Use of DR-18 and oncolytic vaccinia virus in the preparation of antitumor drugs

Abstract

The present disclosure belongs to the field of biological pharmaceuticals and relates to the use of DR-18 and oncolytic vaccinia virus in the preparation of an antitumor drug. The present disclosure is the first to discover that the combination of oncolytic vaccinia virus and DR-18 has a synergistic antitumor effect. The present disclosure also relates to an antitumor pharmaceutical composition comprising oncolytic vaccinia virus and DR-18, and the use of oncolytic vaccinia virus and DR-18 in the preparation of an antitumor drug.

Description

[Technical Field]

[0001] This application claims priority to CN202211566283.7, filed December 7, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure is in the field of biopharmaceuticals and relates to the use of DR-18 and oncolytic vaccinia virus in the preparation of antitumor drugs. [Background technology]

[0003] Malignant tumors are the leading cause of death worldwide. According to the Global Cancer Statistics, 19.29 million new cases of cancer were diagnosed worldwide, and 9.95 million deaths were attributed to cancer (SUNG H, FERLAY J, SIEGEL RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries [J]. CA Cancer J Clin, 2021, 0:1-41.). By 2040, the number of new cancer cases worldwide is expected to reach 28.4 million, a 47% increase from 2020, with the largest increases expected in least developed countries, with increases of 95% and 64%, respectively (Cao Maomao, and Chen Wanqing. Interpretation of GLOBOCAN 2020 global cancer statistics [J]. Chinese Journal of Medical Frontiers (Electronic Edition), 2021, 13(3):63-69.). Therefore, the general public has a very wide demand for effective and economical antitumor drugs (Wanqing Chen, Rongshou Zheng, et al. Cancer Statistics in China, 2015 [J]. CA Cancer J Clin, 2016, 6(6):115-132.).

[0004] Traditional treatments for malignant tumors include surgery, radiation therapy, and chemotherapy, which have made great progress over the past few decades. However, these treatments have yet to significantly improve the long-term survival rates of patients, particularly those with intractable tumors such as liver cancer, glioma, pancreatic cancer, and osteosarcoma. Cytotoxic drugs and molecular-targeted drugs are currently used as first-line antitumor drugs in clinical trials, but they suffer from significant toxicity and side effects, as well as a tendency toward drug resistance. Therefore, new, efficient, and less toxic therapeutic approaches and drugs are urgently needed for the treatment of malignant tumors.

[0005] Immunotherapy is currently showing great promise as a cancer treatment. Unlike conventional direct-action therapies such as surgery, radiation therapy, chemotherapy, and molecular targeted drugs, immunotherapy kills tumors by activating the patient's own immune system, leaving minimal impact on normal tissue. Immunotherapy is effective even against some advanced tumors, and can prevent tumor recurrence and achieve complete cure. Currently, clinical agents such as 1) immune checkpoint inhibitors such as PD-1 antibodies (e.g., nivolumab, pembrolizumab), PD-L1 antibodies (atezolizumab), and CTLA4 antibodies (ipilimumab); 2) several monoclonal antibodies targeting surface tumor-associated antigens, such as the anti-CD20 monoclonal antibody (rituximab); 3) two immune-stimulating cytokines (interferon IFN-α and interleukin 2 (IL-2); 4) immunogenic cell death inducers such as cyclophosphamide and oxaliplatin; 5) chimeric antigen receptor T-cell therapy (CART); 6) bacillus Calmette-Guérin (BCG); 7) dendritic cell-based cancer vaccines; and 8) oncolytic viruses (OVs) have been approved by the US Food and Drug Administration (FDA) for cancer immunotherapy.

[0006] Oncolytic virus therapy is a novel antitumor immunotherapy that combines targeted therapy, immunotherapy, and gene therapy. It selectively infects and kills tumor cells, subsequently activating antitumor immune responses through mechanisms such as tumor / viral antigen exposure and cytokine release, thereby exerting direct or indirect antitumor effects. Furthermore, oncolytic viruses themselves can function as vectors to deliver genes such as suicide genes, immunomodulatory genes, proapoptotic genes, and antiangiogenic genes, further modulating the tumor microenvironment to promote antitumor effects. Compared with current clinical treatments, oncolytic virus therapy offers advantages such as potent killing efficacy, high safety, and low cost.

[0007] Oncolytic virus therapy has multiple unique antitumor pathways, and many scientific teams are devoting themselves to its research and development. However, only four oncolytic virus products have been approved for sale and used in clinical tumor treatment. These are RIGVIR, approved for sale in Latvia in 2003; Oncorine (H101), approved for sale in China in 2005; IMLYGIC (T-Vec), approved for sale in the United States in 2015; and Delytact, approved for sale in Japan in 2021. Additionally, several oncolytic viruses are currently undergoing clinical trials, with adenovirus (ADV), herpes simplex virus (HSV), and vaccinia virus (VV) being the most widely used in clinical trials.

[0008] Vaccinia virus is a relatively well-studied virus, formerly used as a smallpox vaccine and widely administered worldwide. It has a history of approximately 200 years and has demonstrated excellent efficacy and safety. Due to its advantages, including low disease risk, distinct pathogenicity and pathogenicity genes, mature attenuation strategy, stable genome, non-integration, non-latency, potent oncolytic effect, immune response activation, large foreign gene load and stable expression, and intravenous administration, vaccinia virus is widely recognized as one of the ideally designed oncolytic virus scaffolds. Based on the aforementioned development advantages, numerous clinical trials of oncolytic vaccinia viruses have been conducted in China and other countries, demonstrating excellent safety and efficacy. For example, JX-594 has been approved for international Phase III multicenter clinical trials in China.

[0009] Over the past few decades, oncolytic vaccinia viruses engineered through artificial genetic engineering have made great advances in the field of tumor therapy; however, no therapeutic oncolytic vaccinia virus products have yet been successfully approved for marketing. Vaccinia virus strains currently undergoing clinical trials include WR, Lister, Copenhagen, Wyeth, LC16m0, and MVA. The following artificial genetic engineering strategies have been used: T601 / TG6002 was derived from Copenhagen by knocking out the TK and RR genes and loading the FCU1 gene; ASP9801 was derived from LC16m0 by knocking out the VGF, I1L, and B5R genes and loading the IL-7 and IL-12 genes; and JX-594 was derived from Wyeth by knocking out the TK gene and loading the GM-CSF gene. Research into the mechanisms of the tumor-killing effects of oncolytic vaccinia viruses and their applications remains challenging. For example, further research is still needed on issues such as the elimination or evasion of antiviral antibodies, the development of immunotherapy targets, and the combined administration, route of administration, and indications of oncolytic viruses and other anticancer drugs.

[0010] IL-18 is an immunostimulatory cytokine with the ability to stimulate T cells, NK cells, and myeloid cells and activate antitumor immune cells, making it a potential candidate molecule for cancer therapy. However, IL-18BP (IL-18-binding protein) in the tumor microenvironment functions as a secreted immune checkpoint molecule, competitively binding with IL-18 and inhibiting its binding to its receptor, limiting the efficacy of IL-18 immunotherapy. Therefore, immune cells cannot activate antitumor immune responses. Summary of the Invention

[0011] In one aspect, the disclosure provides DR-18 for use in treating tumors by administering DR-18 in combination with an oncolytic vaccinia virus.

[0012] In one aspect, the present disclosure provides an oncolytic vaccinia virus for use in treating tumors by administering the oncolytic vaccinia virus in combination with DR-18.

[0013] In one aspect, the present disclosure provides the use of DR-18 in the manufacture of a tumor therapeutic medicament for use in combination with an oncolytic vaccinia virus. In one aspect, the present disclosure provides the use of an oncolytic vaccinia virus in the preparation of a medicament for treating tumors for use in combination with DR-18.

