Recombinant oncolytic cowpox virus and its use

Abstract

This invention provides a recombinant oncolytic cowpox virus capable of expressing 4-1BBL and in which an exogenous gene with a synonymous mutation is functionally inserted, and also provides its use in the manufacture of pharmaceuticals for the prevention or treatment of tumors and cancer. The beneficial effects are as follows: Recombinant cowpox virus VV-mH4-1BBL based on synonymous mutation can improve safety by inactivating the TK gene while retaining the oncolytic effect and the induction and enhancement of antitumor immune response inherent to oncolytic viruses. 4-1BBL is highly expressed on the surface of tumor cells, and 4-1BBL is present in the tumor microenvironment. + By stimulating the 4-1BB signaling pathway in immune cells (including T cells), anti-tumor immunity can be enhanced, and potential systemic toxicity and side effects can be avoided by localizing to tumor tissue and exerting concentrated effects. By introducing synonymous mutation sites, the expression of therapeutic (exogenous) 4-1BBL genes can be detected during the course of treatment with the virus.

Description

[Technical Field]

[0001] This invention relates to the field of biopharmaceuticals, and more specifically to recombinant oncolytic cowpox virus and its use. [Background technology]

[0002] Stimulating the 4-1BB signaling pathway in immune cells can induce potent and sustained antitumor immunity. Clinical research is currently underway on 4-1BB-stimulating monoclonal antibodies such as urelumab and utomilumab. When such antibodies are used systemically, the difference between the effective dose and the safe dose is small. This often results in either effective antitumor immunity being induced but systemic toxicity and side effects occurring, or no obvious toxicity or side effects but a weak antitumor effect. These contradictions significantly limit the clinical application of such antibodies.

[0003] Soluble 4-1BBL can stimulate the 4-1BB signaling pathway, but it has not been studied for clinical application. Direct systemic use may strongly enhance the systemic immune response and potentially cause serious toxicity and side effects.

[0004] Oncolytic viruses can selectively infect tumor cells, induce immunogenic cell death in tumor cells, stimulate the body's anti-tumor immunity, and induce local inflammation, thus acting as immune adjuvants. However, in tumor treatment using only oncolytic viruses, the virus is often rapidly eliminated by the body's antiviral effects. Therefore, unless the anti-tumor immunity induced after the oncolytic virus has exerted its action is enhanced, a strong and sustained anti-tumor effect cannot be induced. Thus, it is necessary to improve the anti-tumor effect of oncolytic viruses by introducing immune-enhancing factors.

[0005] Studies have reported the construction of recombinant mouse 4-1BBL oncolytic viruses using the mouse 4-1BBL gene and the demonstration of the reliability of their effects in a mouse model. However, new research has revealed that the wild-type 4-1BBL gene is normally expressed in tumor cells such as those of intestinal cancer, and its coding product (i.e., the 4-1BBL molecule) is localized inside the cell rather than on the cell surface. Therefore, when recombinant 4-1BBL oncolytic viruses are constructed using the wild-type 4-1BBL gene, it is not possible to detect or track the expression and location of the therapeutic 4-1BBL during the treatment process.

[0006] Oncolytic viruses are attracting attention in the research and development of onco-immunopharmaceuticals as an effective means of tumor immunotherapy because they can selectively infect tumor cells, induce immunogenic cell death in tumor cells, and stimulate the body's anti-tumor immune response. However, oncolytic viruses themselves are easily eliminated by the body as viruses, and the anti-tumor immunity induced by oncolytic viruses is inhibited by suppressive mechanisms such as immune checkpoints. Therefore, the main design concept in this field is to construct recombinant oncolytic viruses that retain the inherent advantages of oncolytic viruses while avoiding their weaknesses, and further overcome immunosuppressive mechanisms by incorporating immune-enhancing factors, thereby inducing safe, effective, systemic, and sustained anti-tumor immunity in the body.

[0007] The co-stimulatory molecule ligand 4-1BBL acts on 4-1BB molecules on the surface of T cells, NK cells, dendritic cells, etc. (i.e., via the 4-1BB / 4-1BBL pathway) to strongly promote antitumor immunity in the body. Therefore, biological agents developed targeting the 4-1BB / 4-1BBL pathway can become novel antitumor drugs. Stimulating 4-1BB monoclonal antibodies and 4-1BBL recombinant oncolytic viruses are important biological agents being developed in this field, but their shortcomings limit their future applications and must be improved.

[0008] While stimulating 4-1BB monoclonal antibodies can activate the potent antitumor effect via the 4-1BB / 4-1BBL pathway, the difference between the dose that produces effective antitumor activity and the dose that causes toxicity and side effects is small (i.e., the safe dose range is narrow). Therefore, by using the natural ligand 4-1BBL as an immunoactivator for the 4-1BB molecule, the 4-1BB signaling pathway can be activated through a natural stimulating mechanism. Furthermore, treating tumors directly with soluble 4-1BBL can lead to toxicity and side effects due to the systemic effects of cytokines.

[0009] However, research shows that the 4-1BBL molecule is not normally expressed on the cell membrane of tumor cells such as those with colon cancer or pancreatic cancer, but rather resides inside these tumor cells. This allows tumor cells to evade stimulation by the 4-1BB molecule on the surface of immune cells. This is a tumor immunity evasion mechanism. If a recombinant oncolytic virus is constructed using the gene for the natural ligand 4-1BBL, 4-1BBL can be expressed on the surface of tumor cells, inducing anti-tumor immunity. However, in future therapeutic processes, it will be extremely difficult to monitor and evaluate therapeutic effects because it will be impossible to distinguish whether the 4-1BBL gene in tumor tissue originates from the tumor tissue itself or from a therapeutic recombinant 4-1BBL oncolytic virus. [Overview of the project] [Problems that the invention aims to solve]

[0010] This invention has been made in view of the above circumstances, and aims to establish a new 4-1BB signaling method, provide recombinant oncolytic cowpox virus and its use that maintains the immune effect of 4-1BBL through stimulation of the 4-1BB signal, and improve its safety of use, and also provide a novel biological preparation in which the therapeutic process and therapeutic effect can be detected and monitored, thereby overcoming the shortcomings and defects described in the background art. [Means for solving the problem]

[0011] To achieve the above objective, the present invention employs the following technical means.

[0012] The first aspect of the present invention is to provide a recombinant oncolytic cowpox virus in which an exogenous gene is functionally inserted, wherein the exogenous gene is capable of expressing 4-1BBL and has a synonymous mutation introduced. Optionally, in the recombinant oncolytic cowpox virus, the exogenous gene is inserted into the TK gene of the cowpox virus. Optionally, in the recombinant oncolytic cowpox virus, the DNA sequence of the exogenous gene is shown in SEQ ID NOs: 1 to 3.

[0013] The applicant provides examples of gene sequences into which the following three synonymous mutations have been introduced, but these do not limit the technical scope of the present invention. In fact, synonymous mutations are possible at any one or more sites, as long as the sequence of the ultimately encoded peptide / amino acid / protein is identical (i.e., the efficacy is identical or similar).

[0014] The sequence of sequence number 1 (the sequence in which the 540th T is mutated to C) is as follows: atggaatacgcctctgacgcttcactggaccccgaagccccgtggcctcccgcgccccgcgctcgcgcctgccgcgtactgccttgggccctggtcgcggggctgctgctgctgctgctgctcgctgccgcctgcgccgtcttcctcgcctgcccctgggccgtgtccggggctcgcgcctcgcccggctccgcggccagcccgagactccgcgagggtcccgagctttcgcccgacgatcccgccggcctcttggacctgcggcagggcatgtttgcgcagctggtggcccaaaatgttctgctgatcgatgggcccctgagctggtacagtgacccaggcctggcaggcgtgtccctgacggggggcctgagctacaaagaggacacgaaggagctggtggtggccaaggctggagtctactatgtcttctttcaactagagctgcggcgcgtggtggccggcgagggctcaggctccgtttcacttgcgctgcacctgcagccactgcgctctgctgctggggccgccgccctggcCttgaccgtggacctgccacccgcctcctccgaggctcggaactcggccttcggtttccagggccgcttgctgcacctgagtgccggccagcgcctgggcgtccatcttcacactgaggccagggcacgccatgcctggcagcttacccagggcgccacagtcttgggactcttccgggtgacccccgaaatcccagccggactcccttcaccgaggtcggaataa

[0015] The sequence of SEQ ID NO: 2 (the sequence with the 468th G mutated to A) is as follows. atggaatacgcctctgacgcttcactggaccccgaagccccgtggcctcccgcgccccgcgctcgcgcctgccgcgtactgccttgggccctggtcgcggggctgctgctgctgctgctgctcgctgccgcctgcgccgtcttcctcgcctgcccctgggccgtgtccggggctcgcgcctcgcccggctccgcggccagcccgagactccgcgagggtcccgagctttcgcccgacgatcccgccggcctcttggacctgcggcagggcatgtttgcgcagctggtggcccaaaatgttctgctgatcgatgggcccctgagctggtacagtgacccaggcctggcaggcgtgtccctgacggggggcctgagctacaaagaggacacgaaggagctggtggtggccaaggctggagtctactatgtcttctttcaactagagctgcggcgcgtggtggccggcgaAggctcaggctccgtttcacttgcgctgcacctgcagccactgcgctctgctgctggggccgccgccctggctttgaccgtggacctgccacccgcctcctccgaggctcggaactcggccttcggtttccagggccgcttgctgcacctgagtgccggccagcgcctgggcgtccatcttcacactgaggccagggcacgccatgcctggcagcttacccagggcgccacagtcttgggactcttccgggtgacccccgaaatcccagccggactcccttcaccgaggtcggaataa

[0016] The sequence of SEQ ID NO: 3 (the sequence with the 597th C mutated to T) is as follows. atggaatacgcctctgacgcttcactggaccccgaagccccgtggcctcccgcgccccgcgctcgcgcctgccgcgtactgccttgggccctggtcgcggggctgctgctgctgctgctgctcgctgccgcctgcgccgtcttcctcgcctgcccctgggccgtgtccggggctcgcgcctcgcccggctccgcggccagcccgagactccgcgagggtcccgagctttcgcccgacgatcccgccggcctcttggacctgcggcagggcatgtttgcgcagctggtggcccaaaatgttctgctgatcgatgggcccctgagctggtacagtgacccaggcctggcaggcgtgtccctgacggggggcctgagctacaaagaggacacgaaggagctggtggtggccaaggctggagtctactatgtcttctttcaactagagctgcggcgcgtggtggccggcgagggctcaggctccgtttcacttgcgctgcacctgcagccactgcgctctgctgctggggccgccgccctggctttgaccgtggacctgccacccgcctcctccgaggctcggaactcggccttcggtttTcagggccgcttgctgcacctgagtgccggccagcgcctgggcgtccatcttcacactgaggccagggcacgccatgcctggcagcttacccagggcgccacagtcttgggactcttccgggtgacccccgaaatcccagccggactcccttcaccgaggtcggaataa Optionally, in the recombinant oncolytic vaccinia virus, the amino acid sequence of the exogenous gene is shown by SEQ ID NO: 4.

