Use of dr-18 and herpes simplex virus in preparation of antitumor drug

Abstract

The present invention relates to use of DR-18 and herpes simplex virus in the preparation of an antitumor drug. It is found for the first time that the combination of herpes simplex virus and DR-18 has a synergistic antitumor effect. The present invention also relates to an antitumor pharmaceutical composition comprising herpes simplex virus and DR-18, and use of herpes simplex virus and DR-18 in the preparation of an antitumor drug.

Description

Application of DR-18 and herpes simplex virus in the preparation of anti-tumor drugs Technical Field

[0001] The present disclosure belongs to the field of biomedicine and relates to the application of DR-18 and herpes simplex virus in the preparation of anti-tumor drugs. Background Art

[0002] Malignant tumors are the leading cause of death and disease threatening human health. According to the Global Cancer Report, there were 19.29 million new cases of cancer and 9.95 million deaths from cancer in 2020 worldwide (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.). It is estimated that by 2040, the number of new cancer cases worldwide will reach 28.4 million, a 47% increase compared to 2020, with the largest increase in cases in low- and medium-developed countries, at 95% and 64%, respectively (CAO Maomao, CHEN Wanqing. Interpretation of GLOBOCAN 2020 global cancer statistics [J]. Journal of Chinese Medical Frontiers (Electronic Edition), 2021, 13(3): 63-69.). Therefore, the general public has a very wide demand for highly effective and economical anti-tumor drugs (Wanqing Chen, Rongshou Zheng, et al. Cancer Statistics in China, 2015[J]. CA Cancer J Clin, 2016, 6(6): 115-132.).

[0003] Traditional treatments for malignant tumors, including surgery, radiotherapy, and chemotherapy, have made significant progress over the past few decades, but they still fail to substantially improve long-term survival, especially for patients with refractory tumors such as liver cancer, glioma, pancreatic cancer, and osteosarcoma. Cytotoxic drugs and molecularly targeted drugs, currently used as first-line anti-tumor drugs in clinical practice, have limitations such as significant side effects and / or the development of drug resistance. Therefore, new, highly effective and low-toxic treatments and drugs are urgently needed for the treatment of malignant tumors.

[0004] Currently, immunotherapy is a highly sought-after cancer treatment. Unlike traditional surgery, radiotherapy, chemotherapy, and targeted drugs, which work directly, immunotherapy activates the patient's own immune system to attack tumors, minimizing the effects on normal tissue. Immunotherapy is effective for some advanced tumors and can even prevent recurrence, achieving a complete cure. The types of cancer immunotherapy drugs currently approved for clinical use by the U.S. Food and Drug Administration (FDA) include: 1. Immune checkpoint inhibitors, such as PD-1 antibodies (Nivolumab, Pembrolizumab, etc.), PD-L1 antibodies (Atezolizumab), and CTLA4 antibodies (Ipilimumab); 2. Multiple monoclonal antibodies targeting surface tumor-associated antigens, such as the anti-CD20 monoclonal antibody rituximab; 3. Two immunostimulatory cytokines (interferon IFN-α) and interleukin 2 (IL-2); 4. Immunogenic cell death inducers, such as cyclophosphamide and oxaliplatin; 5. Adoptive chimeric antigen receptor T cell therapy (CART); 5. Bacillus Calmette-Guérin (BCG); 6. Dendritic cell-based cancer vaccines; 7. Oncolytic virus (OV), etc.

[0005] Oncolytic virus therapy is a new type of anti-tumor immunotherapy that combines targeted therapy, immunotherapy and gene therapy. It can selectively infect and directly kill tumor cells, and then activate anti-tumor immune responses by exposing tumor / viral antigens and releasing cytokines, thereby directly or indirectly exerting an anti-tumor effect. In addition, the oncolytic virus itself can be used as a carrier to carry suicide genes, immune regulatory genes, pro-apoptosis genes, anti-angiogenesis genes and other genes to further regulate the tumor microenvironment and promote anti-tumor drug efficacy. Compared with conventional treatment methods currently used in clinical practice, oncolytic virus therapy has the advantages of strong killing effect, high safety and low cost.

[0006] Due to its unique multiple anti-tumor pathways, many teams of scholars are committed to researching and developing this therapy. However, to date, only four oncolytic virus products have been approved for marketing and used in the clinical treatment of tumors, including RIGVIR approved for marketing in Latvia in 2003, Ancore (H101) approved for marketing in China in 2005, IMLYGIC (T-Vec) approved for marketing in the United States in 2015, and Delytact approved for marketing in Japan in 2021.

[0007] In addition, a variety of oncolytic viruses are also in the preclinical research stage. From the original natural viruses to the gene-edited viruses, the types of oncolytic viruses have also evolved from the original herpes virus to more than ten commonly used viruses. Among them, adenovirus has become the most commonly used oncolytic virus due to its ease of gene editing and flexibility of use. Other commonly used viruses include herpes simplex virus, vaccinia virus, Newcastle disease virus, measles virus, reovirus, coxsackie virus, poliovirus, etc.

[0008] IL-18 is an immune-activating cytokine that can stimulate T cells, NK cells, and bone marrow cells. It has the ability to activate anti-tumor immune cells and can therefore be used as a candidate molecule for the treatment of cancer. However, IL-18BP (IL-18 binding protein) produced in the tumor microenvironment can act as a secretory immune checkpoint molecule, competitively binding to IL-18 and blocking its binding to the receptor, limiting the effect of IL-18 immunotherapy and preventing immune cells from activating anti-tumor immune responses. DR18 (modified IL-18) was obtained by site-directed mutagenesis of key amino acids in the IL-18BP binding site. DR18 can effectively avoid binding to IL-18BP and then effectively bind to the IL-18 receptor, thereby activating anti-tumor immune responses. By utilizing the characteristics of oncolytic viruses that specifically replicate in tumors, DR18, which has a significant immune-activating effect, can be specifically and highly concentratedly expressed at the tumor site, making its concentration in the tumor significantly higher than that in other normal tissues. This improves the efficacy while also reducing the potential risks of systemic exposure. Summary of the Invention

[0009] In one aspect, the present disclosure provides use of DR-18 in the preparation of an anti-tumor drug for use in combination with herpes simplex virus.