[0014] In one aspect, the present disclosure provides the use of DR-18 in the preparation of an oncolytic vaccinia virus anti-tumor synergist or drug resistance reversal agent. In another aspect, the present disclosure provides the use of oncolytic vaccinia virus in the preparation of a DR-18 anti-tumor synergist or drug resistance reversal agent.

[0015] A drug resistance reversal agent refers to the fact that when an oncolytic virus is used as an antitumor agent in tumor treatment, tumors that are not very sensitive to the oncolytic virus or tumors that are resistant to the oncolytic virus exist, and in this case, the combined use of IL-18 (which functions as a drug resistance reversal agent) and the oncolytic virus can reverse tumor resistance to the oncolytic virus. Conversely, when an antitumor substance is used in tumor treatment, tumors that are not very sensitive to the drug or tumors that are resistant to these substances exist, and in this case, the combined use of an oncolytic virus (which functions as a drug resistance reversal agent) and these substances can reverse tumor resistance to these substances.

[0016] DR-18 is a mutated IL-18 that binds to and activates the IL-18 receptor and its downstream pathway, but does not bind to IL-18BP. For example, U.S. Patent Application No. 2019 / 0070262 and Zhou et al., (Nature (2020) 583:609-614) disclose several human DR-18s and several mouse DR-18s.

[0017] In some embodiments, the DR-18 is human DR-18. In some embodiments, the human DR-18 comprises at least one mutation compared to wild-type human IL-18.

[0018] In some embodiments, human DR-18 comprises one or more mutations selected from the group consisting of M51K, K53S, Q56L, P57A, M60L, S105D, D110S, and N111R compared to wild-type human IL-18. In some embodiments, human DR-18 comprises M51K, K53S, Q56L, P57A, M60L, S105D, D110S, and N111R mutations compared to wild-type human IL-18. In some embodiments, the amino acid sequence of human DR-18 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the amino acid sequence of wild-type human IL-18 comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO:3.

[0019] In some embodiments, the DR-18 is mouse DR-18. In some embodiments, the mouse DR-18 comprises at least one mutation compared to wild-type mouse IL-18.

[0020] In some embodiments, the mouse DR-18 comprises one or more mutations selected from the group consisting of N1H, M50A, K52G, E55R, V56A, and L59K compared to wild-type mouse IL-18. In some embodiments, the mouse DR-18 comprises the following mutations compared to wild-type mouse IL-18: N1H, M50A, K52G, E55R, V56A, and L59K.

[0021] In some embodiments, the amino acid sequence of mouse DR-18 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the amino acid sequence of wild-type mouse IL-18 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 4.

[0022] In some embodiments, the oncolytic vaccinia virus is a type defective in the TK gene. In some embodiments, the TK gene of the oncolytic vaccinia virus is inactivated, under-expressed, or deleted. In some embodiments, the TK gene of the oncolytic vaccinia virus is deleted. In some embodiments, the oncolytic vaccinia virus is one or more selected from the group consisting of WR strain, Wyeth strain, Lister strain, Copenhagen strain, and Tiantan strain. In some embodiments, the oncolytic vaccinia virus is selected from the Wyeth strain.

[0023] As used in this disclosure, "human DR-18" and "DR18" are interchangeable, and "mouse DR-18" and "mDR18" are also interchangeable.

[0024] As used in this disclosure, "mutation," "mutant," or "variant" refers to an alteration in a nucleic acid or polypeptide sequence relative to a reference sequence (which may be a naturally occurring normal or "wild-type" sequence), including translocations, deletions, insertions, and substitutions / point mutations. As used herein, a "mutant" or "variant" refers to a nucleic acid or protein that contains a mutation.

[0025] As used in this disclosure, the term "wild-type" refers to a gene or gene product isolated from a natural source. A wild-type gene is that gene that is most commonly observed in a population and is thus arbitrarily designated as the "normal" or "wild-type" form of the gene. In contrast, the terms "modified," "variant," or "mutant" refer to a gene or gene product with modified sequence and / or functional properties (i.e., altered properties) compared to the wild-type gene or gene product.

[0026] In the present disclosure, the term "functionally defective" as used with respect to a gene of an oncolytic virus refers to the inability of the oncolytic virus to perform the function intended by the gene, i.e., loss of function, which can be achieved, for example, by inserting an exogenous fragment into the gene or knocking out the gene.

[0027] In some embodiments, functional deficiency of a gene can be achieved by inserting an exogenous nucleotide sequence into the gene and / or knocking out the gene.

[0028] In one aspect, the present disclosure provides a combination of DR-18 and an oncolytic vaccinia virus for treating tumors. In some embodiments, the term "combination" is broadly construed as, for example, a pharmaceutical composition, a pharmaceutical kit, or a molecular combination of a DR-18 gene and an oncolytic vaccinia virus gene.

[0029] In one aspect, the present disclosure provides a pharmaceutical combination for treating tumors comprising DR-18 and an oncolytic vaccinia virus.

[0030] As used in this disclosure, the term "pharmaceutical combination" means a product obtained by mixing or combining one or more active ingredients, and includes both fixed and non-fixed combinations of active ingredients. As used herein, terms such as "co-administration" or "co-administration" are intended to encompass the administration of selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.

[0031] The term "fixed combination," as used in this disclosure, means that the active ingredients, eg, DR-18 and an oncolytic vaccine virus, are both administered to a patient simultaneously in the form of a single entity or dosage.

[0032] As used herein, the term "non-fixed combination" refers to the administration of active ingredients, such as DR-18 and an oncolytic vaccine virus, to a patient as separate entities simultaneously, concurrently, or sequentially without any specific time limit. Such administration preferably provides therapeutically effective levels of the two compounds simultaneously in the body. As an example, a non-fixed combination may be two capsules each containing one active ingredient, with the aim of achieving treatment by ingesting both active ingredients together in the patient's body.

[0033] As used herein, the term "co-administration" or "use in combination" means that two or more active agents may be administered to a subject as a mixture, simultaneously in a single formulation, or sequentially in any order in a single formulation.

[0034] As used herein, the term "pharmaceutical combination" includes a recombinant oncolytic vaccinia virus in which a target gene (e.g., DR18 in this disclosure) is recombined into the oncolytic vaccinia virus genome to obtain a recombinant oncolytic vaccinia virus capable of exerting the pharmaceutical activity of both the oncolytic vaccinia virus and DR18.

[0035] In this disclosure, the term "in combination with" when used in reference to administering multiple agents to a subject means administering a first agent and at least one additional (i.e., second, third, fourth, fifth, etc.) agent to a subject. For purposes of the present invention, a first agent (e.g., DR-18) is considered to be administered in combination with a second agent (e.g., an oncolytic vaccinia virus) if the biological effect produced by administration of the first agent persists in the subject at the time of administration of the second agent, such that the therapeutic effects of the first and second agents overlap. A second agent is considered to be administered in combination with a first agent, even if the first agent is administered significantly (e.g., days or weeks) away from the time of administration of the second agent, if the administration of the first agent (e.g., DR-18) provides a therapeutic effect over an extended period of time, and the administration of the second agent (e.g., an oncolytic vaccinia virus) provides a therapeutic effect while the therapeutic effect of the first agent persists. In some embodiments, a first agent is considered to be administered in combination with a second agent if the first and second agents are administered simultaneously (within 30 minutes of each other), contemporaneously, or sequentially. In some embodiments, a first agent is considered to be administered "concurrently" with a second agent if the first and second agents are administered within about 24 hours of each other, preferably within about 12 hours of each other, preferably within about 6 hours of each other, preferably within about 2 hours of each other, or preferably within about 30 minutes of each other. The term "in combination with" should also be understood to apply to situations where the first and second agents are combined into a single pharmaceutically acceptable formulation and the combined formulation is administered to a subject. In certain embodiments, the DR-18 and oncolytic vaccine virus are administered or applied sequentially, e.g., one agent is administered before one or more other agents. In other embodiments, the DR-18 and oncolytic vaccine virus are administered simultaneously, e.g., the two or more agents are administered at about the same time, and the two or more agents may be present in two or more separate formulations or may be combined in a single formulation (i.e., a co-formulation). Whether the agents are administered sequentially or simultaneously, they are considered to be administered in combination for purposes of this disclosure.

