Recombinant oncolytic vaccinia virus and use thereof

Abstract

Provided in the present invention is a recombinant oncolytic vaccinia virus, which is operably inserted into a synonymously mutated exogenous gene capable of expressing 4-1BBL; and also provided is the use of the recombinant oncolytic vaccinia virus in the preparation of a drug for preventing or treating tumors and cancers. The present invention has the following beneficial effects: the synonymous-mutation-based recombinant vaccinia virus VV-mH4-1BBL retains the original oncolytic effect of the oncolytic virus and the functions thereof of initiating and enhancing anti-tumor immune responses, and improves the safety by means of deleting the TK gene; 4-1BBL is highly expressed on the surface of a tumor cell, such that 4-1BBL can enhance the anti-tumor immunity by means of exciting a 4-1BB signal from 4-1BB+ immune cells (including T cells) in the tumor microenvironment, and 4-1BBL is also confined within tumor tissues to exert the function thereof in a centralized manner, thereby avoiding potential systemic toxic side effects; and the introduction of a synonymous mutation site enables the virus to detect the expression of a therapeutic (exogenous) 4-1BBL gene during treatment.

Description

Recombinant oncolytic vaccinia virus and its application Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a recombinant oncolytic vaccinia virus and its application. Background Art

[0002] Stimulating 4-1BB signaling in immune cells can trigger strong and long-lasting anti-tumor immunity. Currently, 4-1BB-stimulating monoclonal antibodies, such as urelumab and utomilumab, are entering clinical trials. However, due to the small difference between the effective and safe doses, systemic toxicity and side effects often occur when these antibodies effectively stimulate anti-tumor immunity, or when there are no significant toxicity but no significant anti-tumor effect. These conflicting issues have greatly limited their clinical application.

[0003] Soluble 4-1BBL can stimulate 4-1BB signaling, which has not been seen in clinical application studies. If used directly systemically, it can strongly enhance the immune response throughout the body and cause serious toxic side effects.

[0004] Oncolytic viruses can selectively infect tumor cells, induce immunogenic cell death, activate the body's anti-tumor immunity, and trigger local inflammation, acting as immune adjuvants. However, the use of oncolytic viruses alone to treat tumors often results in the body's antiviral response quickly clearing the virus. Therefore, after the oncolytic virus takes effect, it is necessary to enhance the anti-tumor immunity it triggers in order to induce a strong and lasting anti-tumor effect. Therefore, it is necessary to introduce immune-enhancing factors to improve the anti-tumor effect of oncolytic viruses.

[0005] Studies have reported the construction of a mouse 4-1BBL recombinant oncolytic virus using the mouse 4-1BBL gene, and the reliability of its effect has been demonstrated using a mouse model. However, recent studies have found that the wild-type 4-1BBL gene is commonly expressed in tumor cells such as colorectal cancer, and its encoded product (i.e., the 4-1BBL molecule) is located intracellularly rather than on the cell surface. Therefore, if a 4-1BBL recombinant oncolytic virus is constructed using the wild-type 4-1BBL gene for treatment, the expression and localization of therapeutic 4-1BBL will not be detected and tracked.

[0006] Oncolytic viruses can selectively infect and cause immunogenic death of tumor cells, thereby triggering the body's anti-tumor immune response, making it a feasible means of tumor immunotherapy and entering the field of tumor immunotherapy drug research and development. However, as viruses themselves, oncolytic viruses are easily cleared by the body, and the anti-tumor immunity triggered by oncolytic viruses faces the common problem of being blocked by negative mechanisms such as immune checkpoints. Therefore, the overall design concept in this field is to construct a recombinant oncolytic virus, retain the advantages of the original oncolytic virus, circumvent its weaknesses, and then recombinantly introduce immune enhancement factors to overcome the negative immune mechanism and trigger the body's safe, effective, systemic and long-lasting anti-tumor immunity.

[0007] The co-stimulatory molecule ligand 4-1BBL acts on the 4-1BB molecule on the surface of T cells, NK cells, DCs, etc., that is, through the 4-1BB / 4-1BBL pathway, it strongly promotes the body's anti-tumor immunity. Therefore, biological agents developed with the 4-1BB / 4-1BBL pathway as a stimulatory target may become new anti-tumor drugs. Stimulating 4-1BB monoclonal antibodies and 4-1BBL recombinant oncolytic viruses are important biological agents developed in this field. However, their shortcomings restrict their future application and need to be improved.

[0008] Stimulating 4-1BB monoclonal antibodies can activate the 4-1BB / 4-1BBL pathway to produce a strong anti-tumor effect. However, the difference in monoclonal antibody dose between effective anti-tumor effects and toxic side effects is small, meaning the safe dose range is narrow. Therefore, the natural ligand 4-1BBL was selected as an immune activator of the 4-1BB molecule to provide a natural stimulation method to activate 4-1BB signaling. In addition, if soluble 4-1BBL is directly used to treat tumors, it may cause toxic side effects due to the systemic effects of cytokines.

[0009] However, existing studies have shown that 4-1BBL molecules are commonly present inside tumor cells such as colorectal cancer and pancreatic cancer, but are not expressed on the cell membrane. In this way, tumor cells can avoid stimulating the 4-1BB molecules on the surface of immune cells. This is a tumor immune escape mechanism. For example, using the natural ligand 4-1BBL to construct a recombinant oncolytic virus can express 4-1BBL on the surface of tumor cells and stimulate anti-tumor immunity. However, in future treatment processes, it will be difficult to distinguish whether the 4-1BBL gene in the tumor tissue comes from the tumor tissue itself or the therapeutic 4-1BBL recombinant oncolytic virus, which will make it extremely difficult to monitor and evaluate the efficacy.

[0010] Summary of the Invention

[0011] In response to the above-mentioned technical limitations, the present invention proposes a recombinant oncolytic vaccinia virus and its application, establishes a new 4-1BB signal stimulation method, retains the immune effect of 4-1BBL stimulating 4-1BB signal, improves its safety of use, and provides a new biological agent that can detect and monitor the treatment process and efficacy, overcoming the shortcomings and defects mentioned in the background technology.

[0012] To achieve the above object, the present invention adopts the following technical solutions:

[0013] The invention of the present invention is to provide a recombinant oncolytic vaccinia virus, into which a synonymously mutated exogenous gene capable of expressing 4-1BBL is operably inserted.

[0014] Optionally, in the above-mentioned recombinant oncolytic vaccinia virus, the exogenous gene is inserted into the TK gene of the vaccinia virus.

[0015] Optionally, in the above-mentioned recombinant oncolytic vaccinia virus, the DNA sequence of the exogenous gene is shown as SEQ ID No.1-SEQ ID No.3.

[0016] The applicant has listed the gene sequences after these three synonymous mutations, but they do not limit the scope of the technology of the present invention. In fact, as long as the final encoded polypeptide / amino acid / protein sequence is the same (with the same or similar efficacy), synonymous mutations at any one or several sites are feasible.

[0017] The sequence of SEQ ID No. 1 (540T mutated to C) is:

[0018] The sequence of SEQ ID No. 2 (468G mutated to A) is:

[0019] The sequence of SEQ ID No. 3 (597C mutated to T) is:

[0020] Optionally, in the above-mentioned recombinant oncolytic vaccinia virus, the amino acid sequence of the exogenous gene is shown as SEQ ID No.4.

[0021] The sequence of SEQ ID No. 4 is:

[0022] Optionally, in the above-mentioned recombinant oncolytic vaccinia virus, the exogenous gene is connected via an IRES sequence.

[0023] IRES, ribosome entry site, is just one of the linker sequences that can be used.