[0017] The sequence of SEQ ID NO: 4 is as follows. MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE Optionally, in the recombinant oncolytic vaccinia virus, the exogenous gene is linked by an IRES sequence.

[0018] The IRES is a ribosome entry site and is one of the available linking sequences. Optionally, in the recombinant oncolytic vaccinia virus, the IRES sequence is shown in SEQ ID NO: ⑤.

[0019] The sequence of SEQ ID NO: ⑤ is as follows. TTATCATCGTGTTTTTCAAAGGAAAACCACGTCCCCGTGGTTCGGGGGGCCTAGACGTTTTTTAACCTCGACTAAACACATGTAAAGCATGTGCACCGAGGCCCCAGATCAGATCCCATACAATGGGGTACCTTCTGGGCATC CTTCAGCCCCTTGTTGAATACGCTTGAGGTGAGCCATTTGACTCTTTCCACAACTATCCAACTCACAACGTGGCACTGGGGTTGTGCCGCCTTTGCAGGTGTATCTTATACACGTGGCTTTTGGCCGCAGAGGCACCTGTCGC CAGGTGGGGGGTTCCGCTGCCTGCAAAGGGTCGCTACAGACGTTGTTTGTCTTCAAGAAGCTTCCAGAGGAACTGCTTCCTTCACGACATTCAACAGACCTTGCATTCCTTTGGCGAGAGGGGAAAGACCCCTAGGAATGCTC GTCAAGAAGACAGGGCCAGGTTTCCGGGCCCTCACATTGCCAAAAGACGGCAATATGGTGGAAAATAACATATAGACAAACGCACACCGGCCTTATTCCAAGCGGCTTCGGCCAGTAACGTTAGGGGGGGGGAGGGAGAGGGG

[0020] The second invention of this invention is to provide a pharmaceutical composition containing the recombinant oncolytic cowpox virus, a pharmaceutically acceptable carrier, and a pharmaceutical excipient.

[0021] Optionally, in the pharmaceutical composition, the administration method of the pharmaceutical composition is direct injection and / or injection in combination with an endoscope, the endoscope being preferably a laparoscope, cholangioscope, thoracoscopy, enteroscope, or neuroendoscope.

[0022] A third invention of the present invention is to provide the use of the recombinant oncolytic cowpox virus or the pharmaceutical composition in the manufacture of a pharmaceutical for the prevention or treatment of tumors and / or cancer.

[0023] Optionally, in the foregoing use, the tumor and / or cancer is a solid tumor, preferably a cold tumor and / or a hot tumor, and its histological types include, but are not limited to, pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, gastric cancer, esophageal cancer, glioma, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, lung cancer, breast cancer, multiple myeloma, lymphoma, and melanoma. [Effects of the Invention]

[0024] The key technical points and advantages of this invention are as follows:

[0025] 1. By using a human 4-1BBL gene sequence into which synonymous mutations have been introduced, it is possible to detect and monitor the expression and distribution of therapeutic (exogenous) 4-1BBL after treating tumor patients with recombinant cowpox virus VV-mH4-1BBL.

[0026] 2. Recombinant cowpox virus VV-mH4-1BBL, which uses a human 4-1BBL gene sequence into which synonymous mutations have been introduced, retains the inherent characteristics and advantages of oncolytic viruses, such as oncolytic activity, anti-tumor immune response induction activity, and immune response enhancement as an adjuvant, while further improving its inherently good safety by inactivating the TK gene. By using recombinant cowpox virus VV-mH4-1BBL, 4-1BBL is highly expressed on the surface of tumor cells in the tumor microenvironment, and 4-1BBL is expressed in the tumor microenvironment. + By stimulating the 4-1BB signaling pathway in immune cells (including T cells) to enhance anti-tumor immunity, and further localizing to tumor tissue to exert a concentrated effect, it is possible to avoid the potential systemic toxicity and side effects associated with treating tumors using stimulating 4-1BB monoclonal antibodies or soluble 4-1BB in the body.

[0027] The cowpox virus has the following special characteristics compared to other oncolytic viruses.

[0028] 1. Its safety has already been proven through large-scale vaccination of the entire human population to prevent smallpox.

[0029] 2. Because the TK gene is expressed at a low level or not at all in normal cells and highly in many tumor cells, VV, which has the TK gene knocked out, is hardly expressed in normal cells. Therefore, products recombinant with VV as the parent virus have extremely high safety.

[0030] In this invention, a human 4-1BBL gene with a synonymous mutation introduced (a synonymous mutation is a mutation in a base pair in a DNA fragment that does not alter the encoded amino acid) is created, and by constructing a recombinant oncolytic cowpox virus VV-mH4-1BBL using this human 4-1BBL gene with a synonymous mutation introduced, the inherent characteristics and advantages of oncolytic viruses, such as oncolytic activity, anti-tumor immune response induction activity, and immune response enhancement activity as an adjuvant, can be maintained, while inactivating the TK gene can further improve the inherently good safety. By using recombinant cowpox virus VV-mH4-1BBL, 4-1BBL is highly expressed on the surface of tumor cells in the tumor microenvironment, and 4-1BBL is expressed in the tumor microenvironment. + By stimulating the 4-1BB signaling pathway in immune cells (including T cells) to enhance anti-tumor immunity, and further localizing to tumor tissue to exert a concentrated effect, it is possible to avoid the potential systemic toxicity and side effects associated with treating tumors using stimulating 4-1BB monoclonal antibodies or soluble 4-1BB in the body.

[0031] The use of mH4-1BBL in the manufacture of tumor treatment agents has the following circumstances and effects.

[0032] 1. Use of mH4-1BBL in improving the antitumor immune status of the tumor microenvironment The interaction between tumor cells and anti-tumor immunity determines tumor development and progression. The number and functional status of immune cells (particularly dendritic cells (DCs) and T cells) in the tumor microenvironment are important factors influencing the effectiveness of tumor immunotherapy and patient outcomes.

[0033] mH4-1BBL primarily lyses local tumor tissue due to its oncolytic virus properties, inducing an immune response and creating an inflammatory environment for tumor immunity at the tumor site. By expressing the mH4-1BBL protein on the surface of tumor cells, the antitumor function of immune cells such as DCs and T cells in the "inflammatory environment" of tumor tissue can be enhanced and maintained. Therefore, VV-mH4-1BBL is effective against tumor tissues in various immune states. Specifically, from the perspective of the immunophenotype of the tumor microenvironment, VV-mH4-1BBL can be used in "immunoinflammatory" tumor tissues (i.e., "hot tumors" in which immune cells such as T cells are infiltrated), making the "hot tumor" even "hotter" (i.e., increasing the number and activity of immune cells in the tumor microenvironment). VV-mH4-1BBL can be used in "immune desert" tumor tissue (i.e., "cold tumors" where immune cells such as T cells are scarce inside and around the tumor tissue), and can convert "cold tumors" into "hot tumors" (i.e., it can enhance the antitumor activity while promoting the infiltration of immune cells such as T cells into the tumor tissue), creating favorable conditions for immunotherapy in combination with PD-1 monoclonal antibodies, etc. VV-mH4-1BBL can be used in "immunely privileged" tumor tissue (i.e., where immune cells such as T cells are present around the tumor tissue but cannot infiltrate the inside of the tumor tissue), and can not only promote the invasion of immune cells into the tumor tissue by overcoming various barriers, but can also enhance the antitumor function of immune cells.

[0034] 2. Treatment of superficial or deep tumors by direct injection of VV-mH4-1BBL or injection in combination with endoscopy. In terms of usage (administration), VV-mH4-1BBL can be used to treat various solid tumors located on the body surface, such as melanoma, by direct injection into the tumor. VV-mH4-1BBL can also be used to accurately inject deep solid tumors into the tumor after clearly observing the tumor via laparoscopy, cholangioscopy, thoracoscopy, enteroscopy, neuroendoscopy, etc., to directly lyse tumor cells and stimulate an anti-tumor immune response.

[0035] 3. Use of VV-mH4-1BBL as a therapeutic agent for solid tumors such as colorectal cancer, pancreatic cancer, and melanoma. In terms of tumor types, VV-mH4-1BBL works by directly lysing tumors and inducing and enhancing the function of immune cells such as T cells. Therefore, the above administration method can be used to treat pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, gastric cancer, esophageal cancer, glioma, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, lung cancer, breast cancer, multiple myeloma, lymphoma, melanoma, and other cancers at various clinical stages.

[0036] In fact, VV-mH4-1BBL can be used to treat all types of solid tumors. The applicant has demonstrated this through a large number of relevant experiments. Due to space limitations, this application presents only relevant validation experiments for representative colorectal cancer, pancreatic cancer, and melanoma. However, these experimental data do not limit the scope of VV-mH4-1BBL's efficacy in all types of solid tumors.

[0037] In this invention, a gene with a synonymous mutation, mH4-1BBL, was created, and recombinant cowpox virus VV-mH4-1BBL was constructed. To express the 4-1BBL molecule, which has the function of stimulating potent antitumor immunity, at a high level on the surface of tumor cells and to detect and monitor the expression of therapeutic 4-1BBL, a primer for introducing a synonymous mutation was designed and synthesized, and the synonymous mutation gene mH4-1BBL was obtained by mutating the 540th base T to the 540th base C by PCR. Then, the gene with the synonymous mutation mH4-1BBL was incorporated into the genome of cowpox virus to construct a novel recombinant cowpox virus containing the mH4-1BBL gene sequence, which was named VV-mH4-1BBL. VV-mH4-1BBL rapidly replicates while retaining oncolytic properties and expresses the protein encoded by the mH4-1BBL gene in large quantities on the surface of tumor cells. Recombinant cowpox virus VV-mH4-1BBL directly lyses tumors as a virus and exerts an effect that induces anti-tumor immunity. The protein 4-1BBL encoded by the mH4-1BBL gene it carries localizes to tumor tissue and exerts a concentrated therapeutic effect by enhancing anti-tumor immunity.