[0036] As used herein, the term "about" or "approximately" means within 10%, more preferably within 5%, of a given value or range.

[0037] In some embodiments, the pharmaceutical combination is a pharmaceutical composition or a pharmaceutical kit. In some embodiments, the pharmaceutical composition comprises a mixture of DR-18 and an oncolytic vaccinia virus. In some embodiments, the pharmaceutical kit comprises separately packaged DR-18 and separately packaged oncolytic vaccinia virus. In some embodiments, the pharmaceutical combination is a recombinant oncolytic vaccinia virus having a DR-18 nucleotide sequence inserted into its genome.

[0038] In one aspect, the present disclosure provides a pharmaceutical composition for treating a tumor, comprising DR-18 and an oncolytic vaccinia virus.

[0039] In one aspect, the present disclosure provides a pharmaceutical kit comprising DR-18 and an oncolytic vaccinia virus.

[0040] In some embodiments, the pharmaceutical kit comprises separately packaged DR-18 and separately packaged oncolytic vaccinia virus.

[0041] The difference between the pharmaceutical kit and the pharmaceutical composition is that the DR-18 is in a different dosage form from the oncolytic vaccine virus and is packaged separately (e.g., one tablet, capsule, tablet, or ampoule contains DR-18, and another tablet, capsule, tablet, or ampoule contains the oncolytic vaccine virus). In some embodiments, the oncolytic vaccinia virus, DR-18, and the combination of oncolytic vaccinia virus and DR-18 may further comprise one or more adjuvants. An adjuvant refers to a component in a pharmaceutical composition that can aid in the efficacy of the drug. The pharmaceutical kit may further comprise separately packaged DR-18 and separately packaged oncolytic vaccinia virus. The DR-18 and oncolytic vaccinia virus in the pharmaceutical kit may be administered simultaneously or sequentially in any order. For example, DR-18 is administered before the oncolytic vaccinia virus, or DR-18 is administered after the oncolytic vaccinia virus, or DR-18 and the oncolytic vaccinia virus are administered simultaneously. In various embodiments, the patient may be a mammal.

[0042] In some embodiments, the composition / pharmaceutical kit further comprises a pharmaceutically acceptable vector.

[0043] In some embodiments, the ratio of DR-18 to oncolytic vaccinia virus is 0.01-200 mg:10 3 -10 9 PFU, preferably 0.1-200 mg: 10 4 -10 9 PFU, more preferably 0.1-100 mg:10 5 -10 9 It is PFU.

[0044] In some embodiments, the dose range of DR-18 is 0.01-10 mg / kg, and the titer of the oncolytic vaccinia virus is an MOI of 10. 3 -10 9PFU / kg, preferably the dose range of DR-18 is 0.1-5 mg / kg, and the titer of oncolytic vaccinia virus is MOI 10 4 -10 9 PFU / kg, more preferably the dose range of DR-18 is 0.05-0.5 mg / kg, and the titer of the oncolytic vaccinia virus is an MOI of 10 5 -10 9 PFU / kg.

[0045] In some embodiments, DR-18 is administered by intraperitoneal injection. In some embodiments, the oncolytic vaccinia virus is administered by intratumoral or intravenous injection. In some embodiments, the pharmaceutical combination of the present disclosure is in the form of an injection, a tablet, a capsule, a patch, or the like. In some embodiments, the pharmaceutical combination of the present disclosure is in the form of an intratumoral, intraperitoneal, or intravenous injection.

[0046] In some embodiments, the tumor is a solid tumor or a hematological tumor.

[0047] In some embodiments, the solid tumor is one or more selected from intestinal cancer, pancreatic cancer, liver cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, nasopharyngeal carcinoma, lung cancer, osteosarcoma, and gastric cancer.

[0048] In one aspect, the present disclosure provides a use of DR-18 in preparing a combination therapy for treating a tumor, the combination therapy comprising combining an oncolytic vaccinia virus with DR-18.

[0049] In one aspect, the present disclosure provides for the use of an oncolytic vaccinia virus in the preparation of a combination therapy for tumor treatment, the combination therapy comprising combining DR-18 with an oncolytic vaccinia virus.

[0050] In one aspect, the present disclosure provides the use of the pharmaceutical combination, pharmaceutical composition, pharmaceutical kit, or combination therapy in the preparation of an anti-tumor medicament.

[0051] In one aspect, the present disclosure provides a recombinant oncolytic vaccinia virus, the genome of which comprises a mutated oncolytic vaccinia virus sequence and a DR-18 sequence.

[0052] In some embodiments, the mutation is a functional defect of the TK gene. In some embodiments, the mutation is inactivation, underexpression, or deletion of the TK gene. In some embodiments, the mutation is a deletion of the TK gene. In some embodiments, the DR-18 sequence is a human DR-18 sequence and / or a mouse DR-18 sequence.

[0053] In some embodiments, the nucleotide sequence of human DR-18 comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the nucleotide sequence set forth in SEQ ID NO:5.

[0054] In some embodiments, the nucleotide sequence of mouse DR-18 comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the nucleotide sequence set forth in SEQ ID NO:6.

[0055] In one aspect, the present disclosure provides a pharmaceutical composition comprising the recombinant oncolytic vaccinia virus and a pharmaceutically acceptable vector.

[0056] In some embodiments, the pharmaceutical composition comprises 10 4-10 6 In some embodiments, the pharmaceutical composition comprises 10 PFU of recombinant oncolytic vaccinia virus. 4 -10 5 In some embodiments, the pharmaceutical composition comprises 10 PFU of recombinant oncolytic vaccinia virus. 5 -10 6 Contains PFU of recombinant oncolytic vaccinia virus.

[0057] The present disclosure further provides the use of said pharmaceutical combination, recombinant oncolytic vaccinia virus or pharmaceutical composition in the preparation of an antitumor medicament.

[0058] In one aspect, the present disclosure provides a method for preventing and / or treating tumors, comprising administering the pharmaceutical combination, recombinant oncolytic vaccinia virus, or pharmaceutical composition to a subject in need thereof. DR-18 can be administered simultaneously with, before, or after administration of an oncolytic vaccine virus described herein. Furthermore, DR-18 and / or oncolytic vaccinia virus can be administered once a week or multiple times a week (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). DR-18 and / or oncolytic vaccinia virus can be administered weekly or multiple weeks (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), or over a period of one or several months (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, and longer).

[0059] In some embodiments, the combination of some oncolytic viruses with DR-18 has produced a variety of unexpected effects. The inventors have found that some combinations have clear mutual antagonism or inhibitory effects. However, in contrast to these phenomena, the combination of oncolytic viruses with DR-18 has shown clear synergistic effects in tests against various tumors, which is a surprising and promising discovery. [Brief explanation of the drawings]

