Application of DR-18 and herpes simplex virus in preparation of antitumor drugs

Abstract

The invention belongs to the field of biological medicine, and relates to application of DR-18 and herpes simplex virus in preparation of antitumor drugs. The invention discovers that the combined use of the herpes simplex virus and the DR-18 has a synergistic anti-tumor effect for the first time. The invention also relates to an anti-tumor medicine composition containing the herpes simplex virus and the DR-18, and application of the herpes simplex virus and the DR-18 in preparation of anti-tumor medicines.

Description

Technical Field

[0001] The invention 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 that threatens human health. According to the Global Cancer Report, there were 19.29 million new cancer cases and 9.95 million cancer deaths worldwide in 2020 (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, an increase of 47% compared to 2020, and the largest increase in cases will be in countries with low or medium levels of development, at 95% and 64%, respectively (CAO Maomao, CHEN Wanqing. Interpretation of GLOBOCAN 2020 global cancer statistics [J]. Chinese Journal of Frontier Medicine (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 include surgery, radiotherapy, and chemotherapy. Significant progress has been made in the past few decades, but they still cannot substantially improve the long-term survival rate of patients, especially for patients with refractory tumors such as liver cancer, glioma, pancreatic cancer, and osteosarcoma. Cytotoxic drugs and molecular targeted drugs are now used as first-line clinical anti-tumor drugs, but they have the limitations of large toxic side effects and / or easy to develop drug resistance. Therefore, the treatment of malignant tumors urgently needs new treatment methods and drugs with high efficiency and low toxicity.

[0004] Currently, immunotherapy is a highly sought-after cancer treatment method. Unlike traditional surgery, radiotherapy, chemotherapy and the direct effects of targeted drugs, immunotherapy activates the patient's own immune system to kill tumors, with little impact on normal tissues. Immunotherapy is still effective for some advanced tumors and can even prevent tumor recurrence, achieving a complete cure. The types of drugs currently approved for clinical use by the U.S. Food and Drug Administration (FDA) for cancer immunotherapy 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, oxaliplatin, etc.; 5. Adoptive chimeric antigen receptor T cell therapy (CART); 5. Bacillus Calmette-Guérin (BCG); 6. Dendritic cell-based cancer vaccines; 7. Oncolytic viruses (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, immunomodulatory genes, pro-apoptosis genes, anti-angiogenesis 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 action 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, Ankorui (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 virus to the gene-edited virus, and the types of oncolytic viruses have also developed from the initial herpes virus to more than ten commonly used viruses, among which 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, polio virus, 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 making it impossible for immune cells to activate anti-tumor immune responses. DR18 (modified IL-18) was obtained by site-directed mutagenesis of the 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 to activate anti-tumor immune responses. By utilizing the characteristics of oncolytic viruses that replicate specifically 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, thereby improving the efficacy while also reducing the potential risks of systemic exposure. Summary of the invention

[0009] In one aspect, the present invention provides the 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 invention 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 invention provides the use of DR-18 in the preparation of an anti-tumor synergist or drug resistance reversal agent for herpes simplex virus. In another aspect, the present invention provides the use of herpes simplex virus in the preparation of an anti-tumor synergist or drug resistance reversal agent for DR-18.

[0012] 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 resistance reversal agents) 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 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 type of mutant IL-18 that binds to and activates the IL-18 receptor and its downstream pathways, and does not bind to IL-18BP. For example, U.S. Patent Publication No. 2019 / 0070262 and Zhou et al., Nature (2020) 583: 609-614 disclose some human DR-18 and some mouse DR-18.

[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; 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; in some embodiments, the amino acid sequence of the human DR-18 is as shown in SEQ ID NO:1; in some embodiments, the amino acid sequence of the wild-type human IL-18 is as shown in SEQ ID NO:3.

[0016] 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.

[0017] 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.

[0018] In some embodiments, the amino acid sequence of the murine DR-18 is shown as SEQ ID NO:2; in some embodiments, the amino acid sequence of the wild-type murine IL-18 is shown as SEQ ID NO:4.

[0019] In some embodiments, the herpes simplex virus is herpes simplex virus type I and / or herpes simplex virus type II; in some embodiments, the herpes simplex virus is selected from a wild-type virus strain or a naturally attenuated strain, a genetically engineered selectively attenuated strain, a gene-loaded virus strain, or a gene transcription-targeted virus strain. An example of the genetically engineered selectively attenuated strain is G207, which has deleted the γ34.5 gene, which is a neurotoxic determinant of HSV-1.