[0024] Optionally, in the above-mentioned recombinant oncolytic vaccinia virus, the IRES sequence is shown as SEQ ID No. 5.

[0025] The sequence of SEQ ID No.5 is:

[0026] The second invention of the present invention is to provide a pharmaceutical composition, which includes the above-mentioned recombinant oncolytic vaccinia virus, a pharmaceutically acceptable carrier and pharmaceutical excipients.

[0027] Optionally, the pharmaceutical composition is administered by direct injection and / or combined with endoscopic injection; the endoscope is preferably a laparoscope, a choledochoscope, a thoracoscope, a colonoscope and a neuroendoscope.

[0028] The third invention of the present invention is to provide the use of the above-mentioned recombinant oncolytic vaccinia virus and the above-mentioned pharmaceutical composition in the preparation of drugs for preventing or treating tumors and / or cancers.

[0029] Optionally, in the above-mentioned application, the tumor and / or cancer is a solid tumor, preferably a cold tumor and / or a hot tumor, and its histological type includes but is not limited to pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, gastric cancer, esophageal cancer, brain glioma, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, lung cancer, breast cancer, multiple myeloma, lymphoma, and melanoma.

[0030] The key technical points and advantages of the present invention are:

[0031] 1. A synonymous mutant human 4-1BBL gene sequence, which can be used to detect and monitor the expression and distribution of therapeutic (exogenous) 4-1BBL in tumor patients after treatment with recombinant vaccinia virus VV-mH4-1BBL;

[0032] 2. The recombinant vaccinia virus VV-mH4-1BBL, based on the human 4-1BBL gene sequence with synonymous mutations, retains the original characteristics and advantages of oncolytic viruses in terms of oncolytic effect, initiation of anti-tumor immune response, and enhancement of immune response as an adjuvant. It also makes it safer on the basis of its original good safety by deleting the TK gene. The recombinant vaccinia virus VV-mH4-1BBL achieves high expression of 4-1BBL on the surface of tumor cells in the tumor microenvironment, enabling 4-1BBL to stimulate 4-1BB signals in 4-1BB+ immune cells (including T cells) in the tumor microenvironment to enhance anti-tumor immunity. It also confines 4-1BBL to tumor tissues to concentrate its effects, avoiding the potential systemic toxic side effects brought about by the systemic use of stimulatory 4-1BB monoclonal antibodies or soluble 4-1BBL to treat tumors.

[0033] Compared with other oncolytic viruses, vaccinia virus has its own characteristics:

[0034] 1. It has been used on a large scale to prevent smallpox in the human population and its safety has been proven;

[0035] 2. The weak expression / no expression of the TK gene in normal cells and the strong expression in many tumor cells determine that VV after TK gene knockout is almost not expressed in normal cells, indicating that the product modified with VV as the parent virus has extremely high safety.

[0036] The present invention constructs a human 4-1BBL synonymous mutation gene (a synonymous mutation is a mutation of a certain base pair in a DNA fragment that does not change the encoded amino acid), and on this basis constructs a recombinant oncolytic poxvirus VV-mH4-1BBL with a human 4-1BBL synonymous mutation gene. This not only retains the original oncolytic effect of the oncolytic virus, the initiation of anti-tumor immunity, and the role of an adjuvant to enhance the immune response, but also makes it safer on the basis of its original good safety by deleting the TK gene; through the recombinant cowpox virus VV-mH4-1BBL, 4-1BBL is highly expressed on the surface of tumor cells in the tumor microenvironment, and 4-1BBL stimulates 4-1BB signals of 4-1BB+ immune cells (including T cells) in the tumor microenvironment to enhance anti-tumor immunity. 4-1BBL is also confined to the tumor tissue to exert its effect, avoiding the potential systemic toxic side effects brought about by the systemic use of stimulating 4-1BB monoclonal antibodies or soluble 4-1BBL to treat tumors.

[0037] The application of mH4-1BBL in the preparation of tumor drugs has the following trends and effects:

[0038] 1. Application of mH4-1BBL in improving the anti-tumor immune status of the tumor microenvironment:

[0039] The interaction between tumor cells and anti-tumor immunity jointly determines the occurrence and progression of tumors. The number and functional status of immune cells (especially dendritic cells (DCs) and T cells) in the tumor microenvironment are one of the key factors affecting the efficacy of tumor immunotherapy and patient outcomes.

[0040] mH4-1BBL mainly dissolves local tumor tissue through the characteristics of oncolytic viruses, initiates immune responses, and establishes an inflammatory environment for tumor immunity in the local tumor. By expressing the mH4-1BBL encoding protein and expressing it on the surface of tumor cells, it enhances and maintains the anti-tumor functional state of immune cells such as DCs and T cells in the "inflammatory environment" of tumor tissue. Therefore, VV-mH4-1BBL has an effect on tumor tissues under different immune states: based on the immunophenotyping perspective of the tumor microenvironment, VV-mH4-1BBL can be used in "immune-inflammatory" tumor tissues (i.e., "hot tumors" with infiltration of immune cells such as T cells), making "hot tumors" even "hotter," that is, further enhancing the number and activity of immune cells in the tumor microenvironment; VV-mH4-1BBL can be used in "immune-desert" tumor tissues (i.e., "cold tumors" with a lack of immune cells such as T cells both inside and around the tumor tissue), converting "cold tumors" into "hot tumors," that is, promoting the infiltration of immune cells such as T cells into tumor tissues while enhancing their anti-tumor activity, and also creating conditions for combined immunotherapy such as PD-1 monoclonal antibodies; VV-mH4-1BBL can be used in "immune-privileged" tumor tissues (immune cells such as T cells exist on the periphery of tumor tissues but cannot infiltrate into tumor tissues), not only promoting immune cells to break through various barriers and enter tumor tissues, but also promoting the anti-tumor function of immune cells.

[0041] 2. VV-mH4-1BBL is used to treat superficial and deep tumors through direct injection and combined with endoscopic injection:

[0042] Based on the application (dosage) method, VV-mH4-1BBL can be used for a series of solid tumors present on the surface of the body, such as melanoma, by direct intratumoral injection; VV-mH4-1BBL can also be accurately injected into deep solid tumors through laparoscopes, choledochoscopes, thoracoscopes, colonoscopies, neuroendoscopes, etc., based on clear observation of the tumor body, to directly dissolve tumor cells while stimulating anti-tumor immune responses.

[0043] 3. VV-mH4-1BBL is used in drugs for the treatment of solid tumors such as colorectal cancer, pancreatic cancer and melanoma:

[0044] From the perspective of tumor type, since VV-mH4-1BBL works by directly dissolving tumors and activating and enhancing the efficacy of immune cells such as T cells, VV-mH4-1BBL can be used through the above-mentioned administration method to treat pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, gastric cancer, esophageal cancer, brain glioma, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, lung cancer, breast cancer, multiple myeloma, lymphoma, melanoma, etc. of various clinical stages.

[0045] In fact, VV-mH4-1BBL can be used to treat all types of solid tumors, and the applicant has conducted a large number of related experiments to prove this. However, due to space limitations, only representative validation experiments on colorectal cancer, pancreatic cancer, and melanoma are listed in this application. This experimental data does not limit the scope of VV-mH4-1BBL's effect on all types of solid tumors.

