hours after YSCH-01 / replication-deficient YSCH-01 / OncoMul-V2 mock treatment.

[0018] Figure 2: Syrian hamster model validates the safety of intracranial injection of YSCH-01. (A) Syrian hamsters allow adenovirus multiplicity of infection. YSCH-01 was injected into the cerebral hemisphere of healthy hamsters. (B) Body weight of male (4) and female (4) hamsters after virus injection. (C-E) YSCH-01 was injected in batches into a group of healthy hamsters and perfused hamsters (6) to detect L-IFN expression and YSCH-01 copy number in the brain and peripheral organs. (F) Representative HE staining images of organ sections after YSCH-01 treatment.

[0019] Figure 3: Inhibition of glioma subcutaneous xenograft growth by intratumoral injection of YSCH-01. (A) Flowchart of efficacy test experiment of YSCH-01 in subcutaneous LN-229 xenograft immunodeficient mouse model. (B) LN-229 tumor volume-time graph of Balb / c-nude mice (5) after drug treatment (Vehicle: OncoMul-V2, YSCH-01). (C) Representative HE staining images of LN-229 xenograft tumor sections (injection site) 13 days after drug treatment. Scale bar: top: 400 pm; bottom: 100 pm. Tumor volume = (long axis x short axis) / 2. (D) Flowchart of efficacy test experiment of YSCH-01 in subcutaneous U-118MG xenograft immunodeficient mouse model. (E) U-118MG tumor volume-time graph of Balb / c-nude mice after drug treatment (Vehicle, OncoMul-V2, YSCH-01). (F) Representative HE staining images of U-118MG xenograft tumor sections (injection site) 13 days after drug treatment. Scale bar: top: 400 pm; bottom: 100 pm. Tumor volume = (long axis x short axis) / 2. 2

[0020] Figure 4: Oncolytic effect of YSCH-01 further validated by intracranial glioma xenograft and patient-derived tumor xenograft. (A) Flowchart of testing YSCH-01 efficacy in intracranial U-87MG xenograft immunodeficient mouse model. (B): Change in tumor fluorescence intensity after drug administration (Vehicle: YSCH-01, YSCH-01 injected three times, 6 per group). (C) Survival curve after drug administration (Vehicle, YSCH-01, YSCH-01 injected three times, 6 per group) (Vehicle-YSCH-01: P = 0.0029, Vehicle-YSCH-01 QW3: P = 0.0014, YSCH-01-YSCH-01 QW3: P = 0.2693), n.s. (not significant), **P < 0.01), Log-rank (Mantel-Cox) test. (D) Representative fluorescence imaging of U-87MG tumors. (E) Volume-time graph of U-87MG tumors in subcutaneous xenograft immunodeficient mouse model after drug treatment (Vehicle, OncoMul-V2, YSCH-01; 5 per group). (F) Flowchart of establishing subcutaneous patient-derived tumor xenograft immunodeficient mouse model. (G) Volume-time graph of PDX tumors of Balb / c-nude mice after drug treatment (Vehicle, TMZ, YSCH-01, 10 per group). (H) Long-term efficacy of YSCH-01 with a single dose of YSCH-01 (5 per group).

[0021] ​Figure 5: Investigation of the anti-tumor mechanism of YSCH-01. (A) Apoptosis of LN-18 tumor cells was detected using flow cytometry after treatment. (B) Comparison of the PI proportion in Annexin V-FITC positive LN-18 cells with 10 MOI virus treatment. (C) Apoptosis of LN-229 tumor cells was detected using flow cytometry after treatment. (D) Comparison of the PI proportion in Annexin V-FITC positive LN-229 cells with 10 MOI virus treatment. (E) Western blotting detection of markers related to cell apoptosis. (F) Representative immunohistochemical staining images of CD31, Ki67 and CD45 in U-118MG xenograft tumor sections 13 days after drug treatment. Scale bar: 100 pm. (G) Representative TUNEL immunofluorescence staining images of U-118MG xenograft tumor sections 13 days after drug treatment. Scale bar: 1 mm. n.s. (not significant), *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001, one-way ANOVA test followed by Tukey’s multiple comparison test. DETAILED DESCRIPTION

[0022] The terms used in the present application have the same meanings as the terms in the prior art. In order to clearly explain the meanings of the terms used, the specific meanings of some terms in the present application are given below. When the definition herein conflicts with the conventional meaning of the term, the definition herein shall prevail.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All publications mentioned herein are incorporated by reference to describe and disclose the cells, vectors, and methods reported in the publications that might be used with the present disclosure. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such publication by virtue of prior application.