[0020] As used in the present invention, "human DR-18" and "DR18" can be used interchangeably; "murine DR-18" and "mDR18" can be used interchangeably.

[0021] As used herein, "mutation", "mutant" or "variant" refers to a change 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 translocation, deletion, insertion and substitution / point mutation. "Mutant" or "variant" as used herein refers to a nucleic acid or protein comprising a mutation.

[0022] As used herein, the term "wild-type" refers to a gene or gene product isolated from a natural source. A wild-type gene is a gene most commonly observed in a population and is therefore arbitrarily designated as the "normal" or "wild-type" form of the gene. In contrast, the term "modified," "variant," or "mutant" refers to a gene or gene product that has a sequence and / or functional property modification (i.e., altered properties) compared to a wild-type gene or gene product.

[0023] In one aspect, the present invention provides a technical solution for treating tumors by combining DR-18 and herpes simplex virus. In some embodiments, the "combination" is interpreted in a broad sense, for example, a pharmaceutical composition, a pharmaceutical set.

[0024] As used herein, the term "pharmaceutical combination" refers to a product obtained by mixing or combining more than one active ingredient, including fixed and non-fixed combinations of active ingredients. As used herein, the terms "co-administration" or "combined administration" and the like are intended to encompass administration of selected therapeutic drugs to a single patient, and are intended to include treatment regimens in which the drugs are not necessarily administered by the same route of administration or at the same time.

[0025] As used herein, 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.

[0026] As used herein, 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 may be two capsules each containing one active ingredient, the purpose of which is to treat the patient with both active ingredients together in vivo.

[0027] As used herein, "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 preparation, or sequentially in any order (e.g., simultaneously, sequentially, intermittently) as a single preparation.

[0028] As used herein, the term "combined with..." refers to administering a plurality of agents to a subject, and refers to administering a first agent to a subject and at least one other (i.e., a second, third, fourth, fifth, etc.) agent. As used herein, if, when administering a second agent, the biological effect produced by administering the first agent persists in the subject, superimposing the therapeutic effects of the first agent and the second agent, then it is considered that an agent (such as DR-18) is administered in combination with a second agent (e.g., herpes simplex virus). The administration of the first agent (such as DR-18) provides a therapeutic effect over a longer period of time, and the administration of the second agent (such as herpes simplex virus) provides its therapeutic effect while the therapeutic effect of the first agent is still ongoing, so 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 far away from the time of the second agent administration (such as a few days or weeks). In some embodiments, if the first and second agents are administered simultaneously (within 30 minutes of each other), concurrently, or sequentially, then it is considered that the first agent is administered in combination with the second agent. 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 the case where the first agent and the second agent are co-formulated in a single pharmaceutically acceptable formulation and the co-formulation is administered to the 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 administering one agent. 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 at the same time; these two or more agents may be present in two or more separate formulations, or combined into a single formulation (i.e., a co-formulation). Whether these agents are administered sequentially or simultaneously, as used in the present invention, they are considered to be administered / used in combination.

[0029] In one aspect, the present invention provides the use of DR-18 in the preparation of an anti-tumor drug combination, wherein the drug combination comprises herpes simplex virus in combination with DR-18.

[0030] In one aspect, the present invention 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.

[0031] In one aspect, the present invention provides a drug combination for treating tumors, comprising:

[0032] DR-18, and

[0033] Herpes simplex virus.

[0034] 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 independently packaged DR-18 and independently packaged herpes simplex virus.

[0035] In one aspect, the present invention provides a pharmaceutical composition for treating tumors, comprising:

[0036] DR-18, and herpes simplex virus.

[0037] In one aspect, the present invention provides a pharmaceutical kit comprising:

[0038] DR-18, and herpes simplex virus.

[0039] In some embodiments, the pharmaceutical kit comprises independently packaged DR-18 and independently packaged herpes simplex virus.

[0040] The difference between the pharmaceutical set and the composition is that DR-18 is different from the dosage form of herpes simplex virus, but is independently packaged (for example: a pill, or capsule, or tablet or ampoule contains DR-18; another pill, or capsule, or tablet or ampoule contains herpes simplex virus). In some embodiments, herpes simplex virus, DR-18, and the combination of herpes simplex virus and DR-18 may also contain one or more adjuvants. The adjuvant refers to an ingredient in the drug composition that can assist the efficacy of the drug. The pharmaceutical set may also include independently packaged DR-18 and independently packaged herpes simplex virus. The administration of DR-18 and herpes simplex virus in the pharmaceutical set may be administered simultaneously or in any order of precedence, such as administering DR-18 before herpes simplex virus, or administering DR-18 after herpes simplex virus, or administering both simultaneously. In various embodiments, the patient may be a mammal.