[0060] [Figure 1]1 shows the genetic structure of Plasmid 1 in Example 1, in which the TK gene is knocked out and which has the LacZ reporter gene. [Figure 2] 1 shows the genetic structure of Plasmid 2 in Example 1, in which the TK gene is knocked out and which also contains the GFP gene. [Figure 3] 1 shows the gene structure of plasmid 3 in Example 1, in which the TK gene is knocked out and the mouse DR18 gene (SEQ ID NO: 6) is expressed. [Figure 4] FIG. 1 shows the gene structures of recombinant oncolytic vaccinia viruses in which the TK gene has been knocked out in Examples 2 and 3. [Figure 5] FIG. 1 shows the gene structure of a recombinant oncolytic vaccinia virus in which the TK gene has been knocked out in Example 4. [Figure 6] FIG. 1 shows the gene structure of recombinant oncolytic vaccinia virus VV-mDR18 in which the TK gene has been knocked out and which expresses the mouse DR18 gene in Examples 4 and 5. [Figure 7] This shows the effect of OVs-SN on the activity of DR18 in Example 2. In Figure 7, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and ns indicates a statistically insignificant difference. [Figure 8] Figure 8 shows the weight change curves of animals treated with a combination of OV and mDR18 in Example 3. In Figure 8, ns indicates a statistically insignificant difference. [Figure 9] 9 shows the tumor volume growth curve of the VV+mDR18 group in Example 3. In Figure 9, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and ns indicates a statistically insignificant difference. [Figure 10] 10 shows the tumor volume growth curve of the ADV+mDR18 group in Example 3. In Figure 10, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and ns indicates a statistically insignificant difference. [Figure 11]11 shows the tumor volume growth curve of the VSV+mDR18 group in Example 3. In Figure 11, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and ns indicates a statistically insignificant difference. [Figure 12] 12 shows the weight change curves of animals treated with different doses of VV-mDR18 in Example 4. In Figure 12, ns indicates a statistically insignificant difference. [Figure 13] 13 shows the tumor volume growth curves of animals treated with different doses of VV-mDR18 in Example 4. In Figure 13, *** indicates P<0.001. [Figure 14] 1 shows the relative tumor growth rate curves of animals treated with different doses of VV-mDR18 in Example 4. [Figure 15] 15 shows the weight change curves of animal models bearing different tumors treated with VV-mDR18 in Example 5. In Figure 15, A shows the MC38 tumor-bearing mouse model of intestinal cancer, B shows the LLC tumor-bearing mouse model of lung cancer, and C shows the H22 tumor-bearing mouse model of liver cancer. [Figure 16] 16 shows tumor volume growth curves of different tumor models treated with VV-mDR18 in Example 5 (** indicates P<0.01). In Figure 16, A shows the MC38 tumor-bearing mouse model of intestinal cancer, B shows the LLC tumor-bearing mouse model of lung cancer, and C shows the H22 tumor-bearing mouse model of liver cancer. [Figure 17] 17 shows the relative tumor growth rate curves of different tumor models treated with VV-mDR18 in Example 5. In Figure 17, A shows the MC38 tumor-bearing mouse model of intestinal cancer, B shows the LLC tumor-bearing mouse model of lung cancer, and C shows the H22 tumor-bearing mouse model of liver cancer. DETAILED DESCRIPTION OF THE INVENTION

[0061] The present disclosure will be further described below with reference to the embodiments. However, the embodiments of the present disclosure are not limited to the following embodiments. Any equivalent changes or modifications made according to the principles or concepts of the present disclosure shall be deemed to fall within the scope of protection of the present disclosure. Unless otherwise specified, the materials and experimental methods employed in the present disclosure are conventional materials and methods.

[0062] Unless otherwise explained, 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. The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly suggests otherwise. It is further understood that all base sizes or amino acid sizes, and all molecular weight or molecular weight values ​​given for nucleic acids or polypeptides are approximate and are provided for illustrative purposes. The use of words such as "approximately," "about," and "substantially" modify values / descriptions, as would be understood by one of ordinary skill in the art based on the context and the parameters being described; a 5% value can be applied when further guidance is needed for understanding. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure; suitable methods and materials are described below. The term "comprises" means "containing." The abbreviation "eg" is derived from the Latin "exempli gratia" and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example." The percent sequence identity listed refers to the percent of nucleic acid or amino acid residues that are identical to the reference sequence within a particular DNA, RNA, or protein, respectively.

[0063] Example 1: Construction of a recombinant oncolytic vaccinia virus in which the TK gene has been knocked out and a recombinant oncolytic vaccinia virus in which the TK gene has been knocked out and which expresses the mouse DR18 gene 1. Experimental Materials 1.1 Experimental viruses: The Wyeth strain of wild vaccine virus was purchased from the American Type Culture Collection (ATCC).

[0064] 1.2 Experimental Plasmids: TK gene, LacZ gene or GFP gene, and mouse DR18 gene were synthesized and recombined into pUC57-Mini plasmid by strain design. This plasmid was purchased from GenScript.

[0065] Plasmid 1 was constructed in which the TK gene was knocked out and which carried the LacZ receptor gene, plasmid 2 in which the TK gene was knocked out and which carried the GFP gene, and plasmid 3 in which the TK gene was knocked out and which expressed the mouse DR18 gene (sequence number 6).

[0066] The genetic structure of Plasmid 1, in which the TK gene is knocked out and which carries the LacZ reporter gene, is shown in FIG.

[0067] The genetic structure of Plasmid 2, in which the TK gene was knocked out and which contained the GFP gene, is shown in Figure 2.

[0068] The gene structure of plasmid 3, in which the TK gene is knocked out and the mouse DR18 gene (SEQ ID NO: 6) is expressed, is shown in FIG.

[0069] 1.3 Experimental Cells: Human thymidine kinase-deficient osteosarcoma cells (143B(TK-)) were purchased from ATCC.

[0070] 2. Experimental Method 2.1 Homologous recombination One day before the experiment, 143B(TK-) cells were inoculated into a cell culture flask and cultured overnight. A virus solution of the Wyeth strain of wild-type vaccine virus was collected and used to infect the cells. Lipofectamine TMThe plasmid transfection system was prepared according to the 3000 Transfection Reagent instructions based on the amount of recombinant plasmid used and added to the cells. After cytopathic effect reached 80% or more, the recombinant virus solution was collected.

[0071] 2.2 Plaque purification One day before the experiment, 143B(TK-) cells were seeded into 12-well plates and cultured overnight. The recombinant virus solution was diluted 10-fold and added to the cells for 60 minutes. The virus-infected supernatant was removed, and 1% low-melting-point agarose-5% FBS MEM solid medium was added and allowed to solidify at room temperature. The cells were then cultured for 24–48 hours. Solid medium containing X-gal was added to the 12-well plates, allowed to solidify at room temperature, and allowed to develop with X-gal for 24–48 hours. Blue plaques were punctured using a pipette tip and subjected to repeated freeze-thaw cycles for subsequent plaque purification. Plaque purification was repeated five times or more. Plaque-purified virus clones were amplified to obtain recombinant oncolytic vaccine virus seed stocks.

[0072] Example 2: Evaluation of the effect of supernatants from different tumor cells infected with various oncolytic viruses on DR18 activity using an IL-18 receptor cell model IL-18 receptor cell model: These cells express IL-18 receptor, IL-18 downstream pathway molecules, and luciferase. IL-18 binds to the receptor and mediates luciferase expression, and cell count indicates pathway activation. This pathway is an important pathway for IL-18 to activate anti-tumor immunity in vivo, and can indirectly reflect the anti-cancer activity of IL-18.

[0073] 1. Experimental Materials 1.1 Oncolytic viruses: VV VV is a recombinant oncolytic vaccinia virus in which the TK gene has been knocked out, and the genome structure of the oncolytic vaccinia virus is shown in Figure 4.

[0074] ADV is an oncolytic adenovirus with knockout of the E1a-CR2-24bp gene and the E3 region gene. VSV is an attenuated oncolytic vesicular stomatitis virus with knockout of the G gene and V48R and M51R mutations in the M gene. REO is a serotype 3 oncolytic reovirus. These viruses are commercially available, customized by conventional methods, or constructed by Guangzhou ViroTech Co., Ltd. by conventional methods.

[0075] 1.2 Experimental cells: IL-18 receptor cells (H_IL18 receptor 293 cell line), prostate cancer cells (DU145), bladder cancer cells (SCaBER and KU-19-19), pancreatic cancer cells (MIA Paca-2), kidney cancer cells (786-O), cervical cancer cells (HCC94 and C-33A), intestinal cancer cells (DLD-1), liver cancer cells (Hep3B), lung cancer cells (SHP-77 and A549), breast cancer cells (HCC38), and osteosarcoma cells (MNNG / HOS) were purchased commercially.