[0024] The term "oncolytic virus" refers to a virus that is capable of selectively replicating in cancer or hyperproliferative cells and slowing their growth or inducing their death, while having no or little effect on normal cells. Exemplary oncolytic viruses include vesicular stomatitis virus (VSV), newcastle disease virus (NDV), herpes simplex virus (HSV), reovirus, measles virus, retrovirus, influenza virus, Sindbis virus, vaccinia virus, and adenovirus.

[0025] The term "interferon" refers to a family of secreted proteins produced by various eukaryotic cells after exposure to various environmental stimuli, including viral infection or exposure to mitogens. In addition to having antiviral properties, interferons have been shown to affect a variety of cellular functions. There are primarily three types of interferons: IFN-a, IFN-β, and IFN-γ. Interferons were initially classified according to their cellular origin (leukocyte, fibroblast, or T cell). Leukocyte-derived interferon is now referred to as IFN-a, fibroblast-derived interferon as IFN-β, and T cell-derived interferon as IFN-γ.

[0026] Consensus interferon (CIFN) is an artificially engineered interferon that mirrors most human type I interferons and shows higher biological activity and antiviral capacity. The present inventors have disclosed an L-IFN having an optimized interferon-like immunocancer gene coding sequence in CN111363726A and WO2020135390 Al, the entire contents of which are incorporated herein by reference. In some embodiments of the present application, the L-IFN has an amino acid sequence set forth in SEQ ID NO: 1.

[0027] MCDLPQTHSLGNRRALILLAQMRRISPFSCLKDRHDFGFPQEEFD GNQFQKAQAISVLHEMIQQTFNLFSTKDSSAAWDESLLEKFYTELYQQ LNDLEACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSTNLQERLRRKE (SEQ ID NO: 1)

[0028] In some embodiments, the L-IFN is encoded by SEQ ID NO: 2, 3, and 4, preferably by SEQ ID NO: 2.

[0029] ATGGCCCTGTCCTTCAGCCTGCTGATGGCCGTGCTGGTGCTGAG CTACAAGTCCATCTGCTCCCTGGGCATGTGTGATCTGCCTCAGACACACTCCCTGGGCAATAGAAGGGCCCTGATCCTGCTGGCCCAGATGAGAAGGATCAGCCCCTTCTCCTGCCTGAAGGATAGACACGATTTTGGCTTCCCTCAGGAGGAGTTCGACGGCAATCAGTTTCAGAAGGCCCAGGCCATCTCCGTGCTGCACGAGATGATCCAGCAGACCTTTAACCTGTTCTCCACAAAGGACTCCAGCGCCGCCTGGGACGAGTCCCTGCTGGAGAAGTTTTACACAGAGCTGTACCAGCAGCTGAACGATCTGGAGGCCTGCGTGATCCAGGAGGTGGGCGTGGAGGAGACCCCCCTGATGAATGTGGATTCCATCCTGGCCGTGAAGAAGTACTTTCAGAGAATCACCCTGTACCTGACCGAGAAGAAGTACAGCCCTTGTGCCTGGGAGGTGGTGAGAGCCGAGATCATGAGATCCTTTTCCCTGAGCACAAACCTGCAGGAGAGGCTGAGAAGGAAGGAGTGA (SEQ ID NO: 2)

[0030] ATGGCCCTGTCCTTCTCCCTGCTGATGGCCGTGCTGGTGCTGAG CTACAAGTCCATCTGCTCCCTGGGCATGTGCGACCTGCCTCAGACACACTCCCTGGGCAATAGGAGAGCCCTGATCCTGCTGGCCCAGATGAGGAGGATCTCCCCTTTTAGCTGCCTGAAGGATAGACACGATTTCGGCT TCCCTCAGGAGGAGTTCGATGGCAATCAGTTCCAGAAGGCCCAGGCCATCAGCGTGCTGCACGAGATGATCCAGCAGACCTTCAATCTGTTTAGCACCAAGGACTCCAGCGCCGCCTGGGACGAGTCCCTGCTGGAGAAGTTCTACACCGAGCTGTACCAGCAGCTGAACGACCTGGAGGCCTGCGTGATCCAGGAGGTGGGCGTGGAGGAGACCCCTCTGATGAATGTGGATAGCATCCTGGCCGTGAAGAAGTACTTTCAGAGAATCACACTGTACCTGACAGAGAAGAAGTACAGCCCCTGCGCCTGGGAGGTGGTGAGGGCTGAGATCATGAGGAGCTTTTCCCTGTCCACAAACCTGCAGGAGAGGCTGAGAAGGAAGGAGTGA (SEQ ID NO:3)