[0041] In some embodiments, the composition / drug kit further comprises a pharmaceutically acceptable carrier.

[0042] In some embodiments, the ratio of DR-18 to herpes simplex virus is: 0.01-200 mg: 10 3 -10 9 PFU; preferably 0.1-200mg: 10 4 -10 9 PFU; preferably 0.1-100:10 5 -10 9 PFU.

[0043] In some embodiments, the dosage is: the dosage range of DR-18 is 0.01-10 mg / kg, and the titer of herpes simplex virus is MOI 10 3 -10 9 PFU / kg; preferably, the dosage range of DR-18 is 0.1-5 mg / kg, and the titer of herpes simplex virus is MOI 10 4 -10 9 PFU / kg; preferably, the dosage range of DR-18 is 0.05-0.5 mg / kg, and the titer of herpes simplex virus is MOI 10 5 -10 9 PFU / kg.

[0044] 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.

[0045] On the one hand, the present invention also provides the use of the drug combination / drug composition / drug kit in the preparation of drugs for treating tumors.

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

[0047] 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 carcinoma, lung cancer, sarcoma or gastric cancer.

[0048] In some embodiments, the nucleotide sequence of the human DR-18 is as shown in SEQ ID NO:5.

[0049] In some embodiments, the nucleotide sequence of the murine DR-18 is as shown in SEQ ID NO:6.

[0050] In some embodiments, when some oncolytic viruses are used in combination with DR-18, different and unpredictable effects occur. The inventors have found that some of them have obvious mutual antagonism or inhibition. However, unlike these phenomena, the combination of herpes simplex virus and DR-18 shows obvious synergistic effects in tests on multiple tumors. This is a surprising discovery that brings good development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The following are the effects of the inactivated supernatants of different tumor cells infected with each oncolytic virus in Example 1 on the activity of DR18. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0052] Figure 2 The effect of each OVs-SN on DR18 activity in Example 1, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, ns indicates that the difference is not statistically significant.

[0053] Figure 3 The figure is the curve of animal body weight change in Example 2, and ns indicates that the difference is not statistically significant.

[0054] Figure 4 This 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.

[0055] Figure 5 This is the 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.

[0056] Figure 6 This is the 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.

[0057] Figure 7 The figure is the curve of animal body weight change in Example 3, and ns indicates that the difference is not statistically significant.

[0058] Figure 8 This 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.

[0059] Fig. 9 This is the 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.

[0060] Fig.10The weight change curve of the animals in Example 4, 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.

[0061] Fig.11 The figure is the 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

[0062] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0063] Unless otherwise specified, the materials and experimental methods used in the present invention are conventional materials and methods.

[0064] Example 1 Using the IL-18 reporter cell model to evaluate the effect of supernatants of different tumor cells infected with various oncolytic viruses on DR18 activity

[0065] IL-18 reporter cell model: This cell expresses IL-18 receptor, IL-18 downstream pathway molecules and luciferase. IL18 binds to the receptor and mediates luciferase expression, and its reading represents the activation effect of the pathway. This pathway is the key pathway for DR18 to activate anti-tumor immunity in vivo, and can indirectly reflect the anti-cancer activity of DR18.

[0066] 1. Experimental Materials

[0067] 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.

[0068] ADV: oncolytic adenovirus with E1a-CR2-24bp gene and E3 region gene knockout; VSV: attenuated oncolytic vesicular stomatitis virus with G gene knockout and M gene V48R&M51R mutation; REO: serotype 3 oncolytic reovirus. The sources are commercially available products, or customized products constructed according to conventional methods, or constructed by Guangzhou Weirongte Pharmaceutical Technology Co., Ltd. according to conventional methods.

[0069] 1.2 Experimental cells: IL-18 reporter cells (H_IL18 Reporter 293Cell Line), 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), lung cancer cells (HCC-827), glioma cells (LN18, U-138MG), osteosarcoma cells (MNNG / HOS). Cells were purchased from ATCC, Cell Bank of the Chinese Academy of Sciences, China Center for Type Culture Collection, Geneo Biotechnology, Saiku Biotechnology, Foxai Biotechnology, Tongpai Biotechnology, etc.