[0076] 1.3 Experimental reagents: human DR18 recombinant protein (SEQ ID NO: 1), DMEM medium, MEM medium, RPMI 1640 medium, McCoy's 5A medium, IMDM medium, EMEM medium, fetal bovine serum, puromycin, blasticidin, G418, and ONE-Glo TM Luciferase assay system.

[0077] 1.4 Laboratory equipment: inverted microscope, biosafety cabinet, carbon dioxide incubator, microplate reader

[0078] 2. Experimental Method 2.1 Construction of tumor cell model The appropriate culture conditions and passage rate for cell growth and subculture were selected according to the manufacturer's instructions. After the cells entered the logarithmic growth phase and the cell number was sufficient, the cells were cultured at a density of 3 × 10 4 pieces / cm 2 The cells were seeded into T25 cell culture flasks at a density of 1000 μg / ml.

[0079] 2.2 Infection of tumor cells with oncolytic viruses 16 to 24 hours after inoculation, complete cell adhesion was observed under a microscope. Depending on the number of cells inoculated, each tumor cell was infected with a virus dose of MOI = 1 PFU / cell.

[0080] 2.3 Collection and inactivation of infected supernatant Tumor cells were infected with oncolytic viruses for 48 hours, after which cytopathic effects were observed under a microscope and photographed. Depending on the virus characteristics, virus-infected cell supernatants (SNs) were inactivated by UV irradiation or filtered through a 0.1 μm filter membrane. The inactivated supernatants were collected in 4.5 mL cryovials and stored in a -80°C refrigerator for later use. Non-infected supernatants were collected in the same manner and used as controls.

[0081] 2. Effect of infection supernatant on the biological activity of 4DR18 1) IL-18 receptor cells were thawed and subcultured. After the cells entered the logarithmic growth phase and reached a sufficient cell number, 1.5 × 10 cells were placed in the center well of a 96-well plate. 4 Cells were seeded at a density of 100 cells / well, and 90 μL of medium was added to each central well, and 200 μL of PBS was added to the surrounding wells to seal the edges.

[0082] 2) The cells were cultured in a 5% CO2, 37°C cell incubator for 16-24 hours, and the 96-well plate was removed and examined under a microscope for complete cell adhesion and good morphology. DR18 protein was diluted 3-fold with the inactivated supernatant and control supernatant obtained in step 2.3, mixed uniformly, and added to the cells at 10 μL / well.

[0083] 3) After adding the DR18 protein, the cells were cultured in a cell incubator at 5% CO 2 and 37°C for 16 hours.

[0084] 4) 100 μL of ONE-Glo TMThe substrate was added, and the reaction was allowed to proceed in the dark for 3 minutes to completely lyse the cells. The cell lysate was then transferred to a 96-well luminescence plate, and luciferase was detected using a microplate reader.

[0085] 2.5 Data Processing Based on the relative light units (RLU) detected in each well, the relative light velocity of the cells in each well was calculated using the following formula: Relative speed of light (% of Max) = (RLU sample -RLU blank ) / (RLU max -RLU blank )×100% The fitted curve was generated using GraphPad Prism 8 software. The half-maximal effective concentration (EC 50 ) values ​​were calculated using the [agonist] vs. response-variable slope (four parameter) analysis equation.

[0086] 2.6 Biological Statistics The detection data were statistically analyzed by T-test, where * represents P<0.05, ** represents P<0.01, and *** represents P<0.001, indicating that the differences between the data are statistically significant.

[0087] 3. Experimental Results The effects of inactivated supernatants of various tumor cells infected with different oncolytic viruses on the activity of 3.1DR18 are shown in Figure 7 and Table 1.

[0088] Table 1: Effect of inactivated supernatants of various tumor cells infected with different oncolytic viruses on the activity of DR18 (* indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001) [Table 1]

[0089] DR18 recombinant protein was gradient-diluted using supernatants from different tumor cells infected with different oncolytic viruses. IL-18 receptor cells were treated for 16 hours, and luciferase light units were detected mechanically to generate fitted curves. The calculated EC50 values ​​for the treatment groups are shown in Figure 7, and the statistical analysis results are shown in Table 1.

[0090] The results show that, compared with uninfected controls (CTLs), oncolytic vaccinia virus (VV) infection of prostate cancer cells (DU145), bladder cancer cells (SCaBER and KU-19-19), pancreatic cancer cells (MIA-Paca-2), kidney cancer cells (786-O), cervical cancer cells (HCC94), intestinal cancer cells (DLD-1), liver cancer cells (Hep3B), lung cancer cells (SHP-77), breast cancer cells (HCC38), and osteosarcoma cells (MNNG / HOS) significantly promoted the biological activity of DR18. Oncolytic vaccinia virus (VV) infection of cervical cancer cells (C-33A) tended to promote the biological activity of DR18, but the difference was not statistically significant.

[0091] Infection of prostate cancer cells (DU145), bladder cancer cells (KU-19-19), pancreatic cancer cells (MIA Paca-2), cervical cancer cells (HCC94), and osteosarcoma cells (MNNG / HOS) with oncolytic vesicular stomatitis virus (VSV) significantly promoted the biological activity of DR18, whereas infection of renal cancer cells (786-O) with VSV inhibited the biological activity of DR18.

[0092] Oncolytic adenovirus (ADV) infection of bladder cancer cells (KU-19-19), pancreatic cancer cells (MIA Paca-2), cervical cancer cells (HCC94), liver cancer cells (Hep3B), lung cancer cells (SHP-77), and osteosarcoma cells (MNNG / HOS) significantly promoted the biological activity of DR18, whereas oncolytic adenovirus (ADV) infection of prostate cancer cells (DU145) and breast cancer cells (HCC38) inhibited the biological activity of DR18.

[0093] Oncolytic reovirus (REO) infection of bladder cancer cells (KU-19-19), cervical cancer cells (HCC94), intestinal cancer cells (DLD-1), liver cancer cells (Hep3B), lung cancer cells (SHP-77), and breast cancer cells (HCC38) significantly promoted the biological activity of DR18, whereas infection of kidney cancer cells (786-O) and cervical cancer cells (C-33A) with REO inhibited the biological activity of DR18.

[0094] As can be seen from the above experimental results, the effects of the supernatants of different tumor cell models infected with various oncolytic viruses are different, and the synergistic effect of VV is higher than that of other oncolytic viruses.

[0095] Example 3: Safety and efficacy study of mDR18 in combination with various oncolytic viruses in an immunocompetent mouse model of liver cancer 1. Experimental Materials 1.1 Oncolytic viruses: VV VV is a recombinant oncolytic vaccinia virus in which the TK gene has been knocked out. The genome structure of the oncolytic vaccinia virus is shown in Figure 4.

[0096] ADV is an oncolytic adenovirus with knockout of the E1a-CR2-24bp gene and the E3 gene. VSV is an attenuated oncolytic vesicular stomatitis virus with knockout of the G gene and V48R and M51R mutations in the M gene. These viruses were purchased from Wuhan BrainVTA, FUBIO, etc., or constructed by Guangzhou ViroTech Co., Ltd.

[0097] 1.2 Recombinant protein: Modified murine interleukin-18 (mDR18) recombinant protein (SEQ ID NO: 2).

[0098] 1.3 Tumor cells: Mouse hepatoma cell line H22 was purchased from the China Center for Type Culture Collection.

[0099] 1.4 Experimental animals: 5-7 week old female Balb / c mice

[0100] 2. Experimental Method 2.1 Construction of tumor-bearing mouse model Tumor cells were thawed and subcultured. After the cells entered the logarithmic growth phase and reached a sufficient cell number, they were harvested, counted, and a cell suspension was prepared. H22 cells were cultured at 2 × 10 6 Balb / c mice were inoculated subcutaneously into the back with an inoculum of 100 cells / mouse.