[0031] ATGGCCCTGTCCTTTTCTTTACTGATGGCCGTGCTGGTGCTCAG CTACAAATCCATCTGTTCTCTGGGCATGTGCGACCTGCCGCAGACCCACTCCCTGGGTAACCGTCGTGCTCTGATCCTGCTGGCTCAGATGCGTCGTATCTCCCCGTTCTCCTGCCTGAAAGACCGTCACGACTTCGGTTTCCCGCAGGAAGAATTCGACGGTAACCAGTTCCAGAAAGCTCAGGCTATCTCCGTTCTGCACGAAATGATCCAGCAGACCTTCAACCTGTTCTCCACCAAAGACTCCTCCGCTGCTTGGGACGAATCCCTGCTGGAAAAATTCTACACCGAACTGTACCAGCAGCTGAACGACCTGGAAGCTTGCGTTATCCAGGAAGTTGGTGTTGAAGAAACCCCGCTGATGAACGTTGACTCCATCCTGGCTGTTAAAAAATACTTCCAGCGTATCACCCTGTACCTGACCGAAAAAAAATACTCCCCGTGCGCTTGGGAAGTTGTTCGTGCTGAAATCATGCGTTCCTTCTCCCTGTCCACCAACCTGCAGGAACGTCTGCGTCGTAAAGAATAA (SEQ ID NO: 4)

[0032] Temozolomide (TMZ) is an anticancer drug used to treat brain tumors, such as glioblastoma and anaplastic astrocytoma.

[0033] Temozolomide (TMZ) resistant glioma refers to glioma that cannot be effectively treated by TMZ. Surgical resection, combined radiotherapy and TMZ chemotherapy are the standard treatments for glioma. However, once the patient becomes resistant to TMZ, they are essentially out of drug options. It is reported that the prognosis of patients with glioblastoma multiforme (GBM) treated with standard therapy is still poor, with a recurrence rate of up to 90% and a 5-year survival rate of less than 10%. In clinical practice, there are two types of TMZ-resistant glioma patients: 1) primary glioma patients who respond poorly to TMZ; 2) patients whose tumors recur after TMZ treatment. The former is due to different tumor molecular subtypes. The latter means that 80% of primary malignant gliomas will have tumor recurrence in situ after surgery, radiotherapy and chemotherapy. The recurrent tumor cells acquire resistance to TMZ through functional mutation mismatch repair or base excision repair. Therefore, the vast majority of patients with recurrent GBM do not benefit significantly from TMZ, and there is no approved and accepted alternative drug treatment.

[0034] Gliomas are the most common type of tumor in the central nervous system (i.e., the brain and spinal cord). They originate from glial cells that grow uncontrollably. Gliomas can affect all ages, but they are most common in adults. About 33% of brain tumors are gliomas. Based on the 2021 World Health Organization (WHO) classification, the three main types of gliomas in adults are astrocytoma, oligodendroglioma, and glioblastoma. Glioblastoma, formerly known as glioblastoma multiforme (GBM), is the most aggressive, is the most common type of cancer originating in the brain, and has a very poor survival prognosis.

[0035] The term "pharmaceutical composition" as used herein means a combination of at least one drug and optionally a pharmaceutically acceptable carrier or excipient brought together for a particular purpose. In certain embodiments, the pharmaceutical composition comprises combinations that are separated in time and / or space as long as they can act together for the purposes of the present disclosure. For example, the components of a pharmaceutical composition can be administered to a subject as a single entity or separately. When the components of a pharmaceutical composition are administered separately to a subject, the components can be administered simultaneously or sequentially. Preferably, the pharmaceutically acceptable carrier is water, aqueous buffer solution, isotonic salt solution (e.g. PBS (phosphate buffered saline)), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid or polyalkylene glycol (e.g. polypropylene glycol, triglycerides, etc.). The type of pharmaceutically acceptable carrier depends in particular on whether the composition according to the present disclosure is formulated for oral, nasal, intratumoral, perfusion, intradermal, subcutaneous, intramuscular or intravenous administration. The composition according to the present disclosure can contain lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts to affect osmotic pressure, buffers, coloring substances, flavoring substances and / or aromatic substances, etc. as additives.

[0036] "Administering" means providing a substance, e.g. a pharmaceutical composition, to a subject in a pharmaceutically acceptable manner.

[0037] The dose of a pharmaceutical composition provided to a subject means the amount sufficient to show its benefit to the subject to whom it is administered, which can also be referred to herein as "pharmaceutically effective amount" or "effective amount". The actual amount administered, as well as the speed and time-period of administration, depend on the condition and severity of the subject being treated. The treatment prescription, e.g. dosage determination, etc., is ultimately the responsibility of the general practitioner and other physicians, and depends on their judgment, which is often made considering the disease to be treated, the individual condition of the patient, the delivery site, the method of administration and other factors known to the physician.