[0010] In one aspect, the present disclosure provides the use of herpes simplex virus in the preparation of an anti-tumor drug for use in combination with DR-18.

[0011] In one aspect, the present disclosure provides the use of DR-18 in preparing a herpes simplex virus anti-tumor synergist or drug resistance reversal agent. In another aspect, the present disclosure provides the use of herpes simplex virus in preparing a DR-18 anti-tumor synergist or drug resistance reversal agent.

[0012] Drug resistance reversal agents mean that when some oncolytic viruses are used as anti-tumor drugs to treat tumors, there are some tumors that are not very sensitive to oncolytic viruses, or these tumors are resistant to oncolytic viruses. At this time, oncolytic viruses can be used in combination with DR-18 (as a drug resistance reversal agent) to reverse the tumor's resistance to the oncolytic virus; or, conversely, when some anti-tumor substances are used to treat tumors, there are some tumors that are not very sensitive to drugs, or these tumors are resistant to the substances. At this time, oncolytic viruses (as drug resistance reversal agents) can be used in combination with these substances to reverse the tumor's resistance to the substances.

[0013] DR-18 is a mutant of IL-18 that binds to and activates the IL-18 receptor and its downstream pathways without binding to IL-18BP. For example, U.S. Patent Publication No. 2019 / 0070262 and Zhou et al., Nature (2020) 583:609-614, disclose several human DR-18s and some murine DR-18s.

[0014] In some embodiments, the DR-18 is human DR-18; in some embodiments, the human DR-18 comprises at least one mutation relative to wild-type human IL-18.

[0015] In some embodiments, the human DR-18 comprises one or more of the following mutations relative to wild-type human IL-18: M51K, K53S, Q56L, P57A, M60L, S105D, D110S, and N111R.

[0016] In some embodiments, the human DR-18 comprises the following mutations relative to wild-type human IL-18: M51K, K53S, Q56L, P57A, M60L, S105D, D110S, and N111R.

[0017] In some embodiments, the human DR-18 comprises a sequence that is at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identical to the sequence shown in SEQ ID NO:3.

[0018] In some embodiments, the amino acid sequence of human DR-18 includes a sequence that is at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identical to the sequence shown in SEQ ID NO:1; in some embodiments, the amino acid sequence of wild-type human IL-18 is as shown in SEQ ID NO:3.

[0019] In some embodiments, the DR-18 is murine DR-18; in some embodiments, the murine DR-18 comprises at least one mutation relative to wild-type murine IL-18.

[0020] In some embodiments, the murine DR-18 comprises one or more of the following mutations relative to wild-type murine IL-18: N1H, M50A, K52G, E55R, V56A, and L59K; in some embodiments, the murine DR-18 comprises the following mutations relative to wild-type murine IL-18: N1H, M50A, K52G, E55R, V56A, and L59K.

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

[0022] In some embodiments, the herpes simplex virus is herpes simplex virus type 1 and / or herpes simplex virus type 2; in some embodiments, the herpes simplex virus is selected from a wild-type strain or a naturally attenuated strain, a genetically engineered selectively attenuated strain, a gene-loaded strain, or a gene transcription-targeted strain. An example of a genetically engineered selectively attenuated strain is G207, which has a deletion of the γ34.5 gene, a neurotoxic determinant of HSV-1.

[0023] As used in this disclosure, “human DR-18” and “DR18” can be used interchangeably; “murine DR-18” and “mDR18” can be used interchangeably.

[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 (the reference sequence may be a naturally occurring normal or "wild-type" sequence), and includes translocations, deletions, insertions, and substitution / point mutations. "Mutant" or "variant" as used herein refers to a nucleic acid or protein comprising 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 the gene most commonly observed in the human population and is therefore 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 that has sequence and / or functional property modifications (i.e., altered properties) compared to the wild-type gene or gene product.

[0026] In one aspect, the present disclosure provides a technical solution for treating tumors by combining DR-18 and herpes simplex virus. In some embodiments, the "combination" is interpreted broadly, for example, a pharmaceutical composition or a pharmaceutical kit.

[0027] As used in this disclosure, the term "pharmaceutical combination" means a product resulting from the mixing or combination of more than one active ingredient, and includes fixed and non-fixed combinations of the active ingredients. As used herein, the terms "co-administration" or "administered in combination" and the like 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.

[0028] As used in this disclosure, the term "fixed combination" means that the active ingredients (eg, herpes simplex virus and DR-18) are administered to a patient simultaneously in the form of a single entity or dosage.

[0029] As used in this disclosure, the term "non-fixed combination" means that the active ingredients (e.g., herpes simplex virus and DR-18) are administered to a patient as separate entities simultaneously, concurrently, or sequentially without specific time limits, wherein such administration provides therapeutically effective levels of both compounds in vivo, preferably at the same time. For example, a non-fixed combination can be two capsules each containing one active ingredient, the purpose of which is to treat the patient with both active ingredients together in vivo.

[0030] As used in this disclosure, "combination" or "combined use" means that two or more active substances can be administered to a subject together in a mixture, simultaneously as a single formulation, or sequentially in any order (e.g., simultaneously, sequentially, spaced apart) as a single formulation.