[0101] 2.2 Grouping and administration After tumor formation, the tumor volume was 100±40mm 3 Mice were selected and randomly divided into eight groups (vehicle control, mDR18, VV, VV+mDR18, ADV, ADV+mDR18, VSV, and VSV+mDR18) with five mice per group. mDR18 was administered intraperitoneally at a dose of 0.32 mg / kg twice a week for a total of four doses. Each oncolytic virus was administered at a dose of 2 × 10 6 The mice were administered once by intratumoral injection at a dose of 100 PFU / mouse. The day of tumor inoculation was designated as D0, and the mice were observed for 14 days after administration.

[0102] 2.3 Mouse weight and tumor measurement During the tumor formation and administration period, mouse body weight and tumor growth were recorded every 3 to 4 days.

[0103] 2.4 Data Processing The measured data were compiled, and tumor volume growth curves for the mice in each group were created.

[0104] The formula for calculating tumor volume (TV) is V=1 / 2×a×b 2 is. where a and b represent the length and width of the tumor, respectively.

[0105] Relative tumor volume (RTV) = V t / V0 where V0 represents the tumor volume measured before grouping and administration, and V t is the tumor volume at each measurement.

[0106] Relative tumor growth rate T / C(%)=T RTV / C RTV ×100% In the formula, T RTV represents the RTV of the single or combined administration groups of each drug, and C RTV represents the RTV of the solvent control group.

[0107] 2.5 Statistical analysis The tumor volume and body weight of the mice were statistically analyzed by repeated measures analysis of variance. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001, all of which indicate that the difference between the data is statistically significant.

[0108] 3. Experimental Results The body weight of each group of animals during the experiment is shown in Figure 8. The body weight of each group of animals maintained a steady increase throughout the experiment. There was no obvious difference between the single-drug or combination-administered groups and the vehicle control group. This is because mDR18 was intraperitoneally injected twice a week at a dose of 0.32 mg / kg, or each oncolytic virus was administered at a dose of 2 × 10 6 We demonstrate that a single intratumoral injection at a dose of 100 PFU / mouse does not cause any obvious drug-related side effects in an immunocompetent mouse model.

[0109] Tumor volume change curves, drawn based on the animal tumor volume growth records, were used to evaluate efficacy and are shown in Figure 10. Compared with the vehicle control group, administration of mDR18 alone (P<0.01) or the combination of mDR18 and VV (P<0.001) significantly inhibited tumor growth in liver cancer tumor-bearing mouse models. Compared with administration of mDR18 alone, the combination of mDR18 and VV (P<0.05) significantly improved tumor inhibition in liver cancer tumor-bearing mouse models.

[0110] Tumor volume change curves, drawn based on the animal tumor volume growth records, were used to evaluate efficacy and are shown in Figure 10. Compared with the vehicle control group, mDR18 alone (P<0.01) or the combination of mDR18 and ADV (P<0.001) significantly inhibited tumor growth in a liver cancer-bearing mouse model. There was no statistically significant difference in the tumor-suppressing effect between mDR18 alone and the combination of mDR18 and ADV, indicating the lack of synergistic effects of the combination of mDR18 and ADV.

[0111] Tumor volume change curves, drawn based on the animal tumor volume growth records, were used to evaluate efficacy and are shown in Figure 11. Compared with the vehicle control group, administration of mDR18 alone (P<0.01) or the combination of mDR18 and VSV (P<0.001) significantly inhibited tumor growth in a liver cancer-bearing mouse model. There was no statistically significant difference in the tumor-suppressing effect between administration of mDR18 alone and the combination of mDR18 and VSV, indicating that the combination of mDR18 and VSV did not have a synergistic effect.

[0112] Example 4: Safety and Efficacy Study of Recombinant Oncolytic Vaccinia Virus Expressing mDR18 in a Tumor-Bearing Immunocompetent Mouse Model of Melanoma 1. Experimental Materials 1.1 Oncolytic viruses: VV and VV-mDR18 VV is a recombinant oncolytic vaccinia virus in which the TK gene has been knocked out. The genome structure of oncolytic vaccinia virus VV (a recombinant oncolytic vaccinia virus in which the TK gene has been knocked out) is shown in Figure 5. VV-mDR18 is a recombinant oncolytic vaccinia virus in which the TK gene has been knocked out and which expresses the mouse DR18 gene. The genomic structure of oncolytic vaccinia virus VV-mDR18 (a recombinant oncolytic vaccinia virus in which the TK gene has been knocked out and which expresses the mouse DR18 gene) is shown in Figure 6. This virus was constructed by Guangzhou ViroTech Co., Ltd.

[0113] FIG. 6 shows the genetic structure of the recombinant oncolytic vaccinia virus VV-mDR18, in which the TK gene has been knocked out and which expresses the mouse DR18 gene.

[0114] 1.2 Tumor cells: Mouse melanoma cells B16-F10 were purchased from the China National Collection of Authenticated Cell Cultures.

[0115] 1.3 Experimental animals: 5-7 week old female C57BL / 6J mice

[0116] 2.1 Construction of tumor-bearing mouse model Tumor cells were thawed and subcultured. After the cells reached the logarithmic growth phase and reached a sufficient cell number, they were harvested, counted, and a cell suspension was prepared. B16-F10 cells were cultured at 5 × 10 5 C57BL / 6J mice were inoculated subcutaneously into the back at an inoculum of 1000 cells / mouse.

[0117] 2.2 Grouping and administration After tumor formation, the tumor volume was 100±40mm 3 Mice were selected and randomly divided into seven groups (vehicle control group, low-dose VV group, medium-dose VV group, high-dose VV group, low-dose VV-mDR18 group, medium-dose VV-mDR18 group, and high-dose VV-mDR18 group) with five mice per group. The doses of the low-dose, medium-dose, and high-dose VV or VV-mDR18 groups were 1 × 10 4 / 5 / 6 The CCID50 / mouse was administered via tail vein injection once. The day of tumor inoculation was designated as D0, and the mice were observed for 14 days after administration.

[0118] 2.3 Mouse weight and tumor measurement During the tumor formation and administration period, mouse body weight and tumor growth were recorded every 3 to 4 days.

[0119] 2.4 Data Processing The measured data were compiled, and tumor volume growth curves for the mice in each group were created.

[0120] The formula for calculating tumor volume (TV) is V=1 / 2×a×b 2 is. where a and b represent the length and width of the tumor, respectively.

[0121] Relative tumor volume (RTV) = V t / V0 where V0 represents the tumor volume measured before grouping and administration, and V t is the tumor volume at each measurement.

[0122] Relative tumor growth rate T / C(%)=T RTV / C RTV ×100% In the formula, T RTV represents the RTV of the single or combined administration groups of each drug, and C RTV represents the RTV of the solvent control group.

[0123] 2.5 Statistical analysis The tumor volume and body weight of the mice were statistically analyzed by repeated measures analysis of variance. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001, all of which indicate that the difference between the data is statistically significant.

[0124] 3. Experimental Results The weight of animals in each group during the experiment is shown in Figure 12. The weight of animals in each group maintained a steady increase throughout the experiment. There was no obvious difference between the drug groups at each dose and the vehicle control group. This indicates that tail vein injection of VV or VV-mDR18 did not cause any obvious drug-related side effects in the immunocompetent mouse model.

[0125] Tumor volume change curves and relative tumor growth rate curves, which were drawn based on the tumor volume growth records of the animals, were used to evaluate efficacy and are shown in Figures 13 and 14, respectively. As a result, the tumor volume change curves and relative tumor growth rate curves were plotted based on the tumor volume growth records of the animals. The ... drawn based on the tumor volume growth records of the animals. The tumor volume change curves and relative tumor growth rate curves were shown in Figures 13 and 14, respectively. As a result, the tumor volume change curves and relative tumor growth rate curves were plotted based on the tumor volume change curves and relative tumor growth rate curves. 5 CCID50) and high-dose group (1 × 106 CCID50) significantly suppressed tumor growth (P<0.001). 4 CCID50), middle dose group (1×10 5 CCID50) and high-dose group (1 × 10 6 The T / C values ​​corresponding to CCID50 were 82.07%, 38.08%, and 26.54%, respectively, indicating that the tumor-inhibitory effect of VV-mDR18 is dose-dependent.