[0038] The term "subject" as used herein means an animal, including a warm-blooded mammal (e.g. humans and primates), a bird, a domesticated livestock or farm animal (e.g. cats, dogs, sheep, goats, cattle, horses and pigs), a laboratory animal (e.g. mice, rats and guinea pigs), a fish, a reptile, a zoo animal and a wild animal, etc.

[0039] It should also be noted that if a specific numerical value is mentioned herein, at least this value is included, unless the context clearly indicates otherwise. When a numerical value represents an approximation, it is understood that this specific numerical value forms another embodiment. As used herein, "about X" (where X is a number) means ±10% (inclusive) of the listed numerical value. If present, all ranges are included and combinable.

[0040] Such terms as "comprising," "including," and "containing" are not limiting. Also, the use of "or" means "and / or" unless stated otherwise.

[0041] In some embodiments, the oncolytic virus is the oncolytic virus having accession number CCTCC NO:V201957. The preservation information is as follows. Preservation agency: China Center for Type Culture Collection; address: Wuhan, China. Preservation date: August 27, 2019. Name / reference: Recombinant human adenovirus type 5 rAd-IFN-1-SP-E1A(Δ24bp)-E1B. Accession number: CCTCC NO:V201957.

[0042] In other embodiments, the oncolytic virus is the oncolytic virus having accession number CCTCC NO:V201958. The preservation information is as follows. Preservation agency: China Center for Type Culture Collection; address: Wuhan, China. Preservation date: August 27, 2019. Name / reference: Recombinant human adenovirus type 5 rAd-IFN-2-SP-E1A(Δ24bp)-E1B. Accession number: CCTCC NO:V201958.

[0043] In other embodiments, the oncolytic virus is the oncolytic virus having accession number CCTCC NO:V201871. The preservation information is as follows. Preservation agency: China Center for Type Culture Collection; address: Wuhan, China. Preservation date: December 12, 2018. Name / reference: Recombinant human adenovirus type 5 rAd-IFN-3-SP-E1A(Δ24bp)-E1B. Accession number: CCTCC NO:V201871.

[0044] Any component, element, attribute, or step disclosed in one embodiment of a method and product can be applied to any other method and product disclosed herein, unless otherwise specified.

[0045] The entire contents of each patent, patent application, cited publication or description related to the present disclosure in this document are incorporated herein by reference.

[0046] The present disclosure is further illustrated in the following examples. It is to be understood that these provided examples are for illustration only and do not purport to be a limitation on the scope of the present disclosure. Those skilled in the art can, based on the teachings and guidance presented in the foregoing disclosure and the examples, make various changes in form and details without departing from the spirit and scope of the present disclosure, and adapt the present disclosure for various uses and circumstances. Examples Materials and Methods

[0047] Ethical Statement

[0048] All human samples were collected under the approval of the Chinese Registered Clinical Trial Ethics Review Committee (ChiECRCT20190201). All patient information and tumor samples were obtained with the informed consent of patients before surgery

[0049] Cell culture

[0050] Glioma cells (including U-138MG, LN-18, T-98G, LN-229, U-118MG, U-87MG, HFF-1 and U-87MG-Luc) were purchased from the National Collection of Authenticated Cell Cultures in Shanghai, China, which also provided STR profile information. Except for U-138MG, which required an additional 1% of non-essential amino acids, all glioma cells were maintained in DMEM containing 10% FBS. In addition, the HFF-1 cell line was maintained in DMEM medium supplemented with 15% FBS. The culture conditions for all cells were 37°C and 5% CO2.

[0051] Construction of oncolytic adenovirus

[0052] The oncolytic adenovirus used in the study was developed by Shanghai Yuansong Biotechnology Co., Ltd., which is currently being used in clinical trials (NCT05180851) for the treatment of various solid tumors, including breast cancer, lung cancer and melanoma. OncoMul-V2 uses the human adenovirus type 5 (Ad5) genome as the backbone16. Its E1A gene deletes a 24-bp sequence17, and its wild-type promoter is replaced with a survivin promoter. Based on the structure of OncoMul-V2, YSCH-01 was constructed by inserting a complete expression cassette of L-IFN gene (modified type I interferon gene) between the packaging signal region of Ad5 and the survivin promoter. The gene sequence of L-IFN was synthesized by a multiplex PCR technique (General Biosystems, Anhui, China), and its expression was controlled by a human cytomegalovirus (HCMV) promoter.

[0053] Detection of L-IFN protein in supernatant

[0054] Briefly, primary tumor cells derived from GBM patients were plated at 1 x 10 5Cells were plated at a density of 4 x 105cells per well in 96-well plates. After overnight incubation, cells were infected with 10 μl of oAd at different MOI. Four days after viral infection, cell viability was determined by CCK8 (Cell Counting Kit-8, #40203ES80, Yeasen Biotechnology, Shanghai, China) assay and IC50values were calculated with GraphPad Prism (version 8.0). All experiments were performed at least three times.