[0046] The present invention constructs a synonymous mutant gene mH4-1BBL, and then constructs a recombinant vaccinia virus VV-mH4-1BBL. In order to express the 4-1BBL molecule, which has a strong anti-tumor immune function, at high levels on the surface of tumor cells and to detect and monitor the expression of therapeutic 4-1BBL, a synonymous mutant gene mH4-1BBL of 4-1BBL was obtained by molecular design and synthesis of synonymous mutant primers, and PCR was used to replace the 540T base with the 540C base. mH4-1BBL was then recombined into the vaccinia virus genome to construct a new recombinant vaccinia virus containing the mH4-1BBL gene sequence, named VV-mH4-1BBL. VV-mH4-1BBL retains oncolytic properties and expresses a large amount of the protein encoded by the mH4-1BBL gene while rapidly replicating and expressing it on the surface of tumor cells. The recombinant vaccinia virus VV-mH4-1BBL, as a virus itself, can directly dissolve oncolytic tumors and then initiate anti-tumor immunity. The protein 4-1BBL encoded by the mH4-1BBL gene it carries is confined to tumor tissues and exerts a therapeutic effect of enhancing anti-tumor immunity.

[0047] If stimulating anti-human 4-1BB monoclonal antibodies or soluble 4-1BBL-acting 4-1BB signaling agonists are used for tumor immunotherapy, there may be a potential risk of systemic toxic side effects. The protein 4-1BBL molecule encoded by the mH4-1BBL gene carried by VV-mH4-1BBL is confined to the tumor microenvironment to exert its effect. This can avoid potential systemic side effects of 4-1BBL and achieve high levels of local 4-1BBL expression in tumor tissue, exerting stronger local anti-tumor efficacy. This is the advantage of the recombinant vaccinia virus VV-mH4-1BBL. In addition, when constructing the recombinant virus, the vaccinia virus thymidine kinase (TK) gene was deleted, making it safer and further enhancing the selectivity of infected tumor cells.

[0048] The present invention has well achieved the technical effect of constructing synonymous mutation genes and recombinant oncolytic viruses, retaining the advantages of the original oncolytic viruses and circumventing their weaknesses. In the present invention, the 540T base of the wild-type human 4-1BBL coding sequence is mutated to a 540C base to construct a synonymous mutation gene mH4-1BBL. Then mH4-1BBL is introduced into vaccinia virus VV by homologous recombination to establish a recombinant vaccinia virus VV-mH4-1BBL. Recombinant vaccinia virus VV-mH4-1BBL is applied to drugs for tumor immunotherapy. While infecting tumor cells and obtaining the effect of lysing tumor cells and promoting the local inflammatory environment of the tumor, the 4-1BBL encoding fusion protein expressed on the cell surface is expressed, so that the 4-1BBL molecular substance is concentrated and confined to the tumor microenvironment, while promoting the anti-tumor immune response, potential systemic toxic and side effects are avoided, and the adverse reactions such as breaking through the bottleneck of tumor immunotherapy in which anti-tumor immune cells are not easy to enter tumor tissue and anti-tumor immunity is easily suppressed by negative immunosuppression in the patient's body.

[0049] The present invention recombines 4-1BBL into an oncolytic virus, which allows 4-1BBL to act locally in the tumor microenvironment and avoid systemic effects. Therefore, the present invention better avoids the potential danger of severe loss of control of the 4-1BB signal caused by artificially prepared antibodies, and also avoids the systemic toxic side effects that may be caused by directly using soluble 4-1BBL.

[0050] The synonymous mutant 4-1BBL recombinant poxvirus constructed by the present invention can monitor the expression of exogenous 4-1BBL, monitor and evaluate the therapeutic effect through molecular biological means such as simple gene sequencing without changing the 4-1BBL protein sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG1 shows a schematic diagram of the gene segments introduced into the recombinant vector PV-1 and the structure of the PV-1 vector.

[0052] FIG2 shows a schematic diagram of the preparation of the F1-2 fragment during site-directed mutagenesis of human 4-1BBL.

[0053] FIG3 shows a schematic diagram of the preparation of the F1-1 fragment during site-directed mutagenesis of human 4-1BBL.

[0054] FIG4 shows a schematic diagram of constructing a synonymous mutant full-length human 4-1BBL (mH4-1BBL) gene by connecting site-directed mutagenesis fragments.

[0055] Figure 5 shows gel electrophoresis images of PCR products resulting from the introduction of synonymous mutations into the human 4-1BBL gene, wherein Figure 5A shows the gel electrophoresis results of PCR products of fragments F1-1 to F3-2, and Figure 5B shows the gel electrophoresis results of PCR products of mH4-1BBL, mH4-1BBL-2, and mH4-1BBL-3 after the synonymous mutations.

[0056] FIG6 shows the expression of 4-1BBL molecules on the cell surface of intestinal cancer cells HCT-116 after infection with VV-ΔTK, VV-mH4-1BBL, VV-mH4-1BBL-2 and VV-mH4-1BBL-3 as detected by flow cytometry.

[0057] FIG7 shows the human 4-1BBL gene sequence expressed in human intestinal cancer cell lines detected by RT-PCR and gene sequencing.

[0058] FIG8 shows the alignment of human 4-1BBL gene sequences expressed in human colorectal cancer cell lines using the BLAST program on the NCBI website.

[0059] FIG9 shows the human 4-1BBL gene sequence expressed in human intestinal cancer cell lines infected with VV-mH4-1BBL detected by RT-PCR and gene sequencing.

[0060] FIG10 shows the human 4-1BBL gene sequence expressed in human intestinal cancer cell lines infected with VV-mH4-1BBL-2 detected by RT-PCR and gene sequencing.

[0061] FIG11 shows the human 4-1BBL gene sequence expressed in human intestinal cancer cell lines infected with VV-mH4-1BBL-3 detected by RT-PCR and gene sequencing.

[0062] FIG12 shows a schematic diagram of the gene segments introduced into the recombinant vector PV-1 and the structure of the PV-1 vector.

[0063] FIG13 shows the identification of the mouse 4-1BBL gene sequence carried by the PV-4-1BBL recombinant vector by gene sequencing.

[0064] FIG14 shows the alignment results of the mouse 4-1BBL gene sequences carried by the PV-4-1BBL recombinant vector.

[0065] FIG15 shows that after MC-38 cells were infected with the recombinant oncolytic poxvirus VV-ΔTK-4-1BBL, high expression of the 4-1BBL gene was detected by RT-PCR.

[0066] FIG16 shows that after MC-38 cells were infected with the recombinant oncolytic poxvirus VV-ΔTK-4-1BBL, the expression of 4-1BBL molecules on the cell surface was detected by immunofluorescence labeling and flow cytometry.

[0067] Figure 17 shows the viral titer method for detecting the proliferation ability of VV-ΔTK-4-1BBL in different tumor cells; wherein, A: mouse pancreatic cancer cell line LTPA; B: mouse melanoma cell line B16-F10; C: mouse pancreatic cancer cell line Panc02 / Luc; D: mouse intestinal cancer cell line MC-38.

[0068] Figure 18 shows the MTT assay to detect the killing ability of VV-ΔTK-4-1BBL against different tumor cells in vitro; wherein, A: mouse pancreatic cancer cell line LTPA; B: mouse melanoma cell line B16-F10; C: mouse pancreatic cancer cell line Panc02 / Luc; D: mouse intestinal cancer cell line MC-38.

[0069] FIG19 shows the mouse 4-1BBL gene sequence expressed in mouse intestinal cancer cells detected by gene sequencing.

[0070] FIG20 shows the alignment results of mouse 4-1BBL gene sequences obtained by RT-PCR.

[0071] FIG21 shows the 4-1BBL gene sequence expressed in mouse intestinal cancer cells in the VV-ΔTK-4-1BBL treatment group detected by gene sequencing.

[0072] FIG22 shows the alignment results of mouse 4-1BBL gene sequences obtained by RT-PCR.