[0070] 1.3 Experimental reagents: human DR18 recombinant protein (SEQ ID NO: 1), DMEM medium, MEM medium, RPMI1640 medium, McCoy's 5A medium, IMDM medium, EMEM medium, Fetal Bovine Serum, Puromycin, Blasticidin, G418, ONE-Glo TM Luciferase Assay System.

[0071] 1.4 Experimental instruments: inverted microscope, biological safety cabinet, carbon dioxide incubator, and microplate reader.

[0072] 2. Experimental Methods

[0073] 2.1 Establishment of tumor cell model:

[0074] According to the instructions of each tumor cell, appropriate culture conditions and subculture ratios were selected for cell amplification and subculture. When the cells entered the logarithmic growth phase and the number was sufficient, the cells were subcultured at 3×10 4 / cm2 Inoculate into T25 cell culture flask.

[0075] 2.2 Oncolytic virus infection of tumor cells:

[0076] 16 to 24 hours after tumor cell inoculation, complete cell attachment was observed under a microscope, and each tumor cell was infected with virus at an MOI of 1 PFU / cells according to the number of cells plated.

[0077] 2.3 Collection and inactivation of infection supernatant:

[0078] After oncolytic virus infection of tumor cells for 48 hours, the cytopathic conditions were observed under a microscope and photographed; the supernatant of virus-infected cells (Supernatant, SN) was inactivated by ultraviolet irradiation or filtered through a 0.1 μm filter membrane according to the characteristics of the virus, and the inactivated supernatant was collected into a 4.5 mL cryopreservation tube and stored in a -80°C refrigerator for later use. The uninfected supernatant was collected in the same way as a control.

[0079] 2.4 Effect of infection supernatant on biological activity of DR18:

[0080] 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 1.5×10 4 / well, 90 μL of culture medium was inoculated into the middle well of the 96-well plate, and 200 μL of PBS / well was added to the surrounding wells for sealing.

[0081] ② Place the cells at 37℃ and 5% CO 2 After culturing in the cell culture incubator for 16-24 hours, the 96-well plate was taken out and the cells were observed under a microscope to show that they were completely attached to the wall and had good morphology. The DR18 protein was diluted 3-fold in a 3-fold gradient with the inactivated supernatant and the control supernatant in step 2.3, and after thorough mixing, 10 μL was added to the cells per well.

[0082] ③After adding DR18 protein, place in 37℃5% CO 2 The cells were cultured in a cell culture incubator for 16 hours.

[0083] ④Add 100 μL ONE-Glo TM Substrate, react in the dark for 3 minutes to allow the cells to completely lyse, transfer the cell lysate to a 96-well luminescent plate, and detect Luciferase with an enzyme reader.

[0084] 2.5 Data processing:

[0085] According to the relative light unit (RLU) of each well obtained by detection, the formula: 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, draw a fitting curve using software, and use [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 .

[0086] 2.6 Biological Statistics:

[0087] * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001, indicating that the data differences are statistically significant.

[0088] 3. Experimental results:

[0089] 3.1 Effects of inactivated supernatants of different tumor cells infected with oncolytic viruses on DR18 activity Figure 1 The DR18 recombinant protein was gradiently diluted with the supernatant of different tumor cells infected with different oncolytic viruses, and the IL-18 reporter cells were treated for 16 hours. The Luciferase luminescence unit was detected on the machine, and the fitting curve was drawn and the EC50 of each treatment group was calculated. Figure 2 , the statistical analysis results are shown in Figure 1 .

[0090] The results showed that compared with the control group (CON) not infected with the virus, oncolytic herpes simplex virus (HSV) infected 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.

[0091] 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.

[0092] 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.

[0093] 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).

[0094] 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 the enhancing effect of HSV is stronger than that of other oncolytic viruses.

[0095] Example 2 Safety and efficacy study of mDR18 combined with various oncolytic viruses in an immune-competent mouse liver cancer model

[0096] 1. Experimental Materials

[0097] 1.1 Oncolytic viruses: HSV: attenuated oncolytic herpes simplex virus type 1 with the neurovirulence factor γ34.5 gene knocked out; ADV: oncolytic adenovirus with E1a-CR2-24bp gene and E3 region gene knocked out; VSV: oncolytic vesicular stomatitis virus with G gene knocked out and M gene V48R&M51R mutations attenuated. Viruses were purchased from Wuhan Shumi, Fubaiao Biological, etc.