[0055] In vitro cytotoxicity assay

[0056] Cells were plated at a density of 4 x 105cells per well in 96-well plates. After overnight incubation, cells were infected with 10 μl of oAd at different MOI. Four days after viral infection, cell viability was determined by CCK8 (Cell Counting Kit-8, #40203ES80, Yeasen Biotechnology, Shanghai, China) assay and IC50values were calculated with GraphPad Prism (version 8.0). All experiments were performed at least three times. 3 Cells were plated at a density of 4 x 105cells per well in 96-well plates. After overnight incubation, cells were infected with 10 μl of oAd at different MOI. Four days after viral infection, cell viability was determined by CCK8 (Cell Counting Kit-8, #40203ES80, Yeasen Biotechnology, Shanghai, China) assay and IC50values were calculated with GraphPad Prism (version 8.0). All experiments were performed at least three times. 3 Cells were plated at a density of 4 x 105cells per well in 96-well plates. After overnight incubation, cells were infected with 10 μl of oAd at different MOI. Four days after viral infection, cell viability was determined by CCK8 (Cell Counting Kit-8, #40203ES80, Yeasen Biotechnology, Shanghai, China) assay and IC50values were calculated with GraphPad Prism (version 8.0). All experiments were performed at least three times. 50 Values. All experiments were performed at least three times.

[0057] In vivo studies to assess OV anti-tumor efficacy

[0058] Four- to five-week-old female Balb / c nude mice were purchased from Shanghai Lingchang Biotechnology Co., Ltd. (Shanghai, China). Mice were housed in a specific pathogen free (SPF) level animal room with an ambient temperature controlled at 20-26 °C and a humidity level controlled at 40-70%, with a 12-hour light, 12-hour dark light cycle. All animal experiments were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.

[0059] Briefly, as with cell-derived xenograft (CDX) tumors, 2 x 105 6 aliquots of glioma cells were injected subcutaneously (s.c.) into both flanks of mice. When the tumor volume reached approximately 120 mm 3 , animals were divided and the tumors on one side were treated. Tumor growth was measured every 3 days. When the mean tumor volume of the vehicle group reached approximately 2000 mm 3 , the experiment was terminated.

[0060] For intracranial glioma model, subcutaneous U-87MG-Luc tumors were taken out and cut into tumor pieces with 0.5-1 mm in diameter. The tumor pieces were transplanted intracranially (i.c) into the right lobe of nude mice. After 7 days, the mice received treatment and bioluminescence imaging (BLI) (Viewworks, KOREA), once every 7 days, for a total of 4 times.

[0061] For patient-derived tumor xenograft (PDX) model, human patient tissue samples were collected under the approval of the Chinese registered clinical trial ethics review committee (ChiECRCT20190201). Recurrent glioblastoma tumor pieces that had been adapted to the fifth generation in vivo in immunodeficient mice were transplanted into the right flank of nude mice. When the tumor volume reached about 150 mm 3 , the nude mice received treatment.

[0062] Detection of apoptosis

[0063] Glioma cells were plated in 12-well plates at a density of 2 x 10 5 cells per well. After overnight incubation, cells were treated with vehicle or infected with OV at 10 MOI (LN-18 and LN-229) in triplicate. After 48 hours, cells were harvested for apoptosis detection (#40302ES60, Yeasen). FACS assay was then performed using a flow cytometer (Agilent Technologies, USA).

[0064] Western blot analysis of cell death-related proteins

[0065] Glioma cells were plated in 12-well plates at a density of 4 x 10 5Cells were plated in 6-well plates at a density of 1 x 105 glioma cells. After overnight incubation, cells were treated with vehicle or infected with OV at 10 MOI (LN-18 and LN-229). Briefly, after 48 hours, total 20 pg of protein mixture was separated equally and transferred to polyvinylidene fluoride (PVDF) membrane. Protein bands were visualized using chemiluminescence system (Tanon, Shanghai, China) combined with enhanced chemiluminescence (ECL) detection reagent (#36208ES60, Yeasen). Primary antibodies included: a-caspase 3 (#19677-1Ap, Proteintech, Wuhan, China), a-caspase 9 (#10380-1Ap, Proteintech), a-poly (ADP-ribose) polymerase (PARP) (#9542, Cell Signaling, Danvers, MA, USA), a-LC3B (#2775s, Cell Signaling) and a-GAPDH (#CY6717, Abways, Shanghai, China). Secondary antibody (#33101ES60, Yeasen) was from Shanghai Yeasen Biotech Co., Ltd. (Shanghai, China).