[0031] As used herein, the term "in combination with" when referring to the administration of multiple agents to a subject refers to the administration of a first agent to a subject and at least one other (i.e., a second, third, fourth, fifth, etc.) agent. As used herein, an agent (e.g., DR-18) is considered to be administered in combination with a second agent (e.g., herpes simplex virus) if, upon administration of the second agent, the biological effect resulting from the administration of the first agent persists in the subject, such that the therapeutic effects of the first and second agents are additive. Administration of the first agent (e.g., DR-18) provides a therapeutic effect over a longer period of time, while administration of the second agent (e.g., herpes simplex virus) provides its therapeutic effect while the therapeutic effect of the first agent is still ongoing, and thus the second agent is considered to be administered in combination with the first agent, even if the first agent may be administered at a time point significantly distant from the time of administration of the second agent (e.g., days or weeks). 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), concurrently, or sequentially. In some embodiments, if the first agent and the second agent are administered within about 24 hours of each other (e.g., within about 12 hours of each other, within about 6 hours of each other, within about 2 hours of each other, or within about 30 minutes of each other), the first agent is considered to be administered "contemporaneously" with the second agent. The term "in combination with..." should also be understood to apply to situations where the first agent and the second agent are co-formulated in a single pharmaceutically acceptable formulation and the co-formulation is administered to a subject. In some embodiments, DR-18 and herpes simplex virus are administered or applied sequentially, for example, one or more other agents are administered after one agent is administered. In some embodiments, DR-18 and herpes simplex virus are administered simultaneously, for example, two or more agents are administered at the same time or approximately the same time; the two or more agents can be present in two or more separate formulations, or combined into a single formulation (i.e., a co-formulation). Regardless of whether these agents are administered sequentially or simultaneously, as used in this disclosure, they are considered to be administered / used in combination.

[0032] In one aspect, the present disclosure provides use of DR-18 in preparing an anti-tumor drug combination, wherein the drug combination comprises herpes simplex virus in combination with DR-18.

[0033] In one aspect, the present disclosure provides use of herpes simplex virus in preparing an anti-tumor drug combination, wherein the drug combination comprises DR-18 in combination with herpes simplex virus.

[0034] In one aspect, the present disclosure provides a drug combination for treating tumors, comprising:

[0035] DR-18, and

[0036] Herpes simplex virus.

[0037] In some embodiments, the pharmaceutical combination is a pharmaceutical composition or a pharmaceutical set; in some embodiments, the pharmaceutical composition comprises a mixture of DR-18 and herpes simplex virus; in some embodiments, the pharmaceutical set comprises separately packaged DR-18 and separately packaged herpes simplex virus.

[0038] In one aspect, the present disclosure provides a pharmaceutical composition for treating tumors, comprising:

[0039] DR-18, and herpes simplex virus.

[0040] In one aspect, the present disclosure provides a pharmaceutical kit comprising:

[0041] DR-18, and herpes simplex virus.

[0042] In some embodiments, the pharmaceutical kit comprises separately packaged DR-18 and separately packaged herpes simplex virus.

[0043] A pharmaceutical kit differs from a composition in that DR-18, unlike the herpes simplex virus, is packaged separately (e.g., a pill, capsule, tablet, or ampoule containing DR-18; another pill, capsule, tablet, or ampoule containing herpes simplex virus). In some embodiments, herpes simplex virus, DR-18, and combinations of herpes simplex virus and DR-18 may also contain one or more adjuvants. An adjuvant refers to an ingredient in a pharmaceutical composition that aids in the efficacy of the drug. A pharmaceutical kit may also include separately packaged DR-18 and separately packaged herpes simplex virus. The DR-18 and herpes simplex virus in the pharmaceutical kit may be administered simultaneously or in any order, such as administering DR-18 before the herpes simplex virus, after the herpes simplex virus, or both simultaneously. In various embodiments, the patient may be a mammal.

[0044] In some embodiments, the composition / drug set further comprises a pharmaceutically acceptable carrier.

[0045] In some embodiments, the ratio of DR-18 to herpes simplex virus is about: 0.01-200 mg: 10 3 -10 9 PFU; In some embodiments, the ratio of DR-18 to herpes simplex virus is about 0.1-200 mg: 10 4 -10 9 PFU; In some embodiments, the ratio of DR-18 to herpes simplex virus is about 0.1-100:10 5 -10 9 PFU.

[0046] In some embodiments, the dosage range of DR-18 is about 0.01-10 mg / kg, and the titer of herpes simplex virus is about MOI 10 3 -10 9 PFU / kg; in some embodiments, the dosage range of DR-18 is about 0.1-5 mg / kg, and the titer of herpes simplex virus is about MOI 10 4 -10 9 PFU / kg; in some embodiments, the dosage range of DR-18 is about 0.05-0.5 mg / kg, and the titer of herpes simplex virus is about MOI 10 5 -10 9 PFU / kg.

[0047] In some embodiments, the DR-18 is administered by intraperitoneal injection; in some embodiments, the herpes simplex virus is administered by intratumoral injection or intravenous injection.

[0048] In one aspect, the present disclosure also provides the use of the drug combination / drug composition / drug kit in the preparation of drugs for treating tumors.

[0049] In some embodiments, the tumor is a solid tumor or a hematological tumor.

[0050] In some embodiments, the solid tumor is selected from one or more of intestinal cancer, pancreatic cancer, liver cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, nasopharyngeal cancer, lung cancer, sarcoma or gastric cancer.

[0051] In some embodiments, the present invention provides a method for treating tumors, comprising: administering any one of the drug combinations to a subject suffering from a tumor.

[0052] In some embodiments, the nucleotide sequence of the human DR-18 is as shown in SEQ ID NO:5.

[0053] In some embodiments, the nucleotide sequence of the murine DR-18 is shown in SEQ ID NO:6.

[0054] In some embodiments, when some oncolytic viruses are used in combination with DR-18, unpredictable and diverse effects occur. The inventors have discovered that some of them exhibit obvious mutual antagonism or inhibition. However, unlike these phenomena, the combination of herpes simplex virus and DR-18 exhibits significant synergistic effects in tests on multiple tumors. This is a surprising discovery that holds great promise for development. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 shows the effects of the inactivated supernatants of different tumor cells infected with various oncolytic viruses in Example 1 on DR18 activity, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0056] Figure 2 shows the effects of various OVs-SNs on DR18 activity in Example 1, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates that the difference is not statistically significant.

[0057] FIG3 is a curve showing changes in animal body weight in Example 2, where ns indicates that the difference is not statistically significant.

[0058] Figure 4 is the tumor volume growth curve of the HSV and mDR18 combination group in Example 2, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates that the difference is not statistically significant.