[0038] When tumor immunotherapy is performed by acting on a 4-1BB signaling agonist with a stimulating anti-human 4-1BB monoclonal antibody or soluble 4-1BBL, there is a potential risk of systemic toxicity and side effects. The 4-1BBL protein molecule encoded by the mH4-1BBL gene carried by VV-mH4-1BBL localizes and exerts its effects in the tumor microenvironment. This avoids the potential systemic side effects of 4-1BBL and enables high expression of 4-1BBL in tumor tissue, resulting in a more potent local antitumor effect. This is an advantage of recombinant cowpox virus VV-mH4-1BBL. Furthermore, by constructing the recombinant virus and simultaneously inactivating the cowpox virus thymidine kinase (TK) gene, safety is improved and the selectivity for infection of tumor cells is further enhanced.

[0039] In this invention, by constructing a gene with a synonymous mutation and a recombinant oncolytic virus, the technical effect of maintaining the inherent advantages of oncolytic viruses while avoiding their weaknesses is successfully achieved. In this invention, the gene mH4-1BBL, in which a synonymous mutation was introduced, was constructed by mutating the 540th base T of the wild-type human 4-1BBL coding sequence to the 540th base C. Then, mH4-1BBL was inserted into cowpox virus VV by homologous recombination to construct recombinant cowpox virus VV-mH4-1BBL. When recombinant cowpox virus VV-mH4-1BBL is used as an immunotumor agent, it infects tumor cells, achieving a lysistic effect on the tumor cells and promoting the inflammatory environment at the tumor site. It also expresses the 4-1BBL coding fusion protein on the cell surface and expresses the 4-1BBL molecular substance intensively and locally in the tumor microenvironment, thereby promoting the antitumor immune response while avoiding potential systemic toxicity and side effects. This overcomes the bottlenecks in immunotumor therapy, where antitumor immune cells have difficulty entering tumor tissue and antitumor immunity is easily inhibited by the patient's immunosuppressive mechanisms.

[0040] In this invention, by incorporating the 4-1BBL gene into an oncolytic virus, 4-1BBL exerts its effects locally within the tumor microenvironment, thus avoiding systemic side effects. Therefore, according to this invention, the potential risk of 4-1BB signaling overactivation caused by artificially produced antibodies can be effectively avoided, and systemic toxicity and side effects associated with the direct use of soluble 4-1BBL can also be avoided.

[0041] By using recombinant cowpox virus incorporating a gene constructed in this invention, which has a synonymous mutation introduced into the 4-1BBL gene, the expression of exogenous 4-1BBL can be monitored using simple molecular biological techniques such as gene sequencing without altering the sequence of the 4-1BBL protein, and the therapeutic effect can be monitored and evaluated. [Brief explanation of the drawing]

[0042]

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[0043] To further clarify the object, technical means, and advantages of the present invention, the present invention will be described in more detail below. However, it should be understood that this description is for interpretation purposes only and not to limit the scope of the present invention.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Terms used herein are for the purpose of describing specific examples and are not intended to limit the invention. All reagents and instruments used herein are commercially available, and all characterization methods described herein are based on prior art and therefore their descriptions are omitted herein.

[0045] To better understand the present invention, it will be described in more detail below based on preferred embodiments.

[0046] Recombinant cowpox virus (abbreviated as VV-mH4-1BBL) with the synonymous mutation introduced into the 4-1BBL gene (mH4-1BBL) is a recombinant cowpox virus in which the gene sequence of the mH4-1BBL gene, with the synonymous mutation introduced, and related regulatory elements are inserted after the 81001 bp position of the parent cowpox virus genome (the gene sequence conforms to GenBank:AY243312.1). Here, the gene sequence inserted into the parent cowpox virus genome is shown as SEQ ID NO: 22.

[0047] Here, the gene mH4-1BBL, into which a synonymous mutation has been introduced, was obtained by selecting the 540th base of the coding sequence of the wild-type human 4-1BBL gene sequence (the gene sequence conforms to the coding sequence of the NCBI Reference Sequence: NM_003811.4), designing a primer, and performing a synonymous mutation by PCR.

[0048] The genetic details of the recombinant virus VV-mH4-1BBL are as follows:

[0049] 1. The gene sequence of the parent cowpox virus used in the construction of recombinant oncolytic virus VV-mH4-1BBL conforms to GenBank:AY243312.1. The gene sequence of the parent cowpox virus referred to below is based on this.

[0050] 2. The gene mH4-1BBL, into which synonymous mutations have been introduced using a recombinant vector (PV-mH4-1BBL), and related regulatory elements are incorporated into the parent cowpox virus by homologous recombination. In this process, the mH4-1BBL gene and related regulatory element sequences are inserted after position 81001 bp in the genome of the parent cowpox virus (i.e., midway between the thymidine kinase (TK) gene). This completely disrupts the TK gene (knocks out the TK gene) and achieves the expression of mH4-1BBL. The related regulatory elements contain the gene for yellow fluorescent protein (YFP) for recombinant virus screening (having loxp sequences on both sides of the gene). By infecting 293T cells transfected with the Cre gene with the above recombinant virus and deleting the YFP, VV-mH4-1BBL, the mH4-1BBL recombinant cowpox virus according to the present invention, is obtained. The VV-mH4-1BBL genome, compared to the genome of the parent cowpox virus (VV), has an inserted gene fragment within the TK gene, whose gene sequence (SEQ ID NO: 22) is shown below (the underlined portion of the sequence is the mH4-1BBL gene; it is in the 3' to 5' direction; the other sequences are regulatory elements such as promoters).

[0051] Sequence ID 22 is as follows: 5'-agatcgataaaaattaattattaccggggtaccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaac TTATTCCGACCTCGGTGAAGGGAGTCCGGCTGGGATTTCGGGGGTCACCCGGAAGAGTCCCAAGACTGTGGCGCCCTGGGTAAGCTGCCAGGCATGGCGTGCCCTGGCCTCAGTGTGAAGATGGACGCCCAGGCGCTGGCCGGCACTCAGGTGCAGCAAGCGGCCCTGGAAACCGAAGGCCGAGTTCCGAGCCTCGGAGGAGGCGGGTGGCAGGTCCACGGTCAAAGCCAGGGCGGCGGCCCCAGCAGCAGAGCGCAGTGGCTGCAGGTGCAGCGCAAGTGAAACGGAGCCTGAGCCCTCGCCGGCCACCACGCGCCGCAGCTCTAGTTGAAAGAAGACATAGTAGACTCCAGCCTTGGCCACCACCAGCTCCTTCGTGTCCTCTTTGTAGCTCAGGCCCCCCGTCAGGGACACGCCTGCCAGGCCTGGGTCACTGTACCAGCTCAGGGGCCCATCGATCAGCAGAACATTTTGGGCCACCAGCTGCGCAAACATGCCCTGCCGCAGGTCCAAGAGGCCGGCGGGATCGTCGGGCGAAAGCTCGGGACCCTCGCGGAGTCTCGGGCTGGCCGCGGAGCCGGGCGAGGCGCGAGCCCCGGACACGGCCCAGGGGCAGGCGAGGAAGACGGCGCAGGCGGCAGCGAGCAGCAGCAGCAGCAGCAGCCCCGCGACCAGGGCCCAAGGCAGTACGCGGCAGGCGCGAGCGCGGGGCGCGGGAGGCCACGGGGCTTCGGGGTCCAGTGAAGCGTCAGAGGCGTATTCCATCCTGCAGGGCCGGCCATTATCATCGTGTTTTTCAAAGGAAAACCACGTCCCCGTGGTTCGGGGGGCCTAGACGTTTTTTAACCTCGACTAAACACATGTAAAGCATGTGCACCGAGGCCCCAGATCAGATCCCATACAATGGGGTACCTTCTGGGCATCCTTCAGCCCCTTGTTGAATACGCTTGAGGTGAGCCATTTGACTCTTTCCACAACTATCCAACTCACAACGTGGCACTGGGGTTGTGCCGCCTTTGCAGGTGTATCTTATACACGTGGCTTTTGGCCGCAGAGGCACCTGTCGCCAGGTGGGGGGTTCCGCTGCCTGCAAAGGGTCGCTACAGACGTTGTTTGTCTTCAAGAAGCTTCCAGAGGAACTGCTTCCTTCACGACATTCAACAGACCTTGCATTCCTTTGGCGAGAGGGGAAAGACCCCTAGGAATGCTCGTCAAGAAGACAGGGCCAGGTTTCCGGGCCCTCACATTGCCAAAAGACGGCAATATGGTGGAAAATAACATATAGACAAACGCACACCGGCCTTATTCCAAGCGGCTTCGGCCAGTAACGTTAGGGGGGGGGGAGGGAGAGGGGCGGGCGCGCCTTAAGTCGACttcgagcttatttatattccaaaaaaaaaaaataaaatttcaatttttaagctttcactaattccaaacccacccgctttttatagtaagtttttcacccataaataataaatacaataattaatttctcgtaaaagtagaaaatatattctaatttattgcacggtaaggaagtagatcataactcgagataacttcgtataatgtatgctatacgaagttatgcggccgcttcctcgctcactgacgctagcgccctatagtgagtcgtattacagatcc-3’

[0052] VV-mH4-1BBL according to the present invention is constructed by the following method.

[0053] 1) Creation of the mH4-1BBL gene with a synonymous mutation: Using the wild-type human 4-1BBL gene (the gene sequence conforms to the coding sequence of the NCBI reference sequence: NM_003811.4) as a template, a primer for introducing a synonymous mutation is designed and synthesized, focusing on the 540th base of its coding sequence. PCR is then used to mutate the 540th base T to the 540th base C. This does not change the encoded peptide product. The 4-1BBL gene with the introduced synonymous mutation is named the mH4-1BBL gene.

[0054] 2) Introduction of restriction enzyme cleavage sites on both sides of the mH4-1BBL gene: After introducing SdaI and HpaI restriction enzyme cleavage sites on both sides of the mH4-1BBL gene by PCR, the gene fragment is isolated.

[0055] 3) Preparation of viscous ends for cowpox virus VV homologous recombination vector: After cleaving the VV homologous recombination vector PV-1 (containing the coding sequence for yellow fluorescent protein YFP) using SdaI and HpaI, the plasmid fragment is isolated.

[0056] 4) Ligation of the mH4-1BBL gene with the viscous end of the VV homologous recombination vector PV-1: The products recovered in steps 2) and 3) are ligated.

[0057] 5) Transformation of competent cells and screening of positive clones: The ligation product from step 4) is incubated with competent E. coli and expanded culture. After that, the recombinant plasmid is extracted, and gene sequencing confirms that the mH4-1BBL gene has been correctly cloned into the homologous recombination vector PV-1, and the plasmid is named PV-mH4-1BBL.