[0126] Example 5: Safety and efficacy study of recombinant oncolytic vaccinia virus carrying mDR18 in a tumor-bearing immunocompetent mouse model 1. Experimental Materials 1.1 Oncolytic virus: VV-mDR18 VV-mDR18 is a recombinant oncolytic vaccinia virus in which the TK gene has been knocked out and which expresses the mouse DR18 gene. The genomic structure of oncolytic vaccinia virus VV-mDR18 (a recombinant oncolytic vaccinia virus in which the TK gene has been knocked out and which expresses the mouse DR18 gene) is shown in Figure 6. This virus was constructed by Guangzhou ViroTech Co., Ltd.

[0127] 1.2 Tumor cells: Mouse intestinal cancer cells MC-38, mouse lung cancer cells LLC, and mouse liver cancer cells H22 were purchased from Shenzhen Luoziman International Institute of Translational Medicine, China Center for Type Culture Collection, and Wuhan Procell Life Science & Technology Co., Ltd.

[0128] 1.3 Experimental animals: 5-7 week old female C57BL / 6N mice and 5-7 week old female Balb / c mice 2. Experimental Method 2.1 Construction of tumor-bearing mouse model The tumor cells were thawed and subcultured. After the cells entered the logarithmic growth phase and reached a sufficient cell number, they were harvested, counted, and a cell suspension was prepared. 1) MC-38 cells were cultured at 2.0 × 10 6 2) LLC cells were inoculated subcutaneously into the back of C57BL / 6N mice at an inoculum dose of 1.5 × 10 cells / mouse. 6 3) H22 cells were inoculated subcutaneously into the back of C57BL / 6N mice at an inoculum size of 1.0 × 10 cells / mouse. 6 Balb / c mice were inoculated subcutaneously into the back at an inoculum of 1000 cells / mouse.

[0129] 2.2 Grouping and administration After tumor formation, the three tumor-bearing models were randomly divided into groups according to tumor volume. Each model had a negative control group and a VV-mDR18 group. Each group contained five mice. The day of tumor inoculation was designated as D0. VV-mDR18 had a tumor volume of 9.88 × 10 5 The dose was 100 PFU / mouse, administered by intratumoral injection once a week for 6 weeks, while the negative control group mice received the same volume of saline by intratumoral injection.

[0130] 2.3 Mouse weight and tumor measurement During the tumor formation and administration period, mouse body weight and tumor growth were recorded every 3 to 4 days.

[0131] 2.4 Data Processing The measured data were compiled, and tumor volume growth curves for the mice in each group were created.

[0132] The formula for calculating tumor volume (TV) is V=1 / 2×a×b 2 is. where a and b represent the length and width of the tumor, respectively.

[0133] Relative tumor volume (RTV) = V t / V0 where V0 represents the tumor volume measured before grouping and administration, and V t is the tumor volume at each measurement.

[0134] Relative tumor growth rate T / C(%)=T RTV / C RTV ×100% In the formula, T RTV represents the RTV of the single or combined administration groups of each drug, and C RTV represents the RTV of the solvent control group.

[0135] 2.5 Statistical analysis The tumor volume and body weight of the mice were statistically analyzed by repeated measures analysis of variance. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001, all of which indicate that the difference between the data is statistically significant.

[0136] 3. Experimental Results The weight of the animals in each group during the experiment is shown in Figure 15. The weight of the animals in each group maintained a steady increase during the experiment. This is because VV-mDR18 was administered at a dose of 9.88 x 10 5 We demonstrate that weekly intratumoral injections at a dose of PFU / mouse do not result in any obvious drug-related side effects in an immunocompetent mouse model.

[0137] Tumor volume change curves and relative tumor growth rate curves, constructed based on the animal tumor volume growth records, are shown in Figures 16 and 17, respectively, and were used to evaluate efficacy. Compared with the negative control group, the recombinant oncolytic vaccinia virus carrying the murine DR18 (VV-mDR18) significantly inhibited tumor growth in the MC38 mouse model of intestinal cancer, the LLC mouse model of lung cancer, and the H22 mouse model of liver cancer (P<0.01). At the test endpoint, the T / C values ​​for the three tumor-bearing models were 22.05%, 3.97%, and 3.35%, respectively.

[0138] Amino acid sequence of human DR-18 (SEQ ID NO: 1) YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSLARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 1)

[0139] Amino acid sequence of mouse DR-18 (SEQ ID NO: 2) HFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYAYGDSRARGKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKRVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 2)

[0140] Amino acid sequence of wild-type human IL-18 (SEQ ID NO: 3) YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 3)

[0141] Amino acid sequence of wild-type mouse IL-18 (SEQ ID NO: 4) NFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKRVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 4)

[0142] Nucleotide sequence of human DR-18 (SEQ ID NO:5) TATTTCGGCAAGCTGGAAAGCAAGCTCTCCGGTGATCAGAAACCTGAATGATCAGGTGCTGTTCATCGACCAGGGCAACCGGCCTCTGTTTGAGGACATGACCGACAGCGATTGCAGAGATAATGCCCCTAGAACCATCTTTATCATTTCTAAGTACAGCGACAGCCTGGCTAGAGGCCTGGCCGTGACAATCAGCGTGAAGTGCGAGAAGATCTCCACCCTGAGCTGCGAGAACAAGAT CATCTCTTTCAAGGAAATGAACCCCCCCGACAACATCAAGGACACAAAGTCCGACATCATCTTCTTCCAGAGAGACGTGCCTGGCCACAGCAGGAAGATGCAGTTCGAGAGCAGCAGCTACGAGGGCTACTTCCTGGCTTGAAAAAAGAACGGGACCTGTTTAAACTGATCCTGAAAAAGGAAGATGAGCTGGGAGATAGAAGCATCATGTTCACCGTGCAGAACGAGGAC (SEQ ID NO: 5)

[0143] Nucleotide sequence of mouse DR-18 (SEQ ID NO: 6) CACTTTGGCCGACTTCACTGTACAACCGCAGTAATACGGAATATAAATGACCAAGTTCTCTTCGTTGACAAAAGACAGCCTGTGTTCGAGGATATGACTGATATTGATCAAAGTGCCAGTGAACCCCAGACCAGACTGATAATATACGCCTACGGCGACAGTAGAGCCAGAGGAAAGGCTGTGACCCTCTCTGTGAAGGATAGTAAAATGTCTACCCTCTCCTGTAAGAACAAGATCAT TTCCTTTGAGGAAATGGATCCACCTGAAAATATTGATGATATACAAAGTGATCTCATATTCTTTCAGAAACGTGTTCCAGGACACAAGATGGAGTTTGAATCTTCACTGTATGAAGGACACTTTCTTGCTTGCCAAAAGGAAGATGATGCTTTCAAACTCATTCTGAAAAAAAAGGATGAAAATGGGGATAAATCTGTAATGTTCACTCTCACTAACTTACATCAAAGT (SEQ ID NO: 6)

[0144] The embodiments described in the present disclosure are merely examples, and the embodiments of the present disclosure are not limited thereto. Any other changes, modifications, substitutions, combinations and simplifications made without departing from the spirit, essence and principles of the present disclosure shall be deemed as equivalent substitutions and fall within the protection scope of the present disclosure.