[0066] Staining of tumor tissue sections

[0067] In the subcutaneous CDX model, tumors were collected 13 days after the initial drug administration. Tissues were fixed and embedded in paraffin, and cut into 3 pm sections. For histological analysis, sections were deparaffinized and stained with hematoxylin and eosin (H&E). Other sections were subjected to immunohistochemical analysis. The primary antibodies used were a-Ki67 (#ab15580, abcam, Cambridge, MA, USA), a-CD45 (#ab208022, abcam) and a-CD31 (#ab182981, abcam). HRP polymer secondary antibody and DAB reagent were obtained from EnVision kit (#K5007, Dako, Denmark). Hematoxylin was used as a stain. In addition, TUNEL staining was also performed on additional sections using In Situ Cell Death Detection Kit (#11684795910, Roche, USA).

[0068] Toxicity and pharmacokinetic evaluation of intracranial injection of YSCH-01

[0069] Four- to five-week-old hamsters were purchased from North Veterinary Lihua Experimental Animal Technology Co., Ltd. and were housed in a SPF environment according to protocols approved by the Institutional Animal Care and Use Committee. Hamsters were acclimated for 1-2 weeks prior to experiments.

[0070] To evaluate the toxicity of YSCH-01, Syrian hamsters were randomly assigned into any of the three groups. Each group included 4 male and 4 female hamsters. After anesthesia, intracranial injection of the drug was performed using a stereotactic device. Body weight was measured throughout the experiment. Brain tissues and major organs from drug-treated hamsters were collected. In addition, peripheral blood samples from hamsters were collected to analyze hemogram and liver function.

[0071] To quantify the copy number of YSCH-01 vector, DNA was extracted from samples using a tissue DNA extraction kit (#DE0596B, Emerther, China). Blood DNA was directly extracted from 200 μl of thawed sample using a blood genomic extraction kit (#DE17002, Emerther) and an automatic nucleic acid extractor. Real-time PCR was performed using Probe qPCR Mix (#RR392A, TAKARA, Japan) to determine the copy number of YSCH-01 vector in tissue or blood DNA.

[0072] To determine the content of L-IFN in the right brain, the tissues collected at different time points were dissolved in PBS (4 ml / g), homogenized and centrifuged at 12000 rpm for 10 minutes at low temperature. Then the supernatant was quantified using a human IFNa enzyme-linked immunosorbent assay kit.

[0073] Statistical analysis

[0074] All data are expressed as mean ± standard deviation (SD). GraphPad Prism (version 8.0) was used for graphing and statistical analysis. Certain specific analyses employed one-way analysis of variance (ANOVA) or log-rank (Mantel-Cox) test. P values less than 0.05 were considered statistically significant.

[0075] Details of the methods can be found in the Supplementary Materials. Example 1 Oncolytic adenovirus YSCH-01 targets glioma cells in vitro

[0076] The engineered oncolytic adenovirus carries a recombinant type I interferon that specifically targets glioma cells and is safe in a Syrian hamster model.

[0077] To enhance the targeting and efficacy of the oncolytic adenovirus, genetic engineering was performed in two aspects: modifying the specific starting conditions of the virus and incorporating a therapeutic gene. Initially, the oncolytic adenovirus (named OncoMul-V2) was engineered by integrating into the survivin promoter and deleting 24 base pairs from the viral E1A genomic region, Figure 1A). This double engineering significantly improved the virus' s targeting ability to glioma cells and tissues. The results showed that the engineered virus had an IC 50 significantly higher to human foreskin fibroblast-1 (HFF-1) compared to human glioma cell lines (U-87MG, U-118MG, LN-229 and LN18) and GBM patient-derived primary tumor cells, indicating that the engineered oncolytic adenovirus was safe to normal cells Figure 1B ). Moreover, using 3D culture on patient-derived glioma tissues, it was found that the engineered oncolytic adenovirus labeled with a green fluorescent protein (GFP) gene expressed itself in the tumor tissues, with the fluorescence intensity reaching a peak at day 14 Figure 1C ). Subsequently, the OncoMul-V2 was further equipped with a previously designed gene encoding a new recombinant type I interferon (L-IFN) (the virus obtained was named YSCH-01) Figure 1A ). This recombinant L-IFN exhibited a higher affinity to the IFNAR1 chain of the IFN1 receptor, resulting in stronger IFN1 signaling and expression of IFN-induced genes in human cells. As the virus infected and propagated, the L-IFN gene was transcribed, translated and released into the extracellular fluid of GBM patient-derived primary tumor cells Figure 1D ). The above triple engineering conferred the adenovirus enhanced targeting to glioma and improved oncolytic potency. Example 2 Safety evaluation of YSCH-01