[0058] 6) Integration of the mH4-1BBL gene into the VV genome: As VV is infected and replicates, homologous recombination occurs with PV-mH4-1BBL; the mH4-1BBL gene fragment and the coding sequence of the yellow fluorescent protein YFP are both integrated into the VV genome by homologous recombination, forming a recombinant virus; because the insertion site of mH4-1BBL into the VV genome is inside the TK gene, the TK gene is destroyed along with it (i.e., the TK gene is inactivated); and then, monoclonal recombinant VV is screened to obtain highly pure recombinant VV.

[0059] 7) High-purity recombinant VV obtained was used to infect 293T cells expressing the cre enzyme. After excising the YFP gene in the recombinant VV genome, monoclonal recombinant VV was screened to obtain the target recombinant VV, which was then purified. Gene sequencing analysis confirmed that the correct mH4-1BBL gene was incorporated into VV. Oncolytic cowpox virus was obtained by recombinant mH4-1BBL, a gene into which a synonymous mutation had been introduced, and named VV-mH4-1BBL.

[0060] The present invention provides for the use of a recombinant cowpox virus (i.e., VV-mH4-1BBL) with a synonymous mutation introduced into the 4-1BBL gene in the manufacture of an antitumor drug. The recombinant cowpox virus VV-mH4-1BBL may be used alone in the manufacture of an antitumor drug, or in combination with other antitumor drugs or means.

[0061] In particular, recombinant cowpox virus VV-mH4-1BBL can be used in the manufacture of pharmaceuticals or products that treat "immunoinflammatory" tumor tissue, "immune desert" tumor tissue, and "immune privileged" tumor tissue.

[0062] In particular, recombinant cowpox virus VV-mH4-1BBL can be used in the manufacture of pharmaceuticals or products that treat superficial or deep tumors by direct injection or injection in combination with endoscopy.

[0063] Superficial tumors, though not limited to them, include solid tumors located on the body surface, such as melanoma.

[0064] Injection therapy combined with endoscopy includes, but is not limited to, treatments in which tumors are clearly observed using laparoscopy, cholangioscopy, thoracoscopy, coloscopy, neuroendoscopy, etc., and then precise intratumoral injections are performed for deep solid tumors.

[0065] In particular, recombinant cowpox virus VV-mH4-1BBL is used in the manufacture of therapeutic drugs and products for treating tumors at various clinical stages. These tumors include, but are not limited to, pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, gastric cancer, esophageal cancer, glioma, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, lung cancer, breast cancer, multiple myeloma, lymphoma, and melanoma.

[0066] Gene sequencing of various tumors, including intestinal cancer cells, using the mH4-1BBL gene with a synonymous mutation revealed that while the tumor cells themselves express wild-type 4-1BBL, its expression product, the 4-1BBL molecule, is not expressed on the cell surface but is localized within the cell. In the treatment of tumor cells using the mH4-1BBL gene with a synonymous mutation, the treatment process and efficacy can be monitored by detecting the gene at the gene level and its expression level.

[0067] <Examples> Example 1 Recombinant oncolytic cowpox virus is constructed as follows: By introducing site-directed mutations by PCR, a specific codon in the wild-type human 4-1BBL gene (the gene sequence conforms to the coding sequence of the NCBI reference sequence: NM_003811.4) is mutated to a synonymous codon (a synonymous mutation means that the nucleotide sequence of the gene codon is modified, but the encoded amino acid is not changed). The human 4-1BBL gene into which the synonymous mutation has been introduced is named mh4-1BBL. mh4-1BBL is incorporated into the cowpox virus genome, and the TK (thymidine kinase) gene is knocked out to construct a novel recombinant cowpox virus, which is named VV-mh4-1BBL. VV-mh4-1BBL retains the oncolytic properties of the parent VV and, after infecting tumor cells, can express the mh4-1BBL molecule and localize it to the surface of the cell membrane. Although the mh4-1BBL molecule has no amino acid sequence difference from the wild-type 4-1BBL molecule, it is possible to distinguish at the mRNA level whether the 4-1BBL expressed in tumor tissue after treatment with VV-mh4-1BBL is endogenous or exogenous. The mh4-1BBL molecule expressed by oncolytic viruses can avoid the potential risks associated with systemic effects when using soluble 4-1BBL and can exert a concentrated therapeutic effect that enhances anti-tumor immunity in tumor tissue (tumor microenvironment).

[0068] Recombinant oncolytic cowpox virus is characterized by the functional insertion of an exogenous gene. This exogenous gene is capable of expressing 4-1BBL and has been introduced with a synonymous mutation. The exogenous gene is inserted into the TK gene of the cowpox virus. The DNA sequences of the exogenous genes are shown in SEQ ID NOs: 1 to 3.

[0069] The applicant provides examples of gene sequences into which the following three synonymous mutations have been introduced, but these do not limit the technical scope of the present invention. In fact, synonymous mutations are possible at any one or more sites, as long as the sequence of the ultimately encoded peptide / amino acid / protein is identical (i.e., the efficacy is identical or similar).

[0070] The sequence of sequence number 1 (the sequence in which the 540th T is mutated to C) is as follows: tggaatacgcctctgacgcttcactggacccccgaagccccgtggcctcccgcgccccgcgctcgcgcctgccgcgtactgccttgggccctggtc gcggggctgctgctgctgctgctgctcgctgccgcctgcgccgtcttcctcgcctgcccctgggccgtgtccggggctcgcgcctcgcccggctcc gcggccagcccgagactccgcgagggtcccgagcttttcgccgacgatcccgccggcctcttggacctgcggcagggcatgtttgcgcagctggt ggcccaaaatgttctgctgatcgatgggcccctgagctggtacagtgacccaggcctggcaggcgtgtccctgacggggggcctgagctacaaaga ggacacgaaggagctggtggtggccaaggctggagtctactatgtcttctttcaactagagctgcggcgcgtggtggccggcgagggctcaggct ccgtttcacttgcgctgcacctgcagccactgcgctctgctgctggggccgccgccctggcCttgaccgtggacctgccacccgcctcctccgagg ctcggaactcggccttcggtttccagggccgcttgctgcacctgagtgccggccagcgcctgggcgtccatcttcacactgaggccagggcacgc catgcctggcagcttacccagggcgccacagtcttgggactcttccgggtgacccccgaaatcccagccggactcccttcaccgaggtcggaataa

[0071] The sequence of sequence number 2 (the sequence in which the 468th G is mutated to A) is as follows: atggaatacgcctctgacgcttcactggaccccgaagccccgtggcctcccgcgccccgcgctcgcgcctgccgcgtactgccttgggccctggtcgcggggctgctgctgctgctgctgctcgctgccgcctgcgccgtcttcctcgcctgcccctgggccgtgtccggggctcgcgcctcgcccggctccgcggccagcccgagactccgcgagggtcccgagctttcgcccgacgatcccgccggcctcttggacctgcggcagggcatgtttgcgcagctggtggcccaaaatgttctgctgatcgatgggcccctgagctggtacagtgacccaggcctggcaggcgtgtccctgacggggggcctgagctacaaagaggacacgaaggagctggtggtggccaaggctggagtctactatgtcttctttcaactagagctgcggcgcgtggtggccggcgaAggctcaggctccgtttcacttgcgctgcacctgcagccactgcgctctgctgctggggccgccgccctggctttgaccgtggacctgccacccgcctcctccgaggctcggaactcggccttcggtttccagggccgcttgctgcacctgagtgccggccagcgcctgggcgtccatcttcacactgaggccagggcacgccatgcctggcagcttacccagggcgccacagtcttgggactcttccgggtgacccccgaaatcccagccggactcccttcaccgaggtcggaataa

[0072] The sequence of SEQ ID NO: 3 (the sequence with the 597th C mutated to T) is as follows. atggaatacgcctctgacgcttcactggaccccgaagccccgtggcctcccgcgccccgcgctcgcgcctgccgcgtactgccttgggccctggtcgcggggctgctgctgctgctgctgctcgctgccgcctgcgccgtcttcctcgcctgcccctgggccgtgtccggggctcgcgcctcgcccggctccgcggccagcccgagactccgcgagggtcccgagctttcgcccgacgatcccgccggcctcttggacctgcggcagggcatgtttgcgcagctggtggcccaaaatgttctgctgatcgatgggcccctgagctggtacagtgacccaggcctggcaggcgtgtccctgacggggggcctgagctacaaagaggacacgaaggagctggtggtggccaaggctggagtctactatgtcttctttcaactagagctgcggcgcgtggtggccggcgagggctcaggctccgtttcacttgcgctgcacctgcagccactgcgctctgctgctggggccgccgccctggctttgaccgtggacctgccacccgcctcctccgaggctcggaactcggccttcggtttTcagggccgcttgctgcacctgagtgccggccagcgcctgggcgtccatcttcacactgaggccagggcacgccatgcctggcagcttacccagggcgccacagtcttgggactcttccgggtgacccccgaaatcccagccggactcccttcaccgaggtcggaataa The amino acid sequence of the exogenous gene is shown in SEQ ID NO: 4.

[0073] The sequence of SEQ ID NO: 4 is as follows. MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYK EDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE Exogenous genes are linked by IRES sequences.

[0074] IRES is a ribosome entry site and one of the usable ligation sequences.

[0075] The IRES sequence is shown in sequence number 5.

[0076] The sequence of sequence number 5 is as follows: TTATCATCGTGTTTTTCAAAGGAAAACCACGTCCCCGTGGTTCGGGGGGCCTAGACGTTTTTTAACCTCGACTAAACACATGTAAAGCATGTGCACCGAGGCCCCAGATCAGATCCCATACAATGGGGTACCTTCTGGGCATC CTTCAGCCCCTTGTTGAATACGCTTGAGGTGAGCCATTTGACTCTTTCCACAACTATCCAACTCACAACGTGGCACTGGGGTTGTGCCGCCTTTGCAGGTGTATCTTATACACGTGGCTTTTGGCCGCAGAGGCACCTGTCGC CAGGTGGGGGGTTCCGCTGCCTGCAAAGGGTCGCTACAGACGTTGTTTGTCTTCAAGAAGCTTCCAGAGGAACTGCTTCCTTCACGACATTCAACAGACCTTGCATTCCTTTGGCGAGAGGGGAAAGACCCCTAGGAATGCTC GTCAAGAAGACAGGGCCAGGTTTCCGGGCCCTCACATTGCCAAAAGACGGCAATATGGTGGAAAATAACATATAGACAAACGCACACCGGCCTTATTCCAAGCGGCTTCGGCCAGTAACGTTAGGGGGGGGGAGGGAGAGGGG

[0077] The present invention further provides a pharmaceutical composition comprising the recombinant oncolytic cowpox virus, a pharmaceutically acceptable carrier, and a pharmaceutical excipient.

[0078] The pharmaceutical composition is administered by direct injection and / or injection in combination with an endoscope, the endoscope being preferably a laparoscope, cholangioscope, thoracoscopy, enteroscope, or neuroendoscope.