[0098] 1.2 Recombinant protein: modified mouse interleukin-18 (mDR18) recombinant protein (SEQ ID NO: 2).

[0099] 1.3 Tumor cells: Mouse hepatoma cell line H22, cells were purchased from China Center for Type Culture Collection.

[0100] 1.4 Experimental animals: Female BALB / c mice, 5-7 weeks old.

[0101] 2. Experimental Methods

[0102] 2.1 Establishment of tumor-bearing mouse model:

[0103] The revived tumor cells were subcultured. When the cells entered the logarithmic growth phase and grew to a sufficient number, the cells were collected, counted and prepared into cell suspension. H22 cells were cultured at 2×10 6 The cells were inoculated subcutaneously on the back of Balb / c mice.

[0104] 2.2 Grouped medication:

[0105] After the tumor is formed, the tumor volume range is 100±40mm 3 The mice were randomly divided into 8 groups, including excipient control group, mDR18 alone group, HSV alone group, HSV+mDR18 combination group, ADV alone group, ADV+mDR18 combination group, VSV alone group, and VSV+mDR18 combination group, with 5 animals in each group; the dosage of mDR18 was 0.32 mg / kg and the administration method was intraperitoneal injection. 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, the administration method was intratumoral injection, and the administration was once. The day of tumor inoculation was defined as D0, and the observation was continued for 21 days after administration.

[0106] 2.3 Mouse body weight and tumor measurement:

[0107] During tumor formation and drug administration, the weight of mice and the growth of tumor were recorded every 3-4 days.

[0108] 2.4 Data processing:

[0109] The measured data were statistically analyzed and the tumor volume growth curve of each group of mice was drawn.

[0110] The calculation formula of tumor volume (TV) is: V = 1 / 2 × a × b 2 , where a and b represent the long and wide diameters of the tumor, respectively.

[0111] 2.5 Statistical analysis

[0112] * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001, indicating that the data differences are statistically significant.

[0113] 3. Experimental results:

[0114] The body weight of animals in each group during the experiment was Figure 3 As shown in the figure, the body weight of each group of animals maintained a steady upward trend during the experiment. Compared with the auxiliary material control group, there was no significant difference in the single or combined use of each drug group, indicating that the intraperitoneal injection of 0.32 mg / kg mDR18 twice a week or a single intratumoral injection of 2×10 6 Each oncolytic virus at PFU / mouse did not cause obvious drug-related side effects in the immunocompetent mouse model.

[0115] Based on the tumor volume growth records of the animals, a tumor volume change curve was drawn. Figure 4 The results showed that compared with the excipient control group, mDR18 alone (P<0.001) or combined with HSV (P<0.001) could significantly inhibit the tumor growth of the mouse liver cancer model; compared with the mDR18 alone group, the combination of HSV and mDR18 (P<0.001) could significantly enhance the tumor inhibition effect on the mouse liver cancer model; compared with the HSV alone group, the combination of HSV and mDR18 (P<0.001) could significantly enhance the tumor inhibition effect on the mouse liver cancer model, indicating that the combination of HSV and mDR18 has a synergistic effect.

[0116] Based on the tumor volume growth records of the animals, a tumor volume change curve was drawn. Figure 5 The results showed that compared with the auxiliary material control group, mDR18 alone (P<0.01) or combined with ADV (P<0.001) could significantly inhibit the tumor growth of the mouse liver cancer model; compared with the mDR18 alone group, the difference in the tumor inhibition effect of the ADV and mDR18 combination group was not statistically significant, indicating that the combination of ADV and mDR18 had no synergistic effect.

[0117] Based on the tumor volume growth records of the animals, a tumor volume change curve was drawn. Figure 6 The results showed that compared with the auxiliary material control group, mDR18 alone (P<0.01) or combined with VSV (P<0.001) could significantly inhibit the tumor growth of the mouse liver cancer model; compared with the mDR18 alone group, the difference in the tumor inhibition effect of the VSV and mDR18 combination group was not statistically significant, indicating that the combination of VSV and mDR18 had no synergistic effect.

[0118] Example 3 Safety and efficacy study of mDR18 combined with various oncolytic viruses in an immune-competent mouse colon cancer model

[0119] 1. Experimental Materials

[0120] 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.