[0078] Given that adenovirus replicates 1000-fold less efficiently in mouse cells than in human cells, intracranial injection of YSCH-01 into the brain of Syrian hamsters (0.25 x 10 9 / 1 x 10 10 VP per hamster) was performed. Syrian hamsters are one of the only two small mammals that support efficient replication of adenovirus and were chosen to precisely evaluate the safety of the virus Figure 2A ). After virus injection, the body weight of the hamsters showed a transient decrease, which gradually returned to normal levels Figure 2B ). In addition, a time-course series of experiments was performed to detect the pharmacokinetics of the virus in the brain and major peripheral organs after injection Figure 2C ). The expression of L-IFN reached a peak at the first day after injection, and then gradually decreased. At day 7, the expression of L-IFN increased again due to virus replication. Subsequently, the expression level gradually decreased, with only a small amount of detectable samples present after day 14 Figure 2DSimilar trends were observed for YSCH-01 genome copy numbers in brain tissue, major peripheral organs, and blood ( Figure 2E ). Overall, the Syrian hamster model confirmed the viral replication capacity and efficacy of L-IFN gene expression after intracranial administration. Example 3 Tumor Killing Efficacy of YSCH-01

[0079] YSCH-01 has good tumor killing efficacy and distal tumor inhibition effects on glioma subcutaneous xenografts.

[0080] To validate the therapeutic efficacy of YSCH-01, we established subcutaneous xenograft models in nude mice using different glioma cell lines. For the LN-229 xenograft model, LN-229 cells were subcutaneously injected into the flanks of nude mice. Once the tumor reached approximately 100 mm 3 At 24 days (after 24 days), the mice were randomly divided into three groups and injected with different drugs (vehicle, OncoMul-V2 and YSCH-01) into the unilateral tumor at a dose of 1×10 10 VP / mouse. Tumor volume was measured every 3 days. Mice were euthanized 27 days after virus injection for further analysis ( Figure 3A The results showed that YSCH-01 significantly reduced the volume of both the injected and contralateral tumors. In contrast, treatment with OncoMul-V2 only slightly inhibited the growth of the injected tumor and had no effect on the contralateral tumor ( Figure 3B Histological examination of tumor sections stained with HE showed that after YSCH-01 treatment, the number of tumor cells was significantly reduced and the infiltration of immune cells in the tissue was increased ( Figure 3C ).

[0081] Similar experiments were performed using U-118MG cells ( Figure 3D ). However, in contrast to the LN-229 model, OncoMul-V2 demonstrated comparable tumor killing efficacy to YSCH-01 at the injection site. Similar to the LN-229 model, OncoMul-V2 failed to inhibit tumor growth at the contralateral site, while YSCH-01 consistently demonstrated superior efficacy ( Figure 3E The findings from the HE staining images were consistent with the tumor growth observed in the animal model. In the tumor located on the injected side, both OncoMul-V2 and YSCH-01 led to a decrease in tumor cells and an increase in immune infiltration. However, only YSCH-01 showed the ability to inhibit tumor cells ( Figure 3F ). Example 4 Tumor Killing Efficacy of YSCH-01

[0082] The oncolytic effect of YSCH-01 was further validated using intracranial glioma xenografts and patient-derived tumor xenografts.

[0083] To further confirm the efficacy of YSCH-01 in situ, luciferase-labeled U-87MG cells were subcutaneously injected into one cerebral hemisphere of nude mice. Eight days after tumor implantation, bioluminescence imaging was performed to monitor tumor growth. The mice were then divided into three groups and treated with vehicle, a single dose of YSCH-01, or three doses of YSCH-01 ( Figure 4A Based on the fluorescence signal intensity and survival time, the results showed that a single injection of YSCH-01 significantly inhibited the growth of intracranial tumors and prolonged the survival time of mice. However, three repeated injections of YSCH-01 did not show a statistically significant advantage over a single injection ( Figures 4B-4D Furthermore, the inhibitory effect of YSCH-01 on distal tumors was confirmed in a bilateral subcutaneous xenograft model ( Figure 4E ).

[0084] Furthermore, to investigate the oncolytic effect of YSCH-01 on heterogeneous tumor components, recurrent glioblastoma patient-derived tumor xenografts were established by transplanting cell suspensions of patient-derived tumor samples into immunodeficient mice ( Figure 4F ). Notably, YSCH-01 exhibited significantly better inhibitory effects on tumor growth compared to TMZ treatment ( Figure 4G ). In addition, to examine the long-term efficacy of YSCH-01, we added a new cohort of PDX animals, and all animals treated with a single dose of YSCH-01 survived for more than 136 days. These animals were then euthanized. Even after a single dose, YSCH-01 showed sustained tumor suppression ( Figure 4H ).