[0079] The present invention further provides the use of the recombinant oncolytic cowpox virus or the pharmaceutical composition in the manufacture of pharmaceuticals for the prevention or treatment of tumors and / or cancer.

[0080] Tumors and / or cancers are solid tumors, preferably cold tumors and / or hot tumors, and their histological types include, but are not limited to, pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, gastric cancer, esophageal cancer, glioma, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, lung cancer, breast cancer, multiple myeloma, lymphoma, and melanoma.

[0081] Example 2 Detection of synonymous mutation sites after infecting intestinal cancer cells with cowpox virus recombinant from human 4-1BBL with synonymous mutations. The biological function of a protein molecule depends on its precise amino acid sequence. The DNA base sequence codes for amino acids using triplet codons. This process involves the biological phenomenon of "degenerate codons" (i.e., one amino acid being coded by one or more triplet codons). Therefore, different codons corresponding to the same amino acid are called synonymous codons. These synonymous codons typically have the same first and second bases, differing only in the third base.

[0082] Based on the biological mechanism described above, synonymous mutations can be introduced into codons in the DNA sequence of genes encoding specific proteins (changing the third base of the codon but not altering the amino acid encoded by that codon). This makes it possible to detect synonymous mutations introduced into cells using molecular biological techniques without affecting the amino acid sequence and function of the gene expression product.

[0083] This embodiment aims to construct and express an oncolytic virus recombinant with a human 4-1BBL gene into which a synonymous mutation has been introduced, thereby exerting the antitumor effect of the oncolytic virus itself, promoting antitumor immunity by expressing the 4-1BBL molecule locally in tumor tissue, and further enabling the detection of therapeutic (exogenous) 4-1BBL by detecting the synonymous mutation site.

[0084] 1. Construction of a recombinant vector containing the human 4-1BBL gene with synonymous mutations. In this example, to verify that the human 4-1BBL mutant gene expressed after infection of tumor cells by cowpox virus recombinant with a synonymous mutation can be detected by molecular biological methods, a human 4-1BBL gene recombinant with a synonymous mutation was created and cloned into the homologous recombination vector PV-1 (Figure 1). The human 4-1BBL gene recombinant with a synonymous mutation was introduced into cowpox virus by homologous recombination, and the tk (thymidine kinase) gene was inactivated to obtain cowpox virus recombinant with the human 4-1BBL gene recombinant with a synonymous mutation.

[0085] Figure 1 shows a schematic diagram of the gene fragment introduced into the recombinant vector PV-1 and the structure of the PV-1 vector. In the vector in Figure 1, the TK5' and TK3' sequences are identical to a portion of the cowpox virus genome and are located on either side of the target gene sequence. By constructing the recombinant vector and introducing it into host cells infected with wild-type cowpox virus, homologous recombination occurs between the TK5' and TK3' sequences and the wild-type cowpox virus gene. The gene sequence between TK5' and TK3' in the vector is introduced into the cowpox virus genome, and the recombinant virus is constructed.

[0086] The terms in Figure 1 are as follows: 1) MCS: A multi-cloning site into which the target gene can be inserted. 2) YFP: The gene for yellow fluorescent protein 3) IRES: Ribosome entry site 4) PV-1: Homologous recombination vector 5) PV-mH4-1BBL: A recombinant vector in which the mH4-1BBL gene is introduced into the multi-cloning site of the PV-1 vector. 6) PV-mH4-1BBL-2: A recombinant vector in which the mH4-1BBL-2 gene is introduced into the multi-cloning site of the PV-1 vector. 7) PV-mH4-1BBL-3: A recombinant vector in which the mH4-1BBL-3 gene is introduced into the multi-cloning site of the PV-1 vector.

[0087] The aforementioned mH4-1BBL gene, mH4-1BBL-2 gene, and mH4-1BBL-3 gene are synonymous 4-1BBL genes, created by introducing synonymous mutations based on the CDS of the wild-type human 4-1BBL gene (NCBI reference sequence: NM_003811.4), in which the base at position 540 (T) is changed to base C, the base at position 468 (G) is changed to base A, and the base at position 597 (C) is changed to base T, respectively.

[0088] 2. Creation of a human 4-1BBL gene with synonymous mutations introduced. 1. Design and production process of synonymous mutations in the CDS of the human 4-1BBL gene, where base 540 T is replaced with base 540 C. To construct a human 4-1BBL gene with synonymous mutations introduced from the coding sequence (CDS) of the wild-type human 4-1BBL gene (H4-1BBL), a mutation primer was designed for base 540 T in the CDS of the wild-type human 4-1BBL gene (NCBI reference sequence: NM_003811.4), and the mutation was performed by PCR to base C. In the CDS of the human 4-1BBL gene, the codon to which base 540 T belongs is "GCT" (the codon that codes for alanine), and even if mutated to "GCC", it still codes for alanine. The primer was designed as follows.

[0089] Pf(Sequence 6): 5'-TATAGTCGACATGGAATACGCCTCTGACGCTTC-3' (including the SalI restriction enzyme cleavage site) Furthermore, we designed and synthesized primers for introducing synonymous mutations, focusing on the 540th base of the coding sequence.

[0090] Pr11 (Sequence ID 7): 5'-CGGGTGGCAGGTCCACGGTCAAGGCCAGGGCGGCGGCCCCAGCAGC-3' Using the wild-type human 4-1BBL gene (H4-1BBL) as a template and Pf and Pr11 as primers, fragments F1-2 were obtained by PCR (the scheme is shown in Figure 2, and the PCR product fragments are shown in Figure 5).

[0091] To construct a human 4-1BBL gene with a synonymous mutation introduced from the wild-type human 4-1BBL gene (H4-1BBL), the coding sequence (CDS) of the wild-type human 4-1BBL gene (NCBI reference sequence: NM_003811.4) was used as the basis. Base T at position 540 of the CDS was selected, a primer was designed, and the mutation to base C was performed by PCR. The primer was designed as follows. Pr(Sequence ID 8): 5'-TATAGGCGCGCCTTATTCCGACCTCGGTGAAGGGAG-3' (including SgsI restriction enzyme cleavage site) Furthermore, we designed and synthesized primers for introducing synonymous mutations, focusing on the 540th base of the coding sequence. Pf12 (Sequence ID 9): 5'-GCTGCTGGGGCCGCCGCCCTGGCCTTGACCGTGGACCTGCCACCCG -3'

[0092] Using the wild-type human 4-1BBL gene (H4-1BBL) as a template, and Pf12 and Pf as primers, fragment F1-1 was obtained by PCR (the scheme is shown in Figure 3, and the PCR product fragment is shown in Figure 5).

[0093] To ligate fragments F1-1 and F1-2, which contain the mutated base, into a full-length human 4-1BBL gene containing the mutation, fragments F1-1 and F1-2 were used as templates, and Pf and Pr were used as primers to obtain a full-length human 4-1BBL gene fragment with the mutated base C at position 540 by PCR. The obtained fragment showed a change in the 540th base of the CDS of the wild-type human 4-1BBL gene compared to the CDS. The mutation is located at the 3rd position of the codon to which it belongs and does not alter the sequence of the encoded peptide product. Therefore, it is called a 4-1BBL gene with a synonymous mutation introduced, and was named mH4-1BBL (the scheme is shown in Figure 4, and the PCR product fragment is shown in Figure 5).

[0094] The details of the primer are as follows: Pf(Sequence 10): 5'-TATAGTCGACATGGAATACGCCTCTGACGCTTC-3' (contains the SalI restriction enzyme cleavage site); Pr(Sequence ID 11): 5'-TATAGGCGCGCCTTATTCCGACCTCGGTGAAGGGAG-3' (including the SgsI restriction enzyme cleavage site). 2. Design and production process of synonymous mutations for base 468 G and base 597 C in the CDS of the human 4-1BBL gene

[0095] To verify the versatility of the synonymous mutation in the 4-1BBL gene in this embodiment, synonymous mutations were performed by selecting base 468 (G) and base 597 (C) of the coding sequence of the wild-type human 4-1BBL gene (gene sequence conforming to NCBI reference sequence: NM_003811.4), similar to the process shown in Figures 2, 3, and 4. Specifically, the procedure is as follows:

[0096] 1) A primer for introducing synonymous mutations was designed, centering on the 468th base of the coding sequence. Pr21 (Sequence ID 12): 5'- CAAGTGAAACGGAGCCTGAGCCTTCGCCGGCCACCACGCGC-3'; Pf22 (Sequence No. 13): 5'- GCGCGTGGTGGCCGGCGAAGGCTCAGGCTCCGTTTCACTTG-3'.

[0097] Using the wild-type human 4-1BBL gene as a template, and with Pf and Pr21 as primer pairs and Pf22 and Pr as primer pairs, a synonymous mutation at position 468 (base 468 G is mutated to base 468 A, but the codon to which it belongs still encodes glutamic acid) was introduced into the human 4-1BBL gene fragment by high-fidelity PCR, and PCR product fragments F2-1 and F2-2 were recovered (Figure 13). Using fragments F2-1 and F2-2 as templates, and F and R as genes, fragments F2-1 and F2-2 were ligated into the complete human 4-1BBL gene with the synonymous mutation at position 468 introduced. The above 468th base mutation does not alter the sequence of the encoded peptide product. The 4-1BBL gene with the introduced synonymous mutation was named the mH4-1BBL-2 gene (PCR product fragment is shown in Figure 5).

[0098] 2) A primer for introducing synonymous mutations was designed, centering on the 597th base of the coding sequence. Pr31 (SEQ ID NO: 14): 5'- GGTGCAGCAAGCGGCCTGAAAACCGAAGGCCGAGTTCCG-3'; Pf32 (Sequence ID 15): 5'- CGGAACTCGGCCTTCGGTTTTCAGGGCCGCTTGCTGCACC-3'.

[0099] Using the wild-type human 4-1BBL gene as a template, and with Pf and Pr31 as primer pairs and Pf32 and Pr as primer pairs, a synonymous mutation at position 597 (base 597 C is mutated to base 597 T, but the codon to which it belongs still encodes phenylalanine) was introduced into the human 4-1BBL gene fragment by high-fidelity PCR, and PCR product fragments F3-1 and F3-2 were recovered (Figure 13). Using fragments F3-1 and F3-2 as templates, and F and R as genes, fragments F3-1 and F3-2 were ligated into the complete human 4-1BBL gene into which the synonymous mutation at position 597 was introduced. The above 597th base mutation does not alter the sequence of the encoded peptide product. The 4-1BBL gene into which the synonymous mutation was introduced was named the mH4-1BBL-3 gene (PCR product fragment is shown in Figure 5).