Claims

1. Use of DR-18 in the preparation of a tumor therapeutic agent in combination with an oncolytic vaccinia virus.

2. Use of an oncolytic vaccinia virus in the preparation of a tumor therapeutic agent in combination with DR-18.

3. DR-18 is human DR-18, Preferably, the human DR-18 comprises at least one mutation compared to wild-type human IL-18; Preferably, the human DR-18 comprises one or more mutations selected from the group consisting of M51K, K53S, Q56L, P57A, M60L, S105D, D110S, and N111R compared to wild-type human IL-18; Preferably, the human DR-18 comprises the following mutations compared to wild-type human IL-18: M51K, K53S, Q56L, P57A, M60L, S105D, D110S, and N111R; Preferably, the amino acid sequence of human DR-18 comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO: 1; The use according to claim 1 or 2, wherein preferably the amino acid sequence of wild-type human IL-18 comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence shown in SEQ ID NO:

3.

4. DR-18 is mouse DR-18, Preferably, the murine DR-18 comprises at least one mutation compared to wild-type murine IL-18; Preferably, the murine DR-18 comprises one or more mutations selected from the group consisting of N1H, M50A, K52G, E55R, V56A, and L59K compared to wild-type murine IL-18; Preferably, the murine DR-18 comprises the following mutations compared to wild-type murine IL-18: N1H, M50A, K52G, E55R, V56A, and L59K; Preferably, the amino acid sequence of mouse DR-18 comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO:2; The use according to any one of claims 1 to 3, wherein the amino acid sequence of wild-type mouse IL-18 preferably comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence shown in SEQ ID NO:

4.

5. the oncolytic vaccinia virus is a type defective in the TK gene, Preferably, the TK gene of the oncolytic vaccinia virus is inactivated, under-expressed or deleted; Preferably, the TK gene of the oncolytic vaccinia virus is deleted; Preferably, the oncolytic vaccinia virus is one or more selected from the WR strain, the Wyeth strain, the Lister strain, the Copenhagen strain, and the Tiantan strain; The use according to any one of claims 1 to 3, wherein the oncolytic vaccinia virus is preferably selected from the Wyeth strain.

6. A pharmaceutical combination for treating tumors comprising DR-18 and an oncolytic vaccinia virus.

7. The pharmaceutical combination is a pharmaceutical composition or a pharmaceutical kit, Preferably, the pharmaceutical composition comprises a mixture of DR-18 and an oncolytic vaccinia virus, Preferably, the pharmaceutical kit comprises an individually packaged DR-18 and an individually packaged oncolytic vaccinia virus; Preferably, the pharmaceutical combination is a recombinant oncolytic vaccinia virus having a DR-18 nucleotide sequence inserted into its genome; Preferably, the DR-18 is human DR-18; Preferably, the human DR-18 comprises at least one mutation compared to wild-type human IL-18; Preferably, the human DR-18 comprises one or more mutations selected from the group consisting of M51K, K53S, Q56L, P57A, M60L, S105D, D110S and N111R compared to wild-type human IL-18; Preferably, the human DR-18 comprises the following mutations compared to wild-type human IL-18: M51K, K53S, Q56L, P57A, M60L, S105D, D110S, and N111R; Preferably, the amino acid sequence of human DR-18 comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO: 1; Preferably, the amino acid sequence of wild-type human IL-18 comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:3; Preferably, the DR-18 is mouse DR-18; Preferably, the murine DR-18 comprises at least one mutation compared to wild-type murine IL-18; Preferably, the amino acid sequence of mouse DR-18 comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO:2; Preferably, the amino acid sequence of wild-type mouse IL-18 comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:4; Preferably, the oncolytic vaccinia virus is a type defective in the TK gene, Preferably, the TK gene of the oncolytic vaccinia virus is inactivated, under-expressed or deleted; Preferably, the TK gene of the oncolytic vaccinia virus is deleted; Preferably, the oncolytic vaccinia virus is one or more selected from the WR strain, the Wyeth strain, the Lister strain, the Copenhagen strain, and the Tiantan strain; Preferably, the oncolytic vaccinia virus is selected from the Wyeth strain, The pharmaceutical combination according to claim 6, wherein the pharmaceutical composition / kit further comprises a pharmaceutically acceptable vector.

8. The ratio of DR-18 to the oncolytic vaccinia virus is 0.01-200 mg:10 ３ -10 ９ PFU, preferably 0.1-200 mg:10 ４ -10 ９ PFU, more preferably 0.1-100 mg:10 ５ -10 ９ PFU, Preferably, the dose range of DR-18 is 0.01-10 mg / kg, and the titer of the oncolytic vaccinia virus is MOI 10 ３ -10 ９ PFU / kg, preferably the dose range of DR-18 is 0.1-5 mg / kg, and the titer of the oncolytic vaccinia virus is MOI 10 ４ -10 ９ PFU / kg, more preferably the dose range of DR-18 is 0.05-0.5 mg / kg, and the titer of the oncolytic vaccinia virus is MOI 10 ５ -10 ９ PFU / kg, Preferably, the pharmaceutical combination is in the form of an injection, a tablet, a capsule or a patch; The pharmaceutical combination according to claim 6 or 7, wherein the form of the pharmaceutical combination comprises intratumoral injection, intraperitoneal injection or intravenous injection.

9. the tumor is a solid tumor or a hematological tumor; 9. The pharmaceutical combination according to any one of claims 1 to 8, wherein the solid tumor is preferably one or more selected from intestinal cancer, pancreatic cancer, liver cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, nasopharyngeal cancer, lung cancer, osteosarcoma, and gastric cancer.

10. 1. A recombinant oncolytic vaccinia virus, comprising: The genome of the recombinant oncolytic vaccinia virus comprises: a mutated oncolytic vaccinia virus sequence; DR-18 sequence, and 1. A recombinant oncolytic vaccinia virus comprising:

11. the mutation is a functional defect of the TK gene; Preferably, the mutation is inactivation, underexpression or deletion of the TK gene; Preferably, the mutation is a deletion in the TK gene; Preferably, the DR-18 sequence is a human DR-18 sequence and / or a mouse DR-18 sequence; Preferably, the nucleotide sequence of human DR-18 comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the nucleotide sequence set forth in SEQ ID NO:5; Preferably, the nucleotide sequence of mouse DR-18 comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the nucleotide sequence set forth in SEQ ID NO:6; Preferably, the oncolytic vaccinia virus is one or more selected from the group consisting of WR strain, Wyeth strain, Lister strain, Copenhagen strain, and Tiantan strain; The recombinant oncolytic vaccinia virus of claim 10, wherein the oncolytic vaccinia virus is preferably selected from the Wyeth strain.

12. A pharmaceutical composition comprising the recombinant oncolytic vaccinia virus of claim 10 or 11 and a pharmaceutically acceptable vector, Preferably, the pharmaceutical composition comprises 10 ４ -10 ６ PFU of a recombinant oncolytic vaccinia virus, Preferably, the pharmaceutical composition comprises 10 ４ -10 ５ PFU of a recombinant oncolytic vaccinia virus, Preferably, the pharmaceutical composition comprises 10 ５ -10 ６ PFU of a recombinant oncolytic vaccinia virus, Preferably, the recombinant oncolytic vaccinia virus is administered by intratumoral injection or intravenous administration.

13. Use of a pharmaceutical combination according to any one of claims 6 to 8, a recombinant oncolytic vaccinia virus according to claim 10 or 11, or a pharmaceutical composition according to claim 12 in the preparation of an antitumor drug.

14. 1. A method for preventing and / or treating a tumor, comprising: A method comprising administering to a subject in need thereof a pharmaceutical combination described in any one of claims 6 to 8, a recombinant oncolytic vaccinia virus described in claim 10 or 11, or a pharmaceutical composition described in claim 12.

15. the tumor is a solid tumor or a hematological tumor; 15. The use or method according to claim 13 or 14, wherein the solid tumor is preferably one or more selected from bowel cancer, pancreatic cancer, liver cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, nasopharyngeal cancer, lung cancer, osteosarcoma, gastric cancer.

Images