[0059] Figure 5 is a tumor volume growth curve of the ADV and mDR18 combination group in Example 2, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates that the difference is not statistically significant.

[0060] Figure 6 is a tumor volume growth curve of the VSV and mDR18 combination group in Example 2, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates that the difference is not statistically significant.

[0061] FIG7 is a curve showing changes in animal body weight in Example 3, where ns indicates that the difference is not statistically significant.

[0062] Figure 8 is the tumor volume growth curve of the HSV and mDR18 combination group in Example 3, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates that the difference is not statistically significant.

[0063] Figure 9 is a tumor volume growth curve of the ADV and mDR18 combination group in Example 3, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates that the difference is not statistically significant.

[0064] Figure 10 shows the weight change curves of the animals in Example 4. ns indicates that the difference was not statistically significant. Among them: A. Mouse lymphoma A20 tumor-bearing model; B. Mouse liver cancer H22 tumor-bearing model; C. Mouse kidney cancer Renca tumor-bearing model; D. Mouse breast cancer 4T1 tumor-bearing model; E. Mouse breast cancer EMT6 tumor-bearing model; F. Mouse colon cancer CT26.WT tumor-bearing model; G. Mouse lymphoma EL4 tumor-bearing model; H. Mouse pancreatic cancer mPAKPC. tumor-bearing model; I. Mouse prostate cancer RM-1 tumor-bearing model; J. Mouse lung cancer LLC1 tumor-bearing model; K. Mouse colon cancer MC38 tumor-bearing model; L. Mouse melanoma B16-F10 tumor-bearing model; M. Mouse pancreatic cancer Pan02 tumor-bearing model; N. Mouse bladder cancer MB49 tumor-bearing model.

[0065] Figure 11 is a tumor volume growth curve in Example 4, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates that the difference is not statistically significant. Among them: A. Mouse lymphoma A20 tumor-bearing model; B. Mouse liver cancer H22 tumor-bearing model; C. Mouse kidney cancer Renca tumor-bearing model; D. Mouse breast cancer 4T1 tumor-bearing model; E. Mouse breast cancer EMT6 tumor-bearing model; F. Mouse colon cancer CT26.WT tumor-bearing model; G. Mouse lymphoma EL4 tumor-bearing model; H. Mouse pancreatic cancer mPAKPC. tumor-bearing model; I. Mouse prostate cancer RM-1 tumor-bearing model; J. Mouse lung cancer LLC1 tumor-bearing model; K. Mouse colon cancer MC38 tumor-bearing model; L. Mouse melanoma B16-F10 tumor-bearing model; M. Mouse pancreatic cancer Pan02 tumor-bearing model; N. Mouse bladder cancer MB49 tumor-bearing model. DETAILED DESCRIPTION

[0066] The following is a detailed description of the technical solution of the present invention, which does not limit the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0067] Unless otherwise specified, the materials and experimental methods used in the present invention are conventional materials and methods.

[0068] Example 1 Using the IL-18 reporter cell model, the effect of supernatants from different tumor cells infected with various oncolytic viruses on DR18 activity was evaluated.

[0069] IL-18 reporter cell model: These cells express the IL-18 receptor, IL-18 downstream pathway molecules, and luciferase. IL-18 binding to the receptor mediates luciferase expression, and the readout represents pathway activation. This pathway is a key pathway for DR18 to activate anti-tumor immunity in vivo and can indirectly reflect DR18's anti-cancer activity.

[0070] 1. Experimental Materials

[0071] 1.1 Oncolytic virus: HSV: attenuated oncolytic herpes simplex virus type 1 with the neurovirulence factor γ34.5 gene knocked out, purchased from Wuhan Shumi.

[0072] ADV: Oncolytic adenovirus with knockout of the E1a-CR2-24bp gene and the E3 region; VSV: Attenuated oncolytic vesicular stomatitis virus with knockout of the G gene and V48R and M51R mutations in the M gene; REO: Oncolytic reovirus serotype 3. These products were commercially available, custom-made using conventional methods, or constructed by Guangzhou Weirongte Pharmaceutical Technology Co., Ltd. using conventional methods.

[0073] 1.2 Experimental cells: IL-18 reporter cells (H_IL18 Reporter 293 Cell Line), prostate cancer cells (DU145), bladder cancer cells (ScaBER, J82, HT1376, 5637), pancreatic cancer cells (MIA-Paca-2, Capan2), cervical cancer cells (HCC94), colorectal cancer cells (DLD-1), hepatocellular carcinoma cells (Hep3B), lung cancer cells (HCC-827), glioma cells (LN18, U-138MG), and osteosarcoma cells (MNNG / HOS) were purchased from ATCC, the Cell Bank of the Committee for Type Culture Collection of the Chinese Academy of Sciences, the China Center for Type Culture Collection, Geneo Biotechnology, Saiku Biotechnology, Foxai Biotechnology, and Tongpai Biotechnology.

[0074] 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, ONE-Glo TM Luciferase Assay System.

[0075] 1.4 Experimental instruments: inverted microscope, biological safety cabinet, carbon dioxide incubator, microplate reader.

[0076] 2. Experimental Methods

[0077] 2.1 Establishment of tumor cell model:

[0078] According to the instructions of each tumor cell, appropriate culture conditions and passage ratios were selected for cell amplification and passage. When the cells entered the logarithmic growth phase and the number was sufficient, the cells were cultured at 3×10 4 / cm 2Inoculate into T25 cell culture flasks.

[0079] 2.2 Oncolytic virus infection of tumor cells:

[0080] 16 to 24 hours after tumor cell inoculation, complete cell attachment was observed under a microscope, and each tumor cell was infected with the virus at an MOI of 1 PFU / cells according to the number of cells plated.

[0081] 2.3 Collection and inactivation of infection supernatant:

[0082] Forty-eight hours after oncolytic virus infection, tumor cells were observed microscopically and photographed to document cytopathic effects. Supernatant (SN) from infected cells was inactivated by UV irradiation or filtered through a 0.1 μm filter, depending on viral characteristics. The inactivated supernatant was collected into 4.5 mL cryovials and stored at -80°C until further use. Uninfected supernatant was collected in the same manner as a control.

[0083] 2.4 Effect of infection supernatant on DR18 biological activity:

[0084] ① IL-18 reporter cells were revived and subcultured. When the cells entered the logarithmic growth phase and the cell number was sufficient, the cells were cultured at a rate of 1.5×10 4 90 μL of culture medium was inoculated into the middle well of a 96-well plate, and 200 μL of PBS was added to the surrounding wells to seal the edges.

[0085] ② After culturing the cells in a 37°C 5% CO2 cell culture incubator for 16-24 hours, remove the 96-well plate and observe under a microscope to ensure that the cells are completely attached to the wall and have good morphology. Use the inactivated supernatant and control supernatant from step 2.3 to make a 3-fold isocratic gradient dilution of the DR18 protein. After thorough mixing, add 10 μL per well to the cells.

[0086] ③ After adding DR18 protein, the cells were cultured in a 37°C 5% CO2 cell culture incubator for 16 hours.

[0087] ④Add 100 μL ONE-Glo TM The substrate was reacted in the dark for 3 minutes to allow the cells to completely lyse. The cell lysate was transferred to a 96-well luminescent plate and Luciferase was detected by a microplate reader.

[0088] 2.5 Data Processing:

[0089] According to the relative luminescence unit (RLU) of each well obtained by detection, the formula is: relative luminescence rate (% of Max) = (RLU 样品 -RLU 空白 ) / (RLU 最大 -RLU 空白)×100%, calculate the relative luminescence rate of cells in each well, use software to draw a fitting curve, and apply the [Agonist] vs. response--Variable slope (four parameters) analysis equation to calculate the half-maximal effect concentration (EC50) value of DR18 biological activity. And normalize the EC50 of each group to obtain the relative EC50 value (Relative Value) = EC50 处理组 / EC50 对照组 .

[0090] 2.6 Biological Statistics:

[0091] * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001, indicating that the data differences are statistically significant.

[0092] 3. Experimental results:

[0093] 3.1 The effect of inactivated supernatants of different tumor cells infected with different oncolytic viruses on DR18 activity is shown in Figure 1. DR18 recombinant protein was serially diluted using the supernatants of different tumor cells infected with different oncolytic viruses and treated with IL-18 reporter cells for 16 hours. Luciferase luminescence units were detected on a microscope, and fitting curves were drawn and the EC50 of each treatment group was calculated (see Figure 2). The statistical analysis results are shown in Figure 1.

[0094] The results showed that compared with the control group (CON) without virus infection, oncolytic herpes simplex virus (HSV) infection of prostate cancer cells (DU145), bladder cancer cells (ScaBER, J82, HT1376, 5637), pancreatic cancer cells (MIA-Paca-2, Capan2), cervical cancer cells (HCC94), intestinal cancer cells (DLD-1), liver cancer cells (Hep3B), glioma cells (LN18, U-138MG), and osteosarcoma cells (MNNG / HOS) could significantly promote the biological activity of DR18.

[0095] Oncolytic vesicular stomatitis virus (VSV) infection of prostate cancer cells (DU145), bladder cancer cells (ScaBER, J82), pancreatic cancer cells (MIA-Paca-2, Capan2), cervical cancer cells (HCC94), glioma cells (LN18, U-138MG), and osteosarcoma cells (MNNG / HOS) can significantly promote the biological activity of DR18.

[0096] Oncolytic adenovirus (ADV) infection of prostate cancer cells (DU145), bladder cancer cells (J82, HT1376, 5637), pancreatic cancer cells (MIA-Paca-2, Capan2), cervical cancer cells (HCC94), liver cancer cells (Hep3B), glioma cells (LN18, U-138MG), and osteosarcoma cells (MNNG / HOS) can significantly promote the biological activity of DR18.

[0097] Oncolytic reovirus (REO) infection of bladder cancer cells (J82, HT1376), pancreatic cancer cells (Capan2), cervical cancer cells (HCC94), intestinal cancer cells (DLD-1), liver cancer cells (Hep3B), and glioma cells (U-138MG) can significantly promote the biological activity of DR18, but inhibit the biological activity of DR18 in prostate cancer cells (DU145) and bladder cancer cells (ScaBER).

[0098] The experimental results show that the supernatants of different tumor cell models infected with various oncolytic viruses have differentiated effects on the biological activity of DR18, among which HSV has a stronger enhancing effect than other oncolytic viruses.

[0099] Example 2 Safety and efficacy study of mDR18 combined with various oncolytic viruses in an immunocompetent mouse liver cancer model

[0100] 1. Experimental Materials

[0101] 1.1 Oncolytic Viruses: HSV: attenuated oncolytic herpes simplex virus type 1 with the neurovirulence factor γ34.5 gene knockout; ADV: oncolytic adenovirus with the E1a-CR2-24bp gene and E3 region gene knockout; VSV: attenuated oncolytic vesicular stomatitis virus with the G gene knockout and the M gene V48R and M51R mutations. Viruses were purchased from Wuhan Shumi, Fubaiao Biotechnology, and other institutions.

[0102] 1.2 Recombinant protein: modified mouse interleukin-18 (mDR18) recombinant protein (SEQ ID NO: 2).

[0103] 1.3 Tumor cells: Mouse hepatoma cell line H22 was purchased from China Center for Type Culture Collection.

[0104] 1.4 Experimental animals: Female BALB / c mice aged 5-7 weeks.

[0105] 2. Experimental Methods

[0106] 2.1 Establishment of tumor-bearing mouse model:

[0107] The revived tumor cells were subcultured. After the cells entered the logarithmic growth phase and grew to a sufficient number, the cells were collected, counted, and cell suspensions were prepared. H22 cells were cultured at 2×10 6 cells / mouse, and inoculated subcutaneously on the back of Balb / c mice.

[0108] 2.2 Grouped Dosage:

[0109] After the tumor is formed, the tumor volume range is 100±40mm 3 The mice were randomly divided into 8 groups, namely, excipient control group, mDR18 single-use group, HSV single-use group, HSV+mDR18 combination group, ADV single-use group, ADV+mDR18 combination group, VSV single-use group, and VSV+mDR18 combination group, with 5 animals in each group; the mDR18 dosage was 0.32 mg / kg and the administration method was intraperitoneal injection, and the administration cycle was 2 times a week for a total of 5 times; the dosage of each oncolytic virus was 2×10 6 PFU / mouse, administered by intratumoral injection, once. The day of tumor inoculation was designated as D0, and the mice were observed for 21 days after administration.

[0110] 2.3 Mouse body weight and tumor measurement:

[0111] During tumor formation and drug administration, the body weight of mice and the growth of tumor were recorded every 3-4 days.

[0112] 2.4 Data Processing:

[0113] The measured data were statistically analyzed and the tumor volume growth curve of each group of mice was drawn.

[0114] The calculation formula for tumor volume (TV) is: V = 1 / 2 × a × b 2 , where a and b represent the long and wide diameters of the tumor, respectively.

[0115] 2.5 Statistical Analysis

[0116] * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001, indicating that the data differences are statistically significant.

[0117] 3. Experimental results:

[0118] The body weights of the animals in each group during the experiment are shown in Figure 3. The body weights of the animals in each group maintained a steady upward trend during the experiment. Compared with the excipient control group, there was no significant difference in the single-drug or combined-drug groups. This indicates that the twice-weekly intraperitoneal injection of 0.32 mg / kg mDR18 or a single intratumoral injection of 2×10 6 Each oncolytic virus at a concentration of PFU / mouse did not cause obvious drug-related side effects in the immunocompetent mouse model.

[0119] Based on the animal tumor volume growth records, a tumor volume change curve was plotted, as shown in Figure 4, for efficacy evaluation. The results showed that compared with the excipient control group, mDR18 alone (P < 0.001) or in combination with HSV (P < 0.001) significantly inhibited tumor growth in the mouse liver cancer model. Compared with the mDR18 alone group, the combination of HSV and mDR18 significantly enhanced the tumor inhibitory effect in the mouse liver cancer model (P < 0.001). Compared with the HSV alone group, the combination of HSV and mDR18 significantly enhanced the tumor inhibitory effect in the mouse liver cancer model (P < 0.001), indicating that the combination of HSV and mDR18 has a synergistic effect.

[0120] Based on the animal tumor volume growth records, a tumor volume change curve was plotted, as shown in Figure 5, for efficacy evaluation. The results showed that compared with the excipient control group, mDR18 alone (P < 0.01) or in combination with ADV (P < 0.001) significantly inhibited tumor growth in the mouse liver cancer model. Compared with the mDR18 alone group, the difference in tumor inhibition between the ADV and mDR18 combination group was not statistically significant, indicating that the combination of ADV and mDR18 had no synergistic effect.

[0121] Based on the animal tumor volume growth records, a tumor volume change curve was plotted, as shown in Figure 6, for efficacy evaluation. The results showed that compared with the excipient control group, mDR18 alone (P < 0.01) or in combination with VSV (P < 0.001) significantly inhibited tumor growth in the mouse liver cancer model. Compared with the mDR18 alone group, the difference in tumor inhibition between the VSV and mDR18 combination group was not statistically significant, indicating that the combination of VSV and mDR18 had no synergistic effect.

[0122] Example 3 Safety and efficacy study of mDR18 combined with various oncolytic viruses in an immunocompetent mouse colorectal cancer model

[0123] 1. Experimental Materials

[0124] 1.1 Oncolytic virus: HSV: attenuated oncolytic herpes simplex virus type 1 with the neurovirulence factor γ34.5 gene knocked out; ADV: oncolytic adenovirus with the E1a-CR2-24bp gene and E3 region gene knocked out.

[0125] 1.2 Recombinant protein: Modified mouse interleukin-18 (mDR18) recombinant protein.

[0126] 1.3 Tumor cells: Mouse colorectal cancer cell line CT26 was purchased from Guangzhou Geneo Biotechnology Co., Ltd.

[0127] 1.4 Experimental animals: Female Balb / c mice aged 5-7 weeks.

[0128] 2. Experimental Methods

[0129] 2.1 Establishment of tumor-bearing mouse model:

[0130] The revived tumor cells were subcultured. After the cells entered the logarithmic growth phase and grew to a sufficient number, the cells were collected, counted, and cell suspensions were prepared. CT26 cells were cultured at 2×10 6 cells / mouse, and inoculated subcutaneously on the back of Balb / c mice.

[0131] 2.2 Grouped Dosage:

[0132] After the tumor is formed, the tumor volume range is 100±40mm 3 The mice were randomly divided into 8 groups, namely, the excipient control group, the mDR18 single-use group, the HSV single-use group, the HSV+mDR18 combination group, the ADV single-use group, and the ADV+mDR18 combination group, with 4 animals in each group; the mDR18 dosage was 0.32 mg / kg and the administration method was intraperitoneal injection, and the administration cycle was 2 times a week for a total of 5 times; the dosage of each oncolytic virus was 2×10 6 PFU / mouse, administered by intratumoral injection, once. The day of tumor inoculation was designated as D0, and the mice were observed for 14 days after administration.

[0133] 2.3 Mouse body weight and tumor measurement:

[0134] During tumor formation and drug administration, the body weight of mice and the growth of tumor were recorded every 3-4 days.

[0135] 2.4 Data Processing:

[0136] The measured data were statistically analyzed and the tumor volume growth curve of each group of mice was drawn.

[0137] The calculation formula for tumor volume (TV) is: V = 1 / 2 × a × b 2 , where a and b represent the long and wide diameters of the tumor, respectively.

[0138] 2.5 Statistical Analysis

[0139] * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001, indicating that the data differences are statistically significant.

[0140] 3. Experimental results:

[0141] The weight of the animals in each group during the experiment is shown in Figure 7. The weight of the animals in each group maintained a steady upward trend during the experiment. Compared with the excipient control group, there was no significant difference in the single or combined use of each drug group. This indicates that the twice-weekly intraperitoneal injection of 0.32 mg / kg mDR18 or a single intratumoral injection of 2×106 Each oncolytic virus at a concentration of PFU / mouse did not cause obvious drug-related side effects in the immunocompetent mouse model.

[0142] Based on the animal tumor volume growth records, a tumor volume change curve was plotted, as shown in Figure 8, for efficacy evaluation. The results showed that compared with the excipient control group, the combination of HSV and mDR18 significantly inhibited tumor growth in the mouse liver cancer model (P < 0.05). Compared with the HSV alone group, the combination of HSV and mDR18 significantly enhanced the tumor inhibitory effect in the mouse liver cancer model (P < 0.05). Compared with the mDR18 alone group, the combination of HSV and mDR18 significantly enhanced the tumor inhibitory effect in the mouse liver cancer model (P < 0.05), indicating that the combination of HSV and mDR18 has a synergistic effect.

[0143] Based on the animal tumor volume growth records, a tumor volume change curve was plotted, as shown in Figure 9, for efficacy evaluation. The results showed that compared with the excipient control group, mDR18 alone (P < 0.001) or in combination with ADV (P < 0.05) significantly inhibited tumor growth in the mouse liver cancer model. Compared with the mDR18 alone group, the difference in tumor inhibition between the ADV and mDR18 combination group was not statistically significant, indicating that the combination of ADV and mDR18 had no synergistic effect.

[0144] Example 4 Safety and efficacy study of mDR18 combined with HSV in an immunocompetent mouse tumor model

[0145] 1. Experimental Materials

[0146] 1.1 Oncolytic virus: HSV: attenuated oncolytic herpes simplex virus type 1 with the neurovirulence factor γ34.5 gene knocked out;

[0147] 1.2 Recombinant protein: Modified mouse interleukin-18 (mDR18) recombinant protein.

[0148] 1.3 Tumor cells: A. Mouse lymphoma cell A20; B. Mouse liver cancer cell H22; C. Mouse renal cancer cell Renca; D. Mouse breast cancer cell 4T1; E. Mouse breast cancer cell EMT6; F. Mouse colon cancer cell CT26.WT; G. Mouse lymphoma cell EL4; H. Mouse pancreatic cancer cell mPAKPC; I. Mouse prostate cancer cell RM-1; J. Mouse lung cancer cell LLC1; K. Mouse colon cancer cell MC38; L. Mouse melanoma cell B16-F10; M. Mouse pancreatic cancer cell Pan02; N. Mouse bladder cancer cell MB49; Cells were obtained from Guangdong Yaokang Biotechnology Co., Ltd.

[0149] 1.4 Experimental animals: Female BALB / c mice, 6-8 weeks old; female C57BL / 6 mice, 6-8 weeks old; male C57BL / 6 mice, 6-8 weeks old.

[0150] 2.1 Establishment of tumor-bearing mouse model:

[0151] The revived tumor cells were subcultured and after the cells entered the logarithmic growth phase and grew to a sufficient number, the cells were collected and counted to prepare a cell suspension: A. A20 cells were cultured at 1×10 6 cells / mouse, were inoculated on the right side of the thigh of BALB / c mice; B.H22 cells were inoculated at 1×10 6 cells / mouse, inoculated on the right back thigh of BALB / c mice; C. Renca cells were inoculated at 5×10 5 cells / mouse, inoculated on the right back thigh of BALB / c mice; D.4T1 cells were inoculated at 5×10 5 E.EMT6 cells were inoculated at 2×10 6 cells / mouse, inoculated on the right back thigh of BALB / c mice; F.CT26.WT cells were inoculated at 3×10 5 cells / mouse, were inoculated on the right back thigh of BALB / c mice; G.EL4 cells were inoculated at 5×10 4 cells / mouse, were inoculated on the right back thigh of BALB / c mice; H.mPAKPC cells were inoculated at 5×10 5 cells / mouse, inoculated into the right forelimb of BALB / c mice; I. RM-1 cells were inoculated at 1×10 6 cells / mouse, inoculated on the right side of the thigh of C57BL / 6 mice; J.LLC1 cells were inoculated at 3×10 5 cells / mouse, inoculated on the right side of the thigh of C57BL / 6 mice; K.MC38 cells were inoculated at 1×10 6 cells / mouse, inoculated into the right forelimb of C57BL / 6 mice; L.B16-F10 cells were inoculated at 5×10 5 cells / mouse, inoculated into the right forelimb of C57BL / 6 mice; M.Pan02 cells were inoculated at 5×10 6 MB49 cells were inoculated subcutaneously into the right forelimb of C57BL / 6 mice; N.MB49 cells were inoculated at 5×10 6 cells / mouse, inoculated subcutaneously into the right forelimb of C57BL / 6 mice.

[0152] 2.2 Grouped Dosage:

[0153] After the tumor is formed, the tumor volume range is 60-100mm 3The mice were randomly divided into two groups: the auxiliary material control group and the HSV+mDR18 group, with 5 animals in each group; the mDR18 dosage was 0.32 mg / kg and the administration method was intraperitoneal injection, and the administration cycle was 2 times a week for a total of 8 times; the HSV dosage was 2×10 6 PFU / mouse, administered via intratumoral injection, once. The day of tumor inoculation was designated D0, and observation was continued for 1 month after administration or at the ethical endpoint.

[0154] 2.3 Mouse body weight and tumor measurement:

[0155] During tumor formation and drug administration, the body weight of mice and the growth of tumor were recorded every 3-4 days.

[0156] 2.4 Data Processing:

[0157] The measured data were statistically analyzed and the tumor volume growth curve of each group of mice was drawn.

[0158] The calculation formula for tumor volume (TV) is: V = 1 / 2 × a × b 2 , where a and b represent the long and wide diameters of the tumor, respectively.

[0159] 2.5 Statistical Analysis

[0160] * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001, indicating that the data differences are statistically significant.

[0161] 3. Experimental results:

[0162] The weight of the animals in each group during the experiment is shown in Figure 10. The weight of the animals in each group maintained a steady upward trend during the experiment. Compared with the auxiliary material control group, there was no significant difference in the HSV and mDR18 combination group, indicating that the twice-weekly intraperitoneal injection of 0.32 mg / kg mDR18 or a single intratumoral injection of 2×10 6 PFU / mouse HSV did not cause obvious drug-related side effects in the immunocompetent mouse model.

[0163] The tumor volume change curve was drawn based on the tumor volume growth record of the animals as shown in Figure 11 for drug efficacy evaluation. The results showed that compared with the excipient control group, the combination of HSV and mDR18 could significantly inhibit the growth of mouse lymphoma cells A20 (P<0.001), mouse liver cancer cells H22 (P<0.001), mouse renal cancer cells Renca (P<0.001), mouse breast cancer cells 4T1 (P<0.001), mouse breast cancer cells EMT6 (P<0.001), mouse colon cancer cells CT26.WT (P<0.001), mouse lymphoma cells EL4 (P<0.001), and mouse ovarian cancer cells EL5 (P<0.001). <0.001), mouse pancreatic cancer cells mPAKPC (P<0.001), mouse prostate cancer cells RM-1 (P<0.001), mouse lung cancer cells LLC1 (P<0.001), mouse colon cancer cells MC38 (P<0.001), mouse melanoma cells B16-F10 (P<0.001), mouse pancreatic cancer cells Pan02 (P<0.001), and mouse bladder cancer cells MB49 (P<0.001) in vivo.

[0164] The embodiments described in the present disclosure are merely illustrative examples, and the embodiments of the present disclosure are not subject to the above limitations. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present disclosure shall be considered equivalent replacement methods and shall be included in the scope of protection of the present disclosure.

Claims

1. Application of DR-18 in the preparation of anti-tumor drugs used in combination with herpes simplex virus.

2. Application of herpes simplex virus in the preparation of anti-tumor drugs used in combination with DR-18.

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

3.

4. The use according to claim 1 or 2, wherein: The DR-18 is mouse DR-18; Preferably, the murine DR-18 comprises at least one mutation relative to wild-type murine IL-18; Preferably, the murine DR-18 comprises one or more of the following mutations relative to wild-type murine IL-18: N1H, M50A, K52G, E55R, V56A and L59K; Preferably, the murine DR-18 comprises the following mutations relative to wild-type murine IL-18: N1H, M50A, K52G, E55R, V56A and L59K; Preferably, the murine DR-18 comprises a sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequence shown in SEQ ID NO:4; Preferably, the amino acid sequence of murine DR-18 comprises a sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequence shown in SEQ ID NO: 2; Preferably, the amino acid sequence of the wild-type murine IL-18 is as shown in SEQ ID NO:

4.

5. The use according to claim 1 or 2, wherein: The herpes simplex virus is herpes simplex virus type I and / or herpes simplex virus type II.

6. A drug combination for treating tumors, comprising: DR-18, and Herpes simplex virus.

7. The pharmaceutical combination according to claim 6, wherein The drug combination is a drug composition or a drug set; Preferably, the pharmaceutical composition comprises a mixture of DR-18 and herpes simplex virus; Preferably, the pharmaceutical kit comprises independently packaged DR-18 and independently packaged herpes simplex virus.

8. The pharmaceutical combination according to any one of claims 6 to 7, wherein: The DR-18 is human DR-18; Preferably, the human DR-18 comprises at least one mutation relative to wild-type human IL-18; Preferably, the human DR-18 comprises one or more of the following mutations relative to wild-type human IL-18: M51K, K53S, Q56L, P57A, M60L, S105D, D110S and N111R; Preferably, the human DR-18 comprises the following mutations relative to wild-type human IL-18: M51K, K53S, Q56L, P57A, M60L, S105D, D110S and N111R; Preferably, the human DR-18 comprises a sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 3; Preferably, the amino acid sequence of human DR-18 comprises a sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 1; Preferably, the amino acid sequence of the wild-type human IL-18 is as shown in SEQ ID NO: 3; Preferably, the DR-18 is mouse DR-18; Preferably, the murine DR-18 comprises at least one mutation relative to wild-type murine IL-18; Preferably, the murine DR-18 comprises a sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequence shown in SEQ ID NO:4; Preferably, the amino acid sequence of murine DR-18 comprises a sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequence shown in SEQ ID NO: 2; Preferably, the amino acid sequence of the wild-type murine IL-18 is as shown in SEQ ID NO: 4; Preferably, the herpes simplex virus is herpes simplex virus type I and / or herpes simplex virus type II; Preferably, the composition / drug kit further comprises a pharmaceutically acceptable carrier.

9. The pharmaceutical combination according to any one of claims 6 to 8, wherein The ratio of DR-18 to herpes simplex virus is about: 0.01-200 mg: 10 3 -10 9 PFU; preferably about 0.1-200mg: 10 4 -10 9 PFU; preferably about 0.1-100:10 5 -10 9 PFU; Preferably, the dosage is: the dosage range of DR-18 is about 0.01-10 mg / kg, and the titer of herpes simplex virus is about MOI 10 3 -10 9 PFU / kg; preferably, the dosage range of DR-18 is about 0.1-5 mg / kg, and the titer of herpes simplex virus is about MOI 10 4 -10 9 PFU / kg; preferably, the dosage range of DR-18 is about 0.05-0.5 mg / kg, and the titer of herpes simplex virus is about MOI 10 5 -10 9 PFU / kg; Preferably, the DR-18 is administered by intraperitoneal injection; Preferably, the herpes simplex virus is administered by intratumoral injection or intravenous injection.

10. The use / drug combination according to any one of claims 1 to 9, wherein: The tumor is a solid tumor or a blood tumor; Preferably, the solid tumor is selected from one or more of intestinal cancer, pancreatic cancer, liver cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, nasopharyngeal carcinoma, lung cancer, sarcoma or gastric cancer.

11. A method for treating a tumor, comprising: Administering the pharmaceutical combination of any one of claims 6 to 9 to a subject suffering from a tumor.

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