[0100] 3. Construction of homologous recombination vectors for human 4-1BBL genes into which synonymous mutations have been introduced. Using the restriction enzymes SalI and SgsI, the vector PV-1, the mH4-1BBL gene, the mH4-1BBL-2 gene, and the mH4-1BBL-3 gene were respectively cleaved, followed by electrophoresis on a 1% gel. The gel was then excised to recover the target fragments. The mH4-1BBL gene, mH4-1BBL-2 gene, and mH4-1BBL-3 gene, which had been reconstituted into the enzymatically cleaved vector PV-1 backbone, were ligated using T4 ligase (ligation overnight at 4°C). The ligation products were transformed into DH5α-competent Escherichia coli, spread on LB plate medium containing 100 μg / mL ampicillin, and cultured at 37°C for 16 hours. Resistance colonies were then collected, and recombinant plasmids were extracted. Gene sequencing confirmed that the mH4-1BBL, mH4-1BBL-2, and mH4-1BBL-3 genes were correctly cloned into the homologous recombination vector PV-1. The recombinant plasmids were named PV-mH4-1BBL, PV-mH4-1BBL-2, and PV-mH4-1BBL-3, respectively. 3. Creation of oncolytic viruses recombinant with human 4-1BBL genes into which synonymous mutations have been introduced.

[0101] Recombinant oncolytic viruses were obtained by infecting CV-1 cells with PV-mH4-1BBL, PV-mH4-1BBL-2, and PV-mH4-1BBL-3, respectively, along with cowpox virus (Vaccinia Virus, VV).

[0102] CV1 cells in the logarithmic growth phase were taken, and the cell density was set to 1 × 10⁻⁶. 5 The cells were adjusted to a concentration of cells / mL and seeded in 24-well plates at 1 mL / well. After 24 hours, CV1 cells were infected with cowpox virus (VV). Two hours after infection, PV-mH4-1BBL, PV-mH4-1BBL-2, and PV-mH4-1BBL-3 were mixed with transfection reagent (Polyplus, France) and incubated at room temperature for 15 minutes. 200 μL of the mixture was taken and transfected into CV1 cells. After 2 hours, DMEM medium containing 10% FCS was added, and the cells were cultured for a further 48 hours. The fluorescence intensity of yellow fluorescent protein (YFP) was detected using a fluorescence microscope to confirm homologous recombination between the plasmid and the virus. After completely disrupting the cells, the viral cell suspension was collected.

[0103] CV1 cells in the logarithmic growth phase were taken, and the cell density was set to 1.5 × 10⁻⁶. 5 The concentration was adjusted to cells / mL, and seeded in 6-well plates at 2 mL / well. After 24 hours, the virus cell suspension was added to infect CV1 cells. After 24 hours of culture, the CV1 cells were digested, and CV1 cells expressing YFP were selected using a flow cytometer to prepare single-cell suspensions. By limiting dilution, YFP + CV1 cells were seeded into 96-well plates pre-coated with CV1 cells. After 48 hours, a single YFP was identified. + Wells containing viral plaques were selected, the cells and supernatant were collected, and a virus suspension was obtained by repeating the freeze-thaw cycle.

[0104] 293T cells expressing the cre enzyme were infected with the viral suspension obtained above, and the YFP gene in the recombinant VV genome was excised. Monoclonal recombinant VV was then screened to obtain the target recombinant VV, which was then purified. Gene sequencing analysis confirmed that the human 4-1BBL genes with synonymous mutations, namely the mH4-1BBL, mH4-1BBL-2, and mH4-1BBL-3 genes, were incorporated into VV. Recombinant oncolytic cowpox viruses with the above-mentioned synonymous mutations in the human 4-1BBL genes mH4-1BBL, mH4-1BBL-2, and mH4-1BBL-3 were named VV-mH4-1BBL, VV-mH4-1BBL-2, and VV-mH4-1BBL-3, respectively.

[0105] HeLa cells were cultured in a 15cm diameter petri dish. When the HeLa cell abundance reached approximately 80%, the above-mentioned virus cell suspension was added to infect the HeLa cells, and the infection was amplified until a sufficient amount of virus cell suspension was obtained.

[0106] Recombinant cowpox virus was purified as follows: The virus cell suspension was collected, centrifuged, and the supernatant was discarded. The cells were lysed by repeating the freeze-thaw cycle three times, and the cells were homogenized using a Dawns homogenizer to release the virus particles. The supernatant containing the virus was collected by centrifugation at 1200 r / min, added to the top of a 36% sucrose solution, and centrifuged at 130,000 × g for 1 hour. The resulting precipitate was resuspended in 10 mM Tris-HCl and then frozen for storage.

[0107] To confirm whether the 4-1BBL molecule is expressed on the surface of tumor cells after infecting them with cowpox virus recombinant with the human 4-1BBL gene containing the synonymous mutation constructed above, the virus was used to infect human intestinal cancer cell lines. The expression of the 4-1BBL molecule on the cell surface was then analyzed and detected using immunofluorescence labeling and flow cytometry. Specifically, the results are as follows:

[0108] Human intestinal cancer cell line HCT-116 was taken during the logarithmic growth phase, and the cell density was set to 3 × 10⁻⁶.5 The cells were adjusted to a concentration of cells / mL and seeded in 24-well plates at 1 mL / well. After the cells had completely adhered to the cell wall, they were divided into VV-ΔTK, VV-mH4-1BBL, VV-mH4-1BBL-2, and VV-mH4-1BBL-3 infection groups, and each group was infected to achieve a Multiplication of Infection (MOI) of 1. After 24 hours, the infected cells were collected, stained with an APC-labeled anti-human 4-1BBL monoclonal antibody, and the expression of 4-1BBL molecules in the cell membrane was analyzed by flow cytometry. As is clear from the results, HCT-116 infected with cowpox virus VV-ΔTK, in which only the tk gene was inactivated, did not express the 4-1BBL molecule on the cell surface, but HCT-116 infected with VV-mH4-1BBL, VV-mH4-1BBL-2, and VV-mH4-1BBL-3 were detected to express the 4-1BBL molecule at high levels on the cell surface (Figure 6).

[0109] Figure 6 shows: VV-ΔTK: cowpox virus with the tk gene inactivated; VV-mH4-1BBL: recombinant cowpox virus with the tk gene inactivated and the mH4-1BBL gene fragment inserted; VV-mH4-1BBL-2: recombinant cowpox virus with the tk gene inactivated and the mH4-1BBL-2 gene fragment inserted; VV-mH4-1BBL-3: recombinant cowpox virus with the tk gene inactivated and the mH4-1BBL-3 gene fragment inserted; and Anti-4-1BBL-APC: anti-human 4-1BBL monoclonal antibody labeled with APC.

[0110] IV. Detection of synonymous mutation sites by molecular biological methods after infecting tumor cells with oncolytic cowpox virus recombinant with the human 4-1BBL gene into which synonymous mutations have been introduced. To investigate whether exogenous 4-1BBL introduced after infection of tumor cells with oncolytic cowpox virus recombinant with a synonymous mutation in the human 4-1BBL gene could be detected, human intestinal cancer cell line HCT-116 was infected with recombinant oncolytic viruses VV-mH4-1BBL, VV-mH4-1BBL-2, and VV-mH4-1BBL-3. A non-infected group was also established as a control. Subsequently, detection and analysis were performed by RT-PCR. The details are as follows.

[0111] Using the method described above, VV-mH4-1BBL, VV-mH4-1BBL-2, and VV-mH4-1BBL-3 were infected with the human intestinal cancer cell line HCT-116 at an MOI of 1. A non-infected group was also established. After 24 hours, the cells were harvested, lysed in 200 μL of Trizol solution, and total RNA was extracted. The RNA was reverse transcribed into cDNA using a Takara RT-PCR kit. In addition, total RNA from uninfected HCT116 cells was extracted and reverse transcribed to obtain cDNA.

[0112] Primers were designed for the CDS region of the human 4-1BBL gene (gene sequence conforming to NCBI reference sequence: NM_003811.4) that covers the mutation site. The primer sequences are as follows. Pif(Sequence ID 16): 5'-TGAGCTACAAAGAGGACACGAAGGAGC-3'; PiR (Sequence ID 17): 5'-TTATTCCGACCTCGGTGAAGGGAGTCC-3'.

[0113] Using the obtained cDNA as a template and Pif and PiR as primers, a human 4-1BBL gene fragment covering the synonymous mutation site was amplified by PCR. After ligating the PCR product into a pUMc-T vector, competent DH5α E. coli was transformed and spread on ampicillin-resistant LB plate medium. After resistant colonies formed, a single colony was randomly selected and single-read sequencing was performed. The sequence of the sequencing primer was 5'-GTTGTAAAACGACGGCCAG-3' (SEQ ID NO: 18). The results are as follows.

[0114] Five colonies were selected from the non-infected group, plasmids were extracted, and gene sequencing was performed. The results are shown in Figure 7. Alignment using the BLAST program on the NCBI website showed that the fragments covered by primers Pif and Pir were 100% identical to the CDS region of the human 4-1BBL gene (NCBI reference sequence: NM_003811.4) (Figure 8). This result indicates that the human 4-1BBL gene expressed in intestinal cancer cells is not mutated.

[0115] 1) Ten colonies from the VV-mH4-1BBL-infected group were selected and single-read sequencing was performed. All ten colonies were sequenced, and the synonymous mutation indicated by the arrow in Figure 9 was detected in all of them, with no mutations in other locations. This result indicates that VV-mH4-1BBL can replicate in large quantities after infecting tumor cells, and that its synonymous mutation (a mutation from base 540 T to base 540 C) can be clearly detected. Therefore, synonymous mutations in the human 4-1BBL gene can provide a means to detect and identify the introduction of exogenous human 4-1BBL genes without altering the encoded amino acid sequence.

[0116] 2) Ten colonies were selected from the VV-mH4-1BBL-1 infected group and single-read sequencing was performed. All ten colonies were sequenced, and the synonymous mutation indicated by the arrow in Figure 10 was detected in all of them, with no mutations in other locations. This result indicates that VV-mH4-1BBL can replicate in large quantities after infecting tumor cells, and that its synonymous mutation (a mutation from base 468 G to base 468 A) can be clearly detected. Therefore, synonymous mutations in the human 4-1BBL gene can provide a means to detect and identify the introduction of exogenous human 4-1BBL genes without altering the encoded amino acid sequence.