[0073] FIG23 shows the gross observation of the spleen of MC-38 tumor-bearing mice after treatment with VV-ΔTK-4-1BBL, as well as the comparison of spleen weight and cell number within the spleen; FIG23A shows the comparison of spleen weight, and FIG23B shows the comparison of cell number within the spleen.

[0074] Figure 24 shows the percentages of CD3+T, CD8+T, and CD4+T cells in the spleen after VV-ΔTK-4-1BBL treatment; Figure 24A shows the change in the percentage of CD3+T cells in the spleen, Figure 24B shows the change in the percentage of CD8+T cells in the spleen, and Figure 24C shows the change in the percentage of CD4+T cells in the spleen.

[0075] Figure 25 shows the effect of VV-ΔTK-4-1BBL treatment on the expression of PD-1 on CD4+T and CD8+T cells in spleen tissue, wherein Figure 25A is the results of immunofluorescence staining and flow cytometry analysis after VV-ΔTK-4-1BBL treatment, Figure 25B is a comparison of the expression of PD-1 on CD8+T cells in the spleen measured by flow cytometry after VV-ΔTK-4-1BBL treatment, and Figure 25C is a comparison of the expression of PD-1 on CD4+T cells in the spleen measured by flow cytometry after VV-ΔTK-4-1BBL treatment.

[0076] Figure 26 shows the percentages of CD3+T, CD8+T, and CD4+T cells in tumor tissues after VV-ΔTK-4-1BBL treatment, wherein Figure 26A shows the percentage of CD3+T cells, Figure 26B shows the percentage of CD8+T cells, and Figure 26C shows the percentage of CD4+T cells.

[0077] Figure 27 shows the changes in immune molecules in tumor tissue after VV-ΔTK-4-1BBL treatment, wherein Figure 27A shows the changes in the immune molecule perforin, Figure 27B shows the changes in the immune molecule granzyme B, Figure 27C shows the changes in the immune molecule IFN-γ, Figure 27D shows the changes in the immune molecule IL-1β, Figure 27E shows the changes in the immune molecule TNF-α, and Figure 27F shows the changes in the immune molecule TGF-β.

[0078] FIG28 shows a comparison of the therapeutic effects of recombinant vaccinia virus in treating mouse peritoneal tumor models of colorectal cancer.

[0079] Figure 29 shows the observation of survival time in the MC-38 peritoneal mouse tumor model treated with recombinant oncolytic poxvirus VV-ΔTK-4-1BBL combined with PD-1 / PD-L1 blockade. DETAILED DESCRIPTION

[0080] To make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below. However, it should be understood that the description herein is only for explaining the present invention and is not intended to limit the scope of the present invention.

[0081] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention pertains. The terms used in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The reagents and instruments used herein are all commercially available. The characterization methods involved can be found in the relevant descriptions in the prior art and will not be described in detail herein.

[0082] In order to further understand the present invention, the present invention is further described in detail below in conjunction with the best embodiment.

[0083] The synonymous mutant gene 4-1BBL (mH4-1BBL) recombinant vaccinia virus, codenamed VV-mH4-1BBL, is obtained by inserting the gene sequence of the synonymous mutant gene mH4-1BBL and related regulatory elements after position 81001 bp of the vaccinia virus parental genome (gene sequence based on GenBank: AY243312.1). The gene sequence inserted into the vaccinia virus parental genome is shown in SEQ ID No. 22.

[0084] Among them, the synonymous mutant gene mH4-1BBL was obtained by selecting the 540th base of its coding sequence based on the wild-type human 4-1BBL gene sequence (the gene sequence is based on the NCBI Reference Sequence: NM_003811.4 coding sequence) and performing synonymous mutation through PCR by designing primers.

[0085] The genetic information of the VV-mH4-1BBL recombinant virus is:

[0086] 1. The gene sequence of the vaccinia virus parent used to construct the VV-mH4-1BBL recombinant oncolytic virus was based on GenBank: AY243312.1. The gene sequence of the vaccinia virus parent mentioned in the following statements is based on this.

[0087] 2. The synonymous mutant gene mH4-1BBL and related regulatory elements were homologously recombined with the vaccinia virus parental vector using a recombinant vector (PV-mH4-1BBL). This process involved inserting the mH4-1BBL gene and related regulatory element sequences after position 81001bp in the vaccinia virus parental genome, located within the thymidine kinase (TK) gene. This not only disrupted the intact TK gene (knockout), but also enabled expression of mH4-1BBL. Because the relevant regulatory elements included the yellow fluorescent protein (YFP) gene (flanked by loxP sequences) used for recombinant virus screening, the recombinant virus was used to infect 293T cells transfected with the Cre gene, deleting YFP and thereby obtaining the mH4-1BBL recombinant vaccinia virus of the present invention: VV-mH4-1BBL. Compared to the genome of the parent vaccinia virus (VV), a gene fragment (SEQ ID No. 22) is inserted into the TK gene in the VV-mH4-1BBL genome. Its gene sequence is as follows (the underlined sequence is the mH4-1BBL gene; from 3' to 5' direction; other sequences are regulatory elements such as the promoter):

[0088] The VV-mH4-1BBL of the present invention was established by the following method:

[0089] 1) Construction of the synonymous mutant gene mH4-1BBL: Using the wild-type human 4-1BBL gene (the gene sequence was based on the coding sequence of NCBI Reference Sequence: NM_003811.4) as a template, synonymous mutant primers were designed and synthesized centered around base 540 of the coding sequence. PCR was used to convert base 540T to base 540C, changing only the encoded polypeptide product. The 4-1BBL gene after the synonymous mutation was named mH4-1BBL gene;

[0090] 2) Introduction of restriction enzyme sites on both sides of the mH4-1BBL gene: SdaI and HpaI restriction enzyme sites were introduced on both sides of the mH4-1BBL gene by PCR, and the gene fragment was isolated;

[0091] 3) Preparation of cohesive ends of the vaccinia virus VV homologous recombination vector: The VV homologous recombination vector PV-1 (containing the yellow fluorescent YFP sequence) was cut with SdaI and HpaI to isolate the plasmid fragment;

[0092] 4) Ligation of the gene mH4-1BBL with the cohesive ends of the VV homologous recombination vector PV-1: ligating the recovered products from steps 2) and 3);

[0093] 5) Transformation of competent cells and screening of positive clones: The ligation product from step 4) was incubated with competent E. coli. After expansion, the recombinant plasmid was extracted and sequenced to confirm that the gene mH4-1BBL was correctly cloned into the homologous recombination vector PV-1. The plasmid was named PV-mH4-1BBL.

[0094] 6) The gene mH4-1BBL is recombined into the VV genome: As VV is infected and replicates, PV-mH4-1BBL undergoes homologous recombination with it. The mH4-1BBL gene fragment and the yellow fluorescent YFP sequence are simultaneously homologously recombined into the VV genome to form a recombinant virus. At the same time, because the mH4-1BBL insertion site in the VV genome is within the TK gene, the TK gene is destroyed (i.e., the TK gene is deleted). Single clones of recombinant VV are then screened to obtain high-purity recombinant VV.

[0095] 7) The obtained high-purity recombinant VV was used to infect 293T cells expressing the Cre enzyme, excising the YFP gene from the recombinant VV genome. Single clones of recombinant VV were then screened to obtain and purify the desired recombinant VV seed. Sequencing analysis confirmed the correct integration of the mH4-1BBL gene into VV, resulting in a recombinant oncolytic vaccinia virus with the synonymous mutant gene mH4-1BBL, designated VV-mH4-1BBL.

[0096] The present invention provides a recombinant vaccinia virus with a synonymous mutation 4-1BBL, namely VV-mH4-1BBL, for use in the preparation of anti-tumor drugs. The recombinant vaccinia virus VV-mH4-1BBL can be used alone in the preparation of anti-tumor drugs or in combination with other anti-tumor drugs or methods.

[0097] Among them, the recombinant vaccinia virus VV-mH4-1BBL can be used to prepare drugs or products for treating "immune inflammatory" tumor tissue, "immune desert" tumor tissue, and "immune exempt" tumor tissue.

[0098] Among them, the recombinant vaccinia virus VV-mH4-1BBL can be used to prepare drugs and products for treating surface tumors and deep tumors through direct injection and combined with endoscopic injection;

[0099] Surface tumors include, but are not limited to, solid tumors such as melanoma that exist on the surface of the body.

[0100] Combined endoscopic injection therapy includes but is not limited to precise intratumoral injection therapy of deep solid tumors based on clear observation of the tumor mass through laparoscope, choledochoscope, thoracoscope, colonoscopy, neuroendoscope, etc.

[0101] Among them, the recombinant vaccinia virus VV-mH4-1BBL can be used to prepare drugs and products for treating tumors of various clinical stages, including but not limited to pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, gastric cancer, esophageal cancer, brain glioma, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, lung cancer, breast cancer, multiple myeloma, lymphoma, and melanoma.

[0102] The application of the synonymous mutant gene mH4-1BBL, through gene sequencing of a series of tumors such as colorectal cancer cells, has shown that tumor cells themselves express wild-type 4-1BBL, but the expression product 4-1BBL molecule is localized intracellularly and not expressed on the cell surface. During the process of using the synonymous mutant gene mH4-1BBL to treat tumor cells, its expression level can be detected at the genetic level and used to monitor the treatment process and efficacy.

[0103] Example 1

[0104] Construction of a recombinant oncolytic vaccinia virus: Through PCR site-directed mutagenesis, the wild-type human 4-1BBL gene (the gene sequence is based on the coding sequence of NCBI Reference Sequence: NM_003811.4) was mutated to a synonymous codon (a synonymous mutation refers to a change in the nucleotide sequence of a gene codon, but the amino acid encoded by it does not change). The synonymous mutated human 4-1BBL was named mh4-1BBL. mh4-1BBL was recombined into the vaccinia virus genome, and the TK (thymidine kinase) gene was knocked out at the same time to construct a new recombinant vaccinia virus named VV-mh4-1BBL. VV-mh4-1BBL retains the oncolytic properties of the parental VV and can express the mh4-1BBL molecule and localize to the cell membrane surface after infecting tumor cells. The amino acid sequence of the mh4-1BBL molecule is identical to that of the wild-type 4-1BBL molecule, but at the mRNA level, it is possible to distinguish whether the 4-1BBL expressed in tumor tissue after VV-mh4-1BBL treatment is endogenous or exogenous. The mh4-1BBL molecule expressed by an oncolytic virus avoids the potential risks of systemic effects associated with the use of soluble 4-1BBL and allows the mh4-1BBL molecule to exert its therapeutic effect of enhancing anti-tumor immunity in tumor tissue (tumor microenvironment).

[0105] A recombinant oncolytic vaccinia virus is operably inserted into a synonymous mutated exogenous gene capable of expressing 4-1BBL.

[0106] The foreign gene is inserted into the TK gene of the vaccinia virus.

[0107] The DNA sequences of the exogenous genes are shown in SEQ ID No. 1 to SEQ ID No. 3.

[0108] The applicant has listed the gene sequences after these three synonymous mutations, but they do not limit the scope of the technology of the present invention. In fact, as long as the final encoded polypeptide / amino acid / protein sequence is the same (with the same or similar efficacy), synonymous mutations at any one or several sites are feasible.

[0109] The sequence of SEQ ID No. 1 (540T mutated to C) is:

[0110] The sequence of SEQ ID No. 2 (468G mutated to A) is:

[0111] The sequence of SEQ ID No. 3 (597C mutated to T) is:

[0112] The amino acid sequence of the exogenous gene is shown in SEQ ID No.4.

[0113] The sequence of SEQ ID No. 4 is:

[0114] Foreign genes are linked via IRES sequences.

[0115] IRES, ribosome entry site, is just one of the linker sequences that can be used.

[0116] The IRES sequence is shown as SEQ ID No.5.

[0117] The sequence of SEQ ID No.5 is:

[0118] The present invention also provides a pharmaceutical composition comprising the above-mentioned recombinant oncolytic vaccinia virus, a pharmaceutically acceptable carrier and pharmaceutical excipients.

[0119] The pharmaceutical composition is administered by direct injection and / or combined with endoscopic injection; the endoscope is preferably a laparoscope, a choledochoscope, a thoracoscope, a colonoscope and a neuroendoscope.

[0120] The present invention also provides the use of the above-mentioned recombinant oncolytic vaccinia virus and the above-mentioned pharmaceutical composition in the preparation of a drug for preventing or treating tumors and / or cancer.

[0121] The tumor and / or cancer is a solid tumor, preferably a cold tumor and / or a hot tumor, and its histological type includes but is not limited to pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, gastric cancer, esophageal cancer, brain glioma, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, lung cancer, breast cancer, multiple myeloma, lymphoma, and melanoma.

[0122] Example 2

[0123] Detection of synonymous mutation sites after infection of colorectal cancer cells with recombinant human 4-1BBL poxvirus:

[0124] The biological function of proteins depends on their correct amino acid sequence. DNA base sequences encode amino acids using triplet codons. This process involves the biological phenomenon of "degenerate codons," whereby a single amino acid can be encoded by more than one triplet codon. Consequently, different codons corresponding to the same amino acid are called synonymous codons. These synonymous codons typically share the first and second bases, differing only in the third.

[0125] Given the aforementioned biological mechanisms, synonymous mutations can be made to the codons of the DNA sequence encoding a specific protein (changing the third base of the codon while keeping the amino acid encoded by that codon unchanged). This approach does not affect the amino acid sequence and function of the gene expression product, and allows the introduction of synonymous mutations into cells to be detected using molecular biological methods.

[0126] In this example, a recombinant oncolytic virus expressing a synonymous mutation of the human 4-1BBL gene was constructed. The purpose was to express the 4-1BBL molecule locally in the tumor tissue while exerting the anti-tumor effect of the oncolytic virus itself, thereby promoting anti-tumor immunity and detecting therapeutic (exogenous) 4-1BBL by detecting the synonymous mutation site.

[0127] 1. Construction of recombinant vector carrying synonymous mutation of human 4-1BBL gene:

[0128] To verify that the mutant human 4-1BBL gene expressed by a synonymous mutant human 4-1BBL recombinant poxvirus infected tumor cells can be detected by molecular biological methods, a synonymous mutant human 4-1BBL gene was constructed in this example and cloned into the homologous recombination vector PV-1 ( FIG1 ). The synonymous mutant human 4-1BBL gene was introduced into the vaccinia virus by homologous recombination, and the tk (thymidine kinase) gene was deleted simultaneously to obtain a synonymous mutant human 4-1BBL recombinant poxvirus.

[0129] Figure 1 schematically illustrates the gene segments introduced into the recombinant PV-1 vector and the structure of the PV-1 vector. In the vector shown in Figure 1, the TK 5' and TK 3' sequences, which are homologous to portions of the vaccinia virus genome, flank the target gene sequence. The recombinant vector is constructed and introduced into host cells infected with wild-type vaccinia virus. The TK 5' and TK 3' sequences undergo homologous recombination with wild-type vaccinia virus genes, allowing the gene sequence between the TK 5' and TK 3' regions of the vector to be introduced into the vaccinia virus genome, thereby constructing the recombinant virus.

[0130] The nouns in Figure 1 are:

[0131] 1) MCS: Multiple cloning site, where the target gene can be inserted;

[0132] 2) YFP: yellow fluorescent protein gene;

[0133] 3) IRES: ribosome entry site;

[0134] 4) PV-1: homologous recombination vector;

[0135] 5) PV-mH4-1BBL: A recombinant vector containing the mH4-1BBL gene introduced into the multiple cloning site of the PV-1 vector;

[0136] 6) PV-mH4-1BBL-2: A recombinant vector containing the mH4-1BBL-2 gene introduced into the multiple cloning site of the PV-1 vector;

[0137] 7) PV-mH4-1BBL-3: A recombinant vector in which the mH4-1BBL-3 gene is introduced into the multiple cloning site of the PV-1 vector.

[0138] The mH4-1BBL, mH4-1BBL-2, and mH4-1BBL-3 genes described above are based on the CDS of the wild-type human 4-1BBL gene (NCBI Reference Sequence: NM_003811.4), with the T at position 540 mutated to a C, the G at position 468 mutated to an A, and the C at position 597 mutated to a T, respectively, encoding the polypeptide product sequences. These are the 4-1BBL genes after synonymous mutations.

[0139] 2. Preparation of Synonymous Mutation Human 4-1BBL Gene:

[0140] 1. Design and preparation process of synonymous mutation of base 540 T to base 540 C in CDS of human 4-1BBL gene:

[0141] In order to construct a synonymous mutant human 4-1BBL gene based on the coding sequence (CDS) of the wild-type human 4-1BBL gene (H4-1BBL), we used the CDS of the wild-type human 4-1BBL gene (NCBI Reference Sequence: NM_003811.4) as a reference, designed mutation primers for the 540th T base of its CDS and mutated it to a C base by PCR. In the CDS of the human 4-1BBL gene, the codon for the 540th T base is "GCT" (encoding alanine), and after mutation to "GCC", it still encodes alanine. The designed primers are as follows:

[0142] Pf (SEQ ID No. 6):

[0143] 5'-TATAGTCGACATGGAATACGCCTCTGACGCTTC-3' (containing the Sal I restriction enzyme site);

[0144] At the same time, synonymous mutation primers were designed and synthesized centered on the 540th base of the coding sequence:

[0145] Pr11 (SEQ ID No. 7):

[0146] 5'-CGGGTGGCAGGTCCACGGTCAAGGCCAGGGCGGCGGCCCCAGCAGC-3'.

[0147] Using the wild-type human 4-1BBL gene (H4-1BBL) as a template and Pf and Pr11 as primers, fragment F1-2 was obtained by PCR (the technical route is shown in Figure 2; the PCR product fragment is shown in Figure 5).

[0148] To construct a synonymous mutant human 4-1BBL gene based on 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 a reference. The 540th T base of the CDS was selected and primers were designed to mutate it to a C base by PCR. The designed primers are as follows:

[0149] Pr (SEQ ID No. 8):

[0150] 5′-TATAGGCGCGCCTTATTCCGACCTCGGTGAAGGGAG-3′ (containing the Sgs I restriction enzyme site);

[0151] At the same time, synonymous mutation primers were designed and synthesized centered on the 540th base of the coding sequence:

[0152] Pf12 (SEQ ID No. 9):

[0153] 5'-GCTGCTGGGGCCGCCGCCCTGGCCTTGACCGTGGACCTGCCACCCG-3'.

[0154] Using the wild-type human 4-1BBL gene (H4-1BBL) as a template and Pf12 and Pr as primers, fragment F1-1 was obtained by PCR (technical route shown in Figure 3; PCR product fragment shown in Figure 5).

[0155] In order to connect the F1-1 and F1-2 fragments containing the mutated bases into a full-length human 4-1BBL gene containing the mutation, the F1-1 and F1-2 fragments were used as templates, Pf and Pr were used as primers, and a full-length human 4-1BBL gene fragment containing a C base mutation at position 540 was obtained by PCR. Compared with the wild-type human 4-1BBL gene coding sequence (CDS), the obtained fragment changed at position 540 of the CDS. Since the sequence of the polypeptide product encoded by it was not changed at the third position of the codon to which it belongs, it is called a 4-1BBL gene with a synonymous mutation and is named mH4-1BBL (see Figure 4 for the technical route; see Figure 5 for the PCR product fragment).

[0156] Primer information is as follows:

[0157] Pf (SEQ ID No. 10):

[0158] 5'-TATAGTCGACATGGAATACGCCTCTGACGCTTC-3' (containing the Sal I restriction enzyme site);

[0159] Pr (SEQ ID No. 11):

[0160] 5'-TATAGGCGCGCCTTATTCCGACCTCGGTGAAGGGAG-3' (containing the Sgs I restriction enzyme site).

[0161] 2. Design and preparation process of synonymous mutations at base 468 G and base 597 C in the CDS of the human 4-1BBL gene:

[0162] To demonstrate the universality of the effects of the synonymous mutations in the 4-1BBL gene in this example, a process similar to that shown in Figures 2, 3, and 4 was used to perform synonymous mutations at bases 468 G and 597 C of the coding sequence of the wild-type human 4-1BBL gene (NCBI Reference Sequence: NM_003811.4). The details are as follows:

[0163] 1) Design synonymous mutation primers centered around base 468 of the coding sequence:

[0164] Pr21 (SEQ ID No. 12):

[0165] 5'-CAAGTGAAACGGAGCCTGAGCCTTCGCCGGCCACCACGCGC-3';

[0166] Pf22 (SEQ ID No. 13):

[0167] 5'-GCGCGTGGTGGCCGGCGAAGGCTCAGGCTCCGTTTCACTTG-3'.

[0168] Using the wild-type human 4-1BBL gene as a template, Pf and Pr21 as primer pairs, and Pf22 and Pr as primer pairs, the 468 synonymous mutation bases (the 468 G base mutated to the 468 A base, and the codon still encoded glutamate) were introduced into the human 4-1BBL gene fragment by high-fidelity PCR, and the PCR product fragments F2-1 and F2-2 were recovered (as shown in Figure 13). Using fragments F2-1 and F2-2 as templates, F and R were used as genes to connect fragments F2-1 and F2-2 into a complete 468-base synonymous mutation of the human 4-1BBL gene. The above-mentioned 468-base mutation did not change the sequence of the polypeptide product it encoded. The 4-1BBL gene after the synonymous mutation was named mH4-1BBL-2 gene (see Figure 5 for the PCR product fragment).

[0169] 2) Design synonymous mutation primers centered around base 597 of the coding sequence:

[0170] Pr31 (SEQ ID No. 14):

[0171] 5'-GGTGCAGCAAGCGGCCCTGAAAACCGAAGGCCGAGTTCCG-3';

[0172] Pf32 (SEQ ID No. 15):

[0173] 5'-CGGAACTCGGCCTTCGGTTTTCAGGGCCGCTTGCTGCACC-3'.

[0174] Using the wild-type human 4-1BBL gene as a template, Pf and Pr31 as primer pairs, and Pf32 and Pr as primer pairs, the 597th synonymous mutation base (the 597th C base mutated to the 597th T base, and the codon still encoded phenylalanine) was introduced into the human 4-1BBL gene fragment by high-fidelity PCR, and the PCR product fragments F3-1 and F3-2 were recovered (Figure 13). Using fragments F3-1 and F3-2 as templates, F and R were used as genes to connect fragments F3-1 and F3-2 into a complete 597-base synonymous mutation of the human 4-1BBL gene. The above-mentioned 597-base mutation did not change the sequence of the polypeptide product it encoded. The 4-1BBL gene after the synonymous mutation was named mH4-1BBL-3 gene (see Figure 5 for the PCR product fragment).

[0175] 3. Preparation of synonymous mutant human 4-1BBL homologous recombination vector:

[0176] The vector PV-1, mH4-1BBL gene, mH4-1BBL-2 gene and mH4-1BBL-3 gene were double-digested with Sal I and Sgs I restriction enzymes, respectively. After 1% gel electrophoresis, the gel was cut and the target fragments were recovered. The digested vector PV-1 backbone was ligated with the recovered mH4-1BBL gene, mH4-1BBL-2 gene, and mH4-1BBL-3 gene using T4 ligase (ligation overnight at 4°C). The ligation products were transformed into DH5α competent Escherichia coli and plated on LB plates containing 100 μg / mL ampicillin. After incubation at 37°C for 16 hours, resistant colonies were selected, and the recombinant plasmids were extracted. Gene sequencing confirmed that the mH4-1BBL gene, mH4-1BBL-2 gene, and mH4-1BBL-3 gene 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.

[0177] 3. Preparation of Synonymous Mutation Human 4-1BBL Recombinant Oncolytic Virus:

[0178] PV-mH4-1BBL, PV-mH4-1BBL-2, and PV-mH4-1BBL-3 were respectively infected with vaccinia virus (VV) to obtain recombinant oncolytic viruses.

[0179] Take CV1 cells in the logarithmic growth phase and adjust the cell density to 1×10 5 PV-mH4-1BBL-2, PV-mH4-1BBL-3, and PV-mH4-1BBL-2 were inoculated into 24-well plates at a rate of 1 mL per well. Twenty-four hours later, CV1 cells were infected with vaccinia 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. CV1 cells were then transfected with 200 μL of the mixture. Two hours later, DMEM medium supplemented with 10% FCS was added and cultured for an additional 48 hours. Yellow fluorescent protein (YFP) fluorescence intensity was measured using a fluorescence microscope to assess homologous recombination between the plasmid and virus. After complete cell disruption, the virus-cell suspension was harvested.

[0180] Take CV1 cells in the logarithmic growth phase and adjust the cell density to 1.5×10 5100 μg / mL, inoculated into 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 YFP-expressing CV1 cells were sorted using flow cytometry to prepare a single-cell suspension. By limiting dilution, YFP+ CV1 cells were inoculated into 96-well plates pre-plated with CV1 cells. After 48 hours, wells with a single YFP+ viral plaque were selected, and the cells and supernatant were harvested. After repeated freeze-thaw cycles, the viral suspension was obtained for later use.

[0181] The virus suspension obtained above was used to infect 293T cells expressing the Cre enzyme, and the YFP gene in the recombinant VV genome was excised. The monoclonal recombinant VV was then screened to obtain the target recombinant VV seed and purified. Gene sequencing analysis confirmed that the synonymous mutant human 4-1BBL gene mH4-1BBL, mH4-1BBL-2, and mH4-1BBL-3 genes were recombined into VV. The recombinant oncolytic poxviruses inserted with the synonymous mutant human 4-1BBL gene mH4-1BBL, mH4-1BBL-2, and mH4-1BBL-3 were named VV-mH4-1BBL, VV-mH4-1BBL-2, and VV-mH4-1BBL-3.

[0182] Hela cells were cultured in a 15 cm diameter culture dish. When the Hela cell abundance was about 80%, the virus cell suspension was added to infect the Hela cells, and the infection was amplified until sufficient virus cell suspension was obtained.

[0183] Purify recombinant poxvirus: Collect the virus-cell suspension and centrifuge to discard the supernatant. Freeze and thaw the cells three times to lyse them. Grind the cells using a Dounce homogenizer to release viral particles. Centrifuge at 1200 rpm to collect the virus-containing supernatant, add it to the top of a 36% sucrose solution, and centrifuge at 130,000 × g for 1 hour. Resuspend the pellet in 10 mM Tris-HCl and freeze for later use.

[0184] To confirm whether the synonymous mutant human 4-1BBL recombinant poxvirus constructed above can express 4-1BBL molecules on the surface of tumor cells after infecting tumor cells, human colorectal cancer cell lines were infected with the above virus, and the expression of 4-1BBL molecules on the cell surface was detected by immunofluorescence labeling and flow cytometry analysis, as follows:

[0185] Human colorectal cancer cell line HCT-116 in logarithmic growth phase was taken and the cell density was adjusted to 3×10 5 / mL, inoculate 1mL to a 24-well plate. After the cells are completely attached, they are divided into VV-ΔTK, VV-mH4-1BBL, VV-mH4-1BBL-2 and VV-mH4-1BBL-3 infection groups, and are infected according to an infection multiplicity (MOI) of 1. After 24 hours, the infected cells are collected, stained with APC-labeled anti-human 4-1BBL monoclonal antibody, and the expression of cell membrane 4-1BBL molecules is analyzed by flow cytometry. The results showed that the 4-1BBL molecule was not expressed on the surface of HCT-116 cells infected with the cowpox virus VV-ΔTK in which only the tk gene was deleted, while the 4-1BBL molecule was expressed at a high level on the surface of HCT-116 cells infected with VV-mH4-1BBL, VV-mH4-1BBL-2 and VV-mH4-1BBL-3 (Figure 6).

[0186] In Figure 6, VV-ΔTK: vaccinia virus with the tk gene deleted; VV-mH4-1BBL: recombinant vaccinia virus with the tk gene deleted and the mH4-1BBL gene fragment inserted; VV-mH4-1BBL-2: recombinant vaccinia virus with the tk gene deleted and the mH4-1BBL-2 gene fragment inserted; VV-mH4-1BBL-3: recombinant vaccinia virus with the tk gene deleted and the mH4-1BBL-3 gene fragment inserted; Anti-4-1BBL-APC: APC-labeled anti-human 4-1BBL monoclonal antibody.

[0187] 4. Synonymous mutations: Detection of synonymous mutation sites by molecular biological methods after infection of tumor cells with human 4-1BBL recombinant oncolytic poxvirus:

[0188] To verify whether the exogenous 4-1BBL introduced after infection of tumor cells with a synonymous mutant human 4-1BBL recombinant oncolytic poxvirus can be detected, the recombinant oncolytic viruses VV-mH4-1BBL, VV-mH4-1BBL-2, and VV-mH4-1BBL-3 were used to infect the human intestinal cancer cell line HCT-116, and an uninfected group was set up as a control. RT-PCR analysis was then performed, as follows:

[0189] The human intestinal cancer cell line HCT-116 was infected with VV-mH4-1BBL, VV-mH4-1BBL-2, and VV-mH4-1BBL-3 at an MOI of 1 as described above. An uninfected control group was also established. After 24 hours, the cells were harvested and lysed in 200 μL of Trizol solution, and total RNA was extracted. RNA was reverse transcribed into cDNA using a Takara RT-PCR kit. Total RNA was also extracted from uninfected HCT116 cells and reverse transcribed to obtain cDNA.

[0190] Primers were designed in the CDS region of the human 4-1BBL gene (NCBI Reference Sequence: NM_003811.4) covering the mutation site. The primer sequences are as follows:

[0191] Pif (SEQ ID No. 16): 5'-TGAGCTACAAAGAGGACACGAAGGAGC-3';

[0192] PiR (SEQ ID No. 17): 5'-TTATTCCGACCTCGGTGAAGGGAGTCC-3'.

[0193] Using the obtained cDNA as a template and Pif and PiR as primers, a human 4-1BBL gene fragment encompassing the synonymous mutation site was amplified by PCR. The PCR product was ligated with the pUMc-T vector and transformed into competent DH5α E. coli. The cells were then plated onto ampicillin-resistant LB plates. After resistant colonies formed, individual colonies were randomly selected for unidirectional sequencing. The sequencing primer sequence was 5'-GTTGTAAAACGACGGCCAG-3' (SEQ ID No. 18). The results are as follows:

[0194] Plasmids were extracted from five colonies in the uninfected group and sequenced. The results are shown in Figure 7. Comparison using the NCBI BLAST program revealed that the fragments covered by primers Pif and Pir matched the CDS region of the human 4-1BBL gene (NCBI Reference Sequence: NM_003811.4) 100% (Figure 8). This result confirms that the human 4-1BBL gene expressed by colorectal cancer cells is unmutated.

[0195] 1) Ten colonies from the VV-mH4-1BBL infection group were selected for unidirectional sequencing. The results showed that a total of 10 colonies were successfully sequenced, and synonymous mutations as indicated by the arrows in Figure 9 were detected in all of them, while no mutations were found at other sites. This result indicates that VV-mH4-1BBL can replicate in large quantities after infecting tumor cells, and the synonymous mutation it carries (mutation of the 540th T base to the 540th C base) can be clearly detected. Therefore, the synonymous mutation of human 4-1BBL can provide a means for detecting and identifying the introduction of exogenous human 4-1BBL genes without changing their encoded amino acid sequence.

[0196] 2) 10 colonies from the VV-mH4-1BBL-2 infection group were selected for unidirectional sequencing. The results showed that a total of 10 colonies were successfully sequenced, and synonymous mutations as indicated by arrows in Figure 10 were detected in all of them, while no mutations were found at other sites. This result indicates that VV-mH4-1BBL can replicate in large quantities after infecting tumor cells, and the synonymous mutation 468 (the 468 G base mutates to the 468 A base) carried by it can be clearly detected. Therefore, the human 4-1BBL synonymous mutation can provide a means for detecting and identifying the introduction of exogenous human 4-1BBL genes without changing their encoded amino acid sequence.

[0197] 3) 10 colonies from the VV-mH4-1BBL-2 infection group were selected for unidirectional sequencing. The results showed that a total of 10 colonies were successfully sequenced, and synonymous mutations as indicated by arrows in Figure 11 were detected in all of them, while no mutations were found at other sites. This result indicates that VV-mH4-1BBL can replicate in large quantities after infecting tumor cells, and the synonymous mutation 597 (597 C base mutated to 597 T base) carried by it can be clearly detected. Therefore, the synonymous mutation of human 4-1BBL can provide a means for detecting and identifying the introduction of exogenous human 4-1BBL genes without changing their encoded amino acid sequence.

[0198] Example 3

[0199] Anti-tumor effect of synonymous mutation 4-1BBL recombinant poxvirus and detection of synonymous mutation sites:

[0200] To further validate the anti-tumor effect of the synonymous mutation 4-1BBL recombinant poxvirus and verify the detection of the synonymous mutation site 4-1BBL during its treatment, this example constructed a synonymous mutant based on mouse 4-1BBL and constructed a synonymous mutation mouse 4-1BBL oncolytic vaccinia virus. At the same time, a peritoneal mouse intestinal cancer model was established for experimental treatment and the synonymous mutation sites of mouse 4-1BBL were detected during the treatment process. The specific implementation process is as follows:

[0201] 1. Construction of synonymous mutant mouse 4-1BBL gene recombinant vector and synonymous mutant mouse 4-1BBL recombinant oncolytic vaccinia virus:

[0202] To verify that the mutant human 4-1BBL gene expressed after tumor cells were infected with a synonymous human 4-1BBL recombinant poxvirus could be detected using molecular biological methods, a synonymous mouse 4-1BBL gene was constructed using methods similar to those described in Example 2 and cloned into the homologous recombination vector PV-1 ( Figure 12 ), designated PV-4-1BBL. Gene sequencing results are shown in Figure 13 . Comparison of the obtained sequence using the NCBI BLAST program revealed that, compared to the CDS of the mouse 4-1BBL gene (NCBI Reference Sequence ID: NM_009404.3), the PV-4-1BBL gene had a C mutation at position 309 in the CDS to a T (indicated by the arrow in Figure 14 ). This mutation, in turn, mutated the corresponding codon from ACC to ACT, although the encoded product in both cases still encodes threonine. Thus, a homologous recombination vector based on the synonymous mouse 4-1BBL gene (referred to as 4-1BBL in this example) was successfully established.

[0203] Using a similar method as in Example 2, the synonymous mutant 4-1BBL gene was recombined into the oncolytic poxvirus using PV-4-1BBL, and its tk gene was knocked out at the same time. The recombinant oncolytic virus was named VV-ΔTK-4-1BBL, and the anti-tumor function of VV-ΔTK-4-1BBL was verified by in vitro and in vivo experiments, and the synonymous mutation sites of 4-1BBL were detected.

[0204] PV-4-1BBL in Figure 12: Recombinant vector after synonymous mutant mouse 4-1BBL gene was introduced into the multiple cloning site of PV-1 vector.

[0205] 2. RT-PCR was used to detect the expression of 4-1BBL at the mRNA level after VV-ΔTK-4-1BBL infected colorectal cancer cells:

[0206] To examine 4-1BBL gene expression in tumor cells infected with VV-ΔTK-4-1BBL, the colorectal cancer cell line MC-38 was used as a model. MC-38 cells were infected with VV-ΔTK-4-1BBL at an MOI of 1, and a VV-ΔTK-infected group served as a control. Twenty-four hours after infection, tumor cells were harvested, total RNA was extracted, and reverse transcribed into cDNA. The 4-1BBL gene was then amplified by PCR.

[0207] The experimental results (Figure 15) showed that the target gene band was prominent in the VV-ΔTK-4L infection group, while the target gene band was only at background level in the VV-ΔTK and non-infected groups. This result confirmed that after VV-ΔTK-4-1BBL infection of tumor cells, high levels of 4-1BBL gene expression were achieved through rapid viral replication. It also demonstrated that tumor cells had background levels of 4-1BBL gene expression (previous studies by the applicants found that the protein encoded by this gene was expressed in the tumor cell membrane without intervention).

[0208] After MC-38 cells were infected with the recombinant oncolytic poxvirus VV-ΔTK-4-1BBL, the results of high expression of the 4-1BBL gene detected by RT-PCR are shown in Figure 15, wherein lane M: DL 2000 DNA Marker; lane 1: uninfected group; lane 2: VV-ΔTK infected group; lane 3: uninfected group; lane 4: VV-ΔTK-4-1BBL infected group.

[0209] 3. Flow cytometry detection of 4-1BBL expression on the surface of colorectal cancer cells after VV-ΔTK-4-1BBL infection:

[0210] To confirm whether the rapid replication of recombinant oncolytic viruses in tumor cells can achieve high expression of 4-1BBL molecules and localize them on the tumor cell surface, the colorectal cancer cell line MC-38 in the logarithmic growth phase was infected with different viral infection multiplicities (MOI) of 0, 0.1, 1, and 5. After 24 hours, the cells were digested and single-cell suspensions were prepared. The cells were stained with fluorescein-labeled anti-4-1BBL monoclonal antibody, and the expression of cell membrane 4-1BBL was analyzed by flow cytometry.

[0211] The experimental results (Figure 16) show that as the multiplicity of infection (MOI) of the virus increases, the expression of yellow fluorescent protein (YFP) carried by the recombinant virus increases. The VV-ΔTK-4L infection group expressed cell membrane 4-1BBL, and this expression increased with increasing MOI. The VV-ΔTK infection group did not express cell membrane 4-1BBL. This result confirms that VV-ΔTK-4-1BBL infection of tumor