[0121] 1.2 Recombinant protein: modified mouse interleukin-18 (mDR18) recombinant protein.

[0122] 1.3 Tumor cells: Mouse colorectal cancer cell line CT26, cells were purchased from Guangzhou Geneo Biotechnology Co., Ltd.

[0123] 1.4 Experimental animals: Female Balb / c mice aged 5-7 weeks.

[0124] 2. Experimental Methods

[0125] 2.1 Establishment of tumor-bearing mouse model:

[0126] The revived tumor cells were subcultured. When the cells entered the logarithmic growth phase and grew to a sufficient number, the cells were collected, counted and prepared into cell suspension. CT26 cells were cultured at 2×10 6 The cells were inoculated subcutaneously on the back of Balb / c mice.

[0127] 2.2 Grouped medication:

[0128] After the tumor is formed, the tumor volume range is 100±40mm 3 The mice were randomly divided into 8 groups, including excipient control group, mDR18 alone group, HSV alone group, HSV+mDR18 combination group, ADV alone group, and ADV+mDR18 combination group, with 4 animals in each group; the dosage of mDR18 was 0.32 mg / kg and the administration method was intraperitoneal injection. 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, the administration method was intratumoral injection, and the administration was once. The day of tumor inoculation was defined as D0, and the observation was continued for 14 days after administration.

[0129] 2.3 Mouse body weight and tumor measurement:

[0130] During tumor formation and drug administration, the weight of mice and the growth of tumor were recorded every 3-4 days.

[0131] 2.4 Data processing:

[0132] The measured data were statistically analyzed and the tumor volume growth curve of each group of mice was drawn.

[0133] The calculation formula of tumor volume (TV) is: V = 1 / 2 × a × b 2 , where a and b represent the long and wide diameters of the tumor, respectively.

[0134] 2.5 Statistical analysis

[0135] * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001, indicating that the data differences are statistically significant.

[0136] 3. Experimental results:

[0137] The body weight of animals in each group during the experiment was Figure 7As shown in the figure, the body weight of each group of animals maintained a steady upward trend during the experiment. Compared with the auxiliary material control group, there was no significant difference in the single or combined use of each drug group, indicating that the intraperitoneal injection of 0.32 mg / kg mDR18 twice a week or a single intratumoral injection of 2×10 6 Each oncolytic virus at PFU / mouse did not cause obvious drug-related side effects in the immunocompetent mouse model.

[0138] Based on the tumor volume growth records of the animals, a tumor volume change curve was drawn. Figure 8 The results showed that compared with the auxiliary material control group, the combination of HSV and mDR18 (P<0.05) could significantly inhibit the tumor growth of the mouse liver cancer model; compared with the HSV alone group, the combination of HSV and mDR18 (P<0.05) could significantly enhance the tumor inhibition effect on the mouse liver cancer model; compared with the mDR18 alone group, the combination of HSV and mDR18 (P<0.05) could significantly enhance the tumor inhibition effect on the mouse liver cancer model, indicating that the combination of HSV and mDR18 has a synergistic effect.

[0139] Based on the tumor volume growth records of the animals, a tumor volume change curve was drawn. Fig. 9 The results showed that compared with the auxiliary material control group, mDR18 alone (P<0.001) or combined with ADV (P<0.05) could significantly inhibit the tumor growth of the mouse liver cancer model; compared with the mDR18 alone group, the difference in the tumor inhibition effect of the ADV and mDR18 combination group was not statistically significant, indicating that the combination of ADV and mDR18 had no synergistic effect.

[0140] Example 4 Safety and efficacy study of mDR18 combined with HSV in an immunocompetent mouse tumor model

[0141] 1. Experimental Materials

[0142] 1.1 Oncolytic virus: HSV: Attenuated oncolytic herpes simplex virus type 1 with the neurovirulence factor γ34.5 gene knocked out;

[0143] 1.2 Recombinant protein: modified mouse interleukin-18 (mDR18) recombinant protein.

[0144] 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; The cells were obtained from Guangdong Yaokang Biotechnology Co., Ltd.

[0145] 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.