[0117] 3) Ten colonies were selected from the VV-mH4-1BBL-2 infected group and single-read sequencing was performed. All ten colonies were sequenced, and the synonymous mutation indicated by the arrow in Figure 11 was detected in all of them, with no mutations in other locations. This result indicates that VV-mH4-1BBL can replicate in large quantities after infecting tumor cells, and that its synonymous mutation (a mutation from base 597 C to base 597 T) can be clearly detected. Therefore, synonymous mutations in the human 4-1BBL gene can provide a means to detect and identify the introduction of exogenous human 4-1BBL gene without altering the encoded amino acid sequence.

[0118] Example 3 Antitumor activity and detection of synonymous mutation sites of cowpox virus recombinant with the 4-1BBL gene containing synonymous mutations. In this example, to verify the antitumor effect of cowpox virus recombinant with a 4-1BBL gene containing a synonymous mutation, and to further investigate the detection of the synonymous mutation site in the 4-1BBL gene during the treatment process, synonymous mutants of the mouse 4-1BBL gene were created, and oncolytic cowpox virus was constructed recombinant with the mouse 4-1BBL gene into which the synonymous mutation was inserted. Furthermore, an experimental treatment was performed on a mouse intestinal cancer model with peritoneal cancer, and the synonymous mutation site in the mouse 4-1BBL gene during the treatment process was detected. The specific procedure is as follows.

[0119] 1. Construction of a vector recombinant with the mouse 4-1BBL gene containing a synonymous mutation and oncolytic cowpox virus recombinant with the mouse 4-1BBL gene containing a synonymous mutation.

[0120] To verify that the human 4-1BBL mutant gene expressed after infection of tumor cells by cowpox virus recombinant with a human 4-1BBL gene into which a synonymous mutation has been introduced can be detected by molecular biological methods, a mouse 4-1BBL gene into which a synonymous mutation has been introduced was constructed using the same method as in Example 2, cloned into the homologous recombination vector PV-1 (Figure 12), and named PV-4-1BBL. The results of gene sequencing identification are shown in Figure 13. When the obtained sequence was aligned using the BLAST program on the NCBI website, the 309th base C of the CDS of the 4-1BBL gene in PV-4-1BBL was mutated to base T compared with the CDS of the mouse 4-1BBL gene (NCBI reference sequence ID: NM_009404.3) (indicated by the arrow in Figure 14). That is, the codon to which it belongs has mutated from ACC to ACT, but the encoded product in both cases is threonine. Therefore, we were able to construct a homologous recombination vector based on the mouse 4-1BBL gene (referred to as 4-1BBL in this example) into which a synonymous mutation had been introduced.

[0121] Using the same method as in Example 2, the 4-1BBL gene, into which a synonymous mutation was introduced using PV-4-1BBL, was incorporated into oncolytic cowpox virus. Simultaneously, the tk gene of the cowpox virus was knocked out to construct a recombinant oncolytic virus, which was named VV-ΔTK-4-1BBL. Furthermore, the antitumor function of VV-ΔTK-4-1BBL and the synonymous mutation site of the 4-1BBL gene were detected through in vivo and in vitro experiments.

[0122] In Figure 12, PV-4-1BBL is a recombinant vector in which a synonymous mutation in the mouse 4-1BBL gene is introduced into the multi-cloning site of the PV-1 vector. 2. Detect 4-1BBL, which is expressed after VV-ΔTK-4-1BBL infects intestinal cancer cells, at the mRNA level using RT-PCR.

[0123] To detect the expression of the 4-1BBL gene after infection of tumor cells with VV-ΔTK-4-1BBL, the intestinal cancer cell line MC-38 was used as a model, and MC-38 cells were infected with VV-ΔTK-4-1BBL at an infection multiplicity of MOI=1. A VV-ΔTK-infected group was also established as a control. After 24 hours of infection, tumor cells were collected, total RNA was extracted, reverse transcribed into cDNA, and the 4-1BBL gene was amplified by PCR.

[0124] As is clear from the experimental results (Figure 15), the target gene band was bright and dense in the VV-ΔTK-4L infected group, while only a background-level target gene band was present in the VV-ΔTK infected group and the uninfected group. This result indicates that VV-ΔTK-4-1BBL expresses the 4-1BBL gene at a high level through rapid viral replication after infecting tumor cells. This result also indicates that tumor cells express the 4-1BBL gene at a background level (according to the applicant's previous research, the gene, if no synonymous mutation is introduced, has its encoding protein expressed within the tumor cell membrane).

[0125] Figure 15 shows the results of detecting high expression of the 4-1BBL gene by RT-PCR after infection of MC-38 cells with recombinant oncolytic cowpox virus VV-ΔTK-4-1BBL. In the figure, lane M: DL2000 DNA marker; lane 1: uninfected group; lane 2: VV-ΔTK infected group; lane 3: uninfected group; lane 4: VV-ΔTK-4-1BBL infected group.

[0126] 3. Detection of 4-1BBL expression on the surface of VV-ΔTK-4-1BBL-infected intestinal cancer cells by flow cytometry To determine whether rapid replication of recombinant oncolytic viruses within tumor cells can achieve high expression of the 4-1BBL molecule and localization to the tumor cell surface, MC-38 intestinal cancer cells in the logarithmic growth phase were infected with different viral infection multiplicities (MOI) of 0, 0.1, 1, and 5. After 24 hours, the cells were digested to prepare single-cell suspensions, stained with fluorescein-labeled anti-4-1BBL monoclonal antibodies, and 4-1BBL expression in the cell membrane was analyzed by flow cytometry.

[0127] As is clear from the experimental results (Figure 16), the expression of yellow fluorescent protein (YFP) carried by recombinant viruses increased with increasing multiplicity of viral infection (MOI); in the VV-△TK-4L infected group, membrane-type 4-1BBL was expressed and its expression level increased with increasing MOI; in the VV-△TK infected group, membrane-type 4-1BBL was not expressed. These results indicate that after infecting tumor cells with VV-△TK-4-1BBL, the expression of the 4-1BBL molecule on the surface of tumor cells can be achieved.

[0128] IV. Detection of VV-ΔTK-4-1BBL proliferative capacity in different tumor cells using viral titer method. To confirm the effect of genetic recombination on the replication and proliferation ability of cowpox virus in tumor cells, each recombinant virus was used to infect LTPA cells, B16-F10 cells, Panc02 / Luc cells, and MC-38 cells, respectively. The cells were harvested at 24, 48, and 72 hours post-infection, diluted through repeated freeze-thaw cycles, and then infected with green monkey kidney cells (CV-1). Viral production was measured by plaque assay.

[0129] As is clear from the experimental results (Figure 17), the 4-1BBL gene, after being incorporated into the cowpox virus, did not alter the replication characteristics of the virus itself in tumor cells.

[0130] V. Detection and comparison of the tumor cell-killing activity of VV-ΔTK-4-1BBL In this experiment, in order to confirm whether the gene recombination in this example affects the oncolytic effect of the virus, each recombinant virus was infected into LTPA cells, B16-F10 cells, Panc02 / Luc cells and MC-38 cells at different MOIs (0, 0.1, 0.5 and 5). After 48 hours, the proportion of surviving cells was detected by the MTT method.

[0131] As is clear from the experimental results (Figure 18), the 4-1BBL gene does not modify the oncolytic effect of the virus itself after being incorporated into the vaccinia virus.

[0132] VI. Detection of 4-1BBL synonymous mutation sites in the process of tumor treatment by oncolytic vaccinia virus recombinant with 4-1BBL gene with synonymous mutations In order to verify whether it is possible to detect an oncolytic vaccinia virus recombinant with the 4-1BBL gene with synonymous mutations introduced during the tumor treatment process, after establishing a mouse intestinal cancer model and performing experimental treatment with VV-ΔTK-4-1BBL, tumor tissues were collected for detection. The specific procedure is as follows.

[0133] C57BL / 6 mice aged 8 - 12 weeks were selected and inoculated intraperitoneally with 5×10 5 / mouse of MC-38 intestinal cancer cells. After 9 days, the mice were randomly divided into 2 groups. One group was injected intraperitoneally with 2×10 8 PFU / mouse of VV-ΔTK-4L, and the other group was injected intraperitoneally with an equal volume of PBS. After 48 hours, the mice were sacrificed, tumor tissues were collected, 10 mg of tumor tissue was dissolved in Trizol, total RNA was extracted, reverse transcribed into cDNA, and then PCR was performed.

[0134] Primers were designed for the CDS region of the mouse 4-1BBL gene (NCBI reference sequence ID: NM_009404.3). The primer sequences are as follows. Pmf (SEQ ID NO: 19): 5‘-TATAAAGCTTATGGACCAGCACACACTTGATG-3’; Pmr (SEQ ID NO: 20): 5'-TATATCTAGATCATTCCCATGGGTTGTCGG-3'.

[0135] Using the obtained cDNA as a template and Pmf and PmR as primers, the full-length fragment covering the CDS of the mouse 4-1BBL gene was amplified by PCR. After ligating the PCR product into a pUMc-T vector, competent DH5α E. coli were transformed and spread on ampicillin-resistant LB plate medium. After resistance colonies formed, a single colony was randomly selected and single-read sequencing was performed. The sequence of the sequencing primer was 5'-GTTGTAAAACGACGGCCAG-3' (SEQ ID NO: 21).

[0136] As is clear from the experimental results, when three colonies were randomly selected from the PBS treatment group and sequenced, the resulting 4-1BBL gene fragments were all wild-type 4-1BBL genes, i.e., they were 100% identical to the CDS region of the mouse 4-1BBL gene (NCBI reference sequence ID: NM_009404.3) (Figures 19 and 20). There was no mutation at base 309 C of the CDS region of the mouse 4-1BBL gene, as indicated by the arrow in Figure 19. When 10 colonies were randomly selected from the VV-△TK-4-1BBL group and sequenced, one of the colonies had a 4-1BBL gene sequence that was 100% identical to the CDS region of the mouse 4-1BBL gene (NCBI reference sequence ID: NM_009404.3), but the remaining nine colonies all had a mutation at base 309 C of the CDS region, with no mutations in other areas (Figures 21 and 22). In Figure 21, the 309th base C in the CDS of the mouse 4-1BBL gene, indicated by the arrow, was mutated to base T, and in Figure 22, the 309th base C in the CDS of the mouse 4-1BBL gene, indicated by the arrow, was mutated to base T. The wild-type 4-1BBL gene originates from tumor-bearing mice, while the highly expressed 4-1BBL gene with synonymous mutations originates from the oncolytic virus VV-△TK-4-1BBL.

[0137] These results indicate that tumor cells express the 4-1BBL gene at background levels. Furthermore, these results demonstrate that during in vivo treatment with VV-△TK-4-1BBL, tumor cells express the 4-1BBL gene with synonymous mutations introduced through rapid viral replication after infection at high levels, and that this can be detected by molecular biological means.

[0138] 7. Detection of the effects and mechanisms of oncolytic cowpox virus on peripheral immune organs, recombinant with the 4-1BBL gene into which synonymous mutations have been introduced. 1. To comprehensively consider the in vivo antitumor effect and mechanism of action of VV-ΔTK-4-1BBL and to verify its effects on peripheral immune tissues and organs, 8-12 week old C57BL / 6 mice were selected and MC-38 intestinal cancer cells were injected in 5 × 10⁶ cells. 5 Intraperitoneal inoculation was performed in mice. After 9 days, the mice were randomly divided into groups of 2 × 10⁶. 8 PFU / mice were intraperitoneally injected with VV-ΔTK-4L, VV-ΔTK, and an equal volume of PBS. After 5 days, the mice were sacrificially killed, their spleens were harvested and weighed, and single-cell suspensions were prepared and counted.

[0139] As is evident from the experimental results (Figure 23), treatment of MC-38 tumor-bearing mice with VV-ΔTK-4-1BBL induced an increase in spleen weight (Figure 23A) and spleen cell count (Figure 23B), indicating that the proliferation of peripheral immune cells and the enlargement of peripheral immune tissue organs were promoted.

[0140] As is clear from the experimental results (Figure 24), after treating MC-38 tumor-bearing mice with VV-ΔTK-4-1BBL, CD3 in spleen cells + T cell (Figure 24A), CD4 + T cells (Figure 24B) and CD8 + An increase in the proportion of T cells (Figure 24C) is promoted. This indicates that VV-ΔTK-4-1BBL can promote the proliferation of T cells, their subpopulations, and NK cells in peripheral immune tissues and organs through a synergistic effect between the oncolytic virus and the 4-1BBL molecule expressed by it.

[0141] 2. Comprehensively examine the in vivo antitumor effect and mechanism of action of VV-ΔTK-4-1BBL, and identify its major antitumor immune cell, CD4. + T, CD8 + To investigate the effects on the expression of immune checkpoint molecules such as PD-1 in T cells, we selected 8-12 week old C57BL / 6 mice and administered MC-38 intestinal cancer cells in 5 × 10⁶ cells. 5 Intraperitoneal inoculation was performed in mice. After 9 days, the mice were randomly divided into groups of 2 × 10⁶. 8 PFU-enhanced mice were intraperitoneally injected with VV-ΔTK-4L, VV-ΔTK, and an equal volume of PBS. After 5 days, the mice were sacrificially killed, and their spleens were collected to prepare single-cell suspensions, which were then analyzed by immunofluorescence staining and flow cytometry.

[0142] As is clear from the experimental results (Figure 25), VV-ΔTK-4-1BBL induces an antitumor immune response after treating the tumor, and CD4 + T, CD8 + The expression of PD-1 molecules on the surface of immune cells such as T cells can be increased through feedback control. This result suggests that there is sufficient evidence to support the concept that combination therapy with VV-ΔTK-4-1BBL and PD-1 / PD-L1 pathway inhibitors improves the efficacy of antitumor treatment. 8. Detection of the effects and mechanisms of oncolytic cowpox virus on the tumor microenvironment, recombinant with the 4-1BBL gene into which synonymous mutations have been introduced.

[0143] 1. To investigate the effect of VV-ΔTK-4-1BBL on the antitumor immune status in the tumor microenvironment, 8-12 week old C57BL / 6 mice were selected, and MC-38 intestinal cancer cells were introduced in 5 × 10⁶ cells. 5 Intraperitoneal inoculation was performed in mice. After 9 days, the mice were randomly divided into groups of 2 × 10⁶. 8 PFU / mice were intraperitoneally injected with VV-ΔTK-4L, VV-ΔTK, and equivalent volumes of PBS. After 5 days, the mice were sacrificially killed, tumor tissue was collected, and single-cell suspensions were prepared and analyzed by immunofluorescence staining and flow cytometry.

[0144] As is clear from the experimental results (Figure 26), after treating MC-38 tumor-bearing mice with VV-ΔTK-4-1BBL, the CD3 in the tumor tissue was reduced. + T, CD8 + T, CD4 + The proportion of T cells increased significantly. This indicates that VV-ΔTK-4-1BBL can modify the anti-tumor immune status by promoting an increase in the number of T cells and their subpopulations in tumor tissue through the synergistic effect of the oncolytic virus and the 4-1BBL molecule expressed by it.

[0145] 2. To investigate the effect of VV-ΔTK-4-1BBL on the antitumor immune status in the tumor microenvironment, 8-12 week old C57BL / 6 mice were selected, and MC-38 intestinal cancer cells were introduced in 5 × 10⁶ cells. 5 Intraperitoneal inoculation was performed in mice. After 9 days, the mice were randomly divided into groups of 2 × 10⁶. 8 PFU / mice were intraperitoneally injected with VV-ΔTK-4L, VV-ΔTK, and equivalent volumes of PBS. After 5 days, the mice were sacrificially killed, and 50 mg of each tumor tissue was dissolved in Trizol. Total RNA was then extracted, reverse transcribed into cDNA, and analyzed by real-time PCR (real-time quantitative PCR).

[0146] As is clear from the experimental results (Figure 27), antitumor immune molecules such as perforin, granzyme B, IFN-γ, IL-1β, and TNF-α were significantly increased in the tumor tissue of the VV-ΔTK-4-1BBL treatment group, while suppressive immune molecules such as TGF-β were significantly decreased compared to the oncolytic virus blank control (VV-ΔTK) group. These results further indicate that VV-ΔTK-4-1BBL can modify the immune status in tumor tissue and promote antitumor immune effects through synergistic action with the oncolytic virus and the 4-1BBL molecule expressed by it.

[0147] IX. Verification of the in vivo antitumor effect of oncolytic cowpox virus recombinant with the 4-1BBL gene into which synonymous mutations have been introduced. In this study, a novel recombinant cowpox virus was constructed by incorporating the 4-1BBL gene into the cowpox virus genome. To compare its in vivo antitumor activity, a colon cancer peritoneal tumor model was created by injecting mouse colon cancer cells MC-38 into the peritoneal cavity of C57 / BL6 mice. After 9 days, the mice were randomly divided into groups, each recombinant virus was injected intraperitoneally, and the survival time of the mice was observed. As is clear from the results, the survival time of tumor-bearing mice in the VV-ΔTK (VV with the TK gene knocked out) treatment group and the VV-ΔTK-4-1BBL (VV with the TK gene knocked out and the 4-1BBL gene incorporated) treatment group was longer than that of the phosphate-buffered (PBS) treatment group, and the survival time of tumor-bearing mice in the VV-ΔTK-4-1BBL treatment group was longer than that of the VV-ΔTK treatment group.

[0148] As is clear from the experimental results (Figure 28), when used for tumor treatment, VV-ΔTK-4-1BBL exhibits a remarkable antitumor effect and can extend the survival time of the treated patient. 10. Verification of in vivo antitumor activity by combining oncolytic cowpox virus recombinant with a 4-1BBL gene containing a synonymous mutation with a PD-1 monoclonal antibody. To investigate the efficacy of combination therapy with VV-ΔTK-4-1BBL and immune checkpoint inhibitors, this experiment created a peritoneal intestinal cancer tumor model by injecting mouse intestinal cancer cells MC-38 into the peritoneal cavity of C57 / BL6 mice. After 9 days, the mice were randomly divided into four groups: a PBS treatment group, a VV-ΔTK-4-1BBL treatment group, a PD-1 inhibitory monoclonal antibody treatment group, and a combination therapy group of VV-ΔTK-4-1BBL and a PD-1 inhibitory monoclonal antibody. Each group was treated by intraperitoneal injection, and the survival time of the mice was observed.

[0149] As is clear from the experimental results (Figure 29), the survival time of tumor-bearing mice in both the VV-ΔTK-4-1BBL treatment group and the PD-1 inhibitory monoclonal antibody treatment group was longer than that of the PBS treatment group, and the therapeutic effects of the VV-ΔTK-4-1BBL treatment group and the PD-1 inhibitory monoclonal antibody treatment group were equivalent. The therapeutic effect of the combination therapy group of VV-ΔTK-4-1BBL and the PD-1 inhibitory monoclonal antibody was significantly superior to that of the VV-ΔTK-4-1BBL treatment group and the PD-1 inhibitory monoclonal antibody treatment group.

[0150] The results described above indicate that VV-ΔTK-4-1BBL can be an effective means of enhancing the tumor therapeutic effect of immune checkpoint inhibitors such as PD-1 monoclonal antibodies. The combination of VV-ΔTK-4-1BBL and immune checkpoint inhibitors can yield more pronounced antitumor effects and improve the survival rate and survival time of the treated patients.

[0151] The foregoing are merely preferred embodiments of the present invention and do not limit it. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be within the scope of protection of the present invention.

Claims

1. Recombinant oncolytic cowpox virus in which an exogenous gene is functionally inserted, The aforementioned exogenous gene is capable of expressing 4-1BBL and is characterized by having a synonymous mutation introduced into it, thereby enabling recombinant oncolytic cowpox virus.

2. The recombinant oncolytic cowpox virus according to claim 1, characterized in that the exogenous gene is inserted into the TK gene of the cowpox virus.

3. The recombinant oncolytic cowpox virus according to claim 1, characterized in that the DNA sequence of the exogenous gene is represented by SEQ ID NOs: 1 to 3.

4. The recombinant oncolytic cowpox virus according to claim 1, characterized in that the amino acid sequence of the exogenous gene is shown in SEQ ID NO:

4.

5. The recombinant oncolytic cowpox virus according to claim 1, characterized in that the exogenous gene is linked by an IRES sequence.

6. The recombinant oncolytic cowpox virus according to claim 5, characterized in that the IRES sequence is represented by Sequence ID No.

5.

7. A pharmaceutical composition characterized by containing recombinant oncolytic cowpox virus according to any one of claims 1 to 6, a pharmaceutically acceptable carrier, and a pharmaceutical excipient.

8. The pharmaceutical composition according to claim 7, wherein the administration method of the pharmaceutical composition is direct injection and / or injection in combination with an endoscope, the endoscope being preferably a laparoscope, cholangioscope, thoracoscopy, enteroscope, or neuroendoscope.

9. Use of recombinant oncolytic cowpox virus according to any one of claims 1 to 6, or a pharmaceutical composition according to any one of claims 7 to 8, in the manufacture of a pharmaceutical product for preventing or treating tumors and / or cancer.

10. The use according to claim 9, wherein the tumor and / or cancer is a solid tumor, preferably a cold tumor and / or a hot tumor, and its histological types include, but are not limited to, pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, gastric cancer, esophageal cancer, glioma, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, lung cancer, breast cancer, multiple myeloma, lymphoma, and melanoma.

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