[0085] Results from orthotopic cell-derived xenografts and subcutaneous patient-derived xenografts further confirmed the therapeutic efficacy of YSCH-01. The incorporation of dual promoter modifications on the adenovirus and the addition of the L-IFN gene not only enhanced the killing effect in glioma models but also demonstrated potential advantages over TMZ treatment for recurrent glioblastoma. Example 5: YSCH-01 increases tumor cell apoptosis and inhibits tumor proliferation and angiogenesis

[0086] In this study, we successfully verified the safety and efficacy of YSCH-01 as a potential treatment for malignant glioma both in vitro and in vivo. In addition, molecular biochemical studies were conducted to elucidate the underlying mechanism by which YSCH-01 inhibits tumor cells. It was found that YSCH-01 induced a significant increase in apoptosis of LN-18 human glioma cells, as this was also demonstrated by the results of flow cytometry Figure 5A and 5B ). However, this effect was significantly less pronounced in LN-229 cells Figure 5C and D). The observed results were consistent with the results obtained from Western blot analysis. Specifically, YSCH-01 treatment resulted in upregulation of cleaved caspase 3 and PARP cleavage in LN-18 cells, indicating activation of the apoptosis pathway. In contrast, these effects were not evident in LN-229 cells Figure 5E . Furthermore, immunohistochemical staining images taken 13 days after YSCH-01 injection showed a significant decrease in Ki67-positive U-118MG tumor cells Figure 5F , indicating that tumor cell proliferation was inhibited. In addition, YSCH-01 treatment resulted in a decrease in CD31-positive cells, indicating that angiogenesis within the tumor tissue at the injection site was significantly reduced Figure 5F . There was an increase in CD45-positive tumor cells after oncolytic virus treatment Figure 5F . TUNEL testing demonstrated that tumor cell apoptosis was enhanced after YSCH-01 treatment Figure 5G .

[0087] Although the above describes the present disclosure, it is understood by those skilled in the art that various modifications and changes can be made to the present disclosure without departing from the spirit and scope of the present disclosure. The scope of the present disclosure is not limited to the above description, but is as shown in the claims.

Claims

1. A method of treating temozolomide (TMZ)-resistant glioma comprising administering to a subject in need thereof a therapeutically effective amount of an oncolytic adenovirus expressing consensus interferon.

2. The method of claim 1, wherein the consensus interferon is encoded by SEQ ID NO: 2, 3, or 4.

3. The method of claim 1 or 2, wherein the oncolytic adenovirus is administered by intratumoral injection, intravenous injection, intraperitoneal injection, or perfusion, preferably by intratumoral injection.

4. A combination therapy method of treating glioma comprising administering to a subject in need thereof a therapeutically effective amount of an oncolytic adenovirus expressing consensus interferon and TMZ.

5. The method of claim 4, wherein the consensus interferon is encoded by SEQ ID NO: 2, 3, or 4.

6. The method of claim 4 or 5, wherein the oncolytic adenovirus is administered by intratumoral injection, intravenous injection, intraperitoneal injection, or perfusion, preferably by intratumoral injection; and TMZ is administered by the oral route.

7. The method of any one of claims 4 to 6, wherein the oncolytic adenovirus and TMZ are administered in the same treatment cycle or in different treatment cycles.

8. A combination of an oncolytic adenovirus and TMZ for use in the treatment of glioma, wherein the oncolytic adenovirus expresses consensus interferon.

9. The combination of claim 8, wherein the consensus interferon is encoded by SEQ ID NO: 2, 3, or 4.

10. The combination of claim 8 or 9, wherein the oncolytic adenovirus is administered by intratumoral injection, intravenous injection, intraperitoneal injection, or perfusion, preferably by intratumoral injection; and TMZ is administered by the oral route.

11. The combination of any one of claims 8 to 10, wherein the oncolytic adenovirus and TMZ are administered in the same treatment cycle or in different treatment cycles.

12. A kit for use in the treatment of glioma comprising an oncolytic adenovirus and TMZ, wherein the oncolytic adenovirus expresses consensus interferon, the oncolytic adenovirus and TMZ are in different containers.

13. The kit of claim 12, wherein the consensus interferon is encoded by SEQ ID NO: 2, 3, or 4.

14. A composition for the manufacture of a medicament for the treatment of glioma, wherein the composition comprises an oncolytic adenovirus and TMZ, and the oncolytic adenovirus expresses consensus interferon.

15. The composition of claim 14, wherein the consensus interferon is encoded by SEQ ID NO: 2, 3, or 4.

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