[0146] 2.1 Establishment of tumor-bearing mouse model:

[0147] The revived tumor cells were subcultured, and when the cells entered the logarithmic growth phase and grew to a sufficient number, the cells were collected, counted, and prepared into a cell suspension: A. A20 cells were counted at 1×10 6 B.H22 cells were inoculated at 1×10 6 5×105 cells / mouse were inoculated on the right back thigh of BALB / c mice; C. 5×105 cells / mouse were inoculated on the right back thigh of BALB / c mice; D. 5×105 cells / mouse were inoculated on the right back thigh of BALB / c mice; E. 2×10 6 10 cells / mouse were inoculated on the right back thigh of BALB / c mice; 3×105 cells / mouse were inoculated on the right back thigh of BALB / c mice; 5×104 cells / mouse were inoculated on the right back thigh of BALB / c mice; 5×105 cells / mouse were inoculated on the right forelimb of BALB / c mice; 1×10 6 cells / mouse, inoculated on the upper right thigh of C57BL / 6 mice; J.LLC1 cells at 3×105 cells / mouse, inoculated on the upper right thigh of C57BL / 6 mice; K.MC38 cells at 1×10 6 5×105 cells / mouse were inoculated into the right forelimb of C57BL / 6 mice; 5×105 cells / mouse were inoculated into the right forelimb of C57BL / 6 mice; 5×10 6MB49 cells were inoculated subcutaneously in the right forelimb of C57BL / 6 mice at 5×10 6 Cells / mouse were inoculated subcutaneously into the right forelimb of C57BL / 6 mice.

[0148] 2.2 Grouped medication:

[0149] After the tumor is formed, the tumor volume range is 60-100mm 3 The mice were randomly divided into two groups, namely 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, the administration cycle was 2 times a week, for a total of 8 times; the HSV dosage was 2×10 6 PFU / mouse, administered by intratumoral injection, once in total. The day of tumor inoculation was defined as D0, and observation was continued for 1 month after administration or at the ethical end point.

[0150] 2.3 Mouse body weight and tumor measurement:

[0151] During tumor formation and drug administration, the weight of mice and the growth of tumor were recorded every 3-4 days.

[0152] 2.4 Data processing:

[0153] The measured data were statistically analyzed and the tumor volume growth curve of each group of mice was drawn.

[0154] The calculation formula of tumor volume (TV) is: V = 1 / 2 × a × b 2 , where a and b represent the long and wide diameters of the tumor, respectively.

[0155] 2.5 Statistical analysis

[0156] * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001, indicating that the data differences are statistically significant.

[0157] 3. Experimental results:

[0158] The body weight of animals in each group during the experiment was Fig.10 As shown in the figure, the body weight of each group of animals maintained a steady upward trend during the experiment. Compared with the auxiliary material control group, there was no significant difference in the HSV + mDR18 combination group, indicating that 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.

[0159] Based on the tumor volume growth records of the animals, a tumor volume change curve was drawn. Fig.11The results showed that compared with the auxiliary material 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 CT26.WT (P<0.001). <0.001), mouse pancreatic cancer cell mPAKPC (P<0.001), mouse prostate cancer cell RM-1 (P<0.001), mouse lung cancer cell LLC1 (P<0.001), mouse colon cancer cell MC38 (P<0.001), mouse melanoma cell B16-F10 (P<0.001), mouse pancreatic cancer cell Pan02 (P<0.001), and mouse bladder cancer cell MB49 (P<0.001) in vivo tumor growth.

[0160] The embodiments described in the present invention are only illustrative examples, and the embodiments of the present invention 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 invention should be equivalent replacement methods and are included in the protection scope of the present invention.

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, characterized in that: 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 amino acid sequence of the human DR-18 is as 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, characterized in that: The DR-18 is mouse-derived 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 amino acid sequence of the murine DR-18 is as 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, characterized in that: 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, characterized in that 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 claim 6, characterized in that 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 amino acid sequence of the human DR-18 is as 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 amino acid sequence of the murine DR-18 is as 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 claim 6, characterized in that The ratio of DR-18 to herpes simplex virus is: 0.01-200 mg: 10 3 -10 9 PFU; preferably 0.1-200mg: 10 4 -10 9 PFU; preferably 0.1-100:10 5 -10 9 PFU; Preferably, the dosage is: DR-18 dosage range is 0.01-10 mg / kg, and the herpes simplex virus titer is MOI 10 3 -10 9 PFU / kg; preferably, the dosage range of DR-18 is 0.1-5 mg / kg, and the titer of herpes simplex virus is MOI 10 4 -10 9 PFU / kg; preferably, the dosage range of DR-18 is 0.05-0.5 mg / kg, and the titer of herpes simplex virus is 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, characterized in that: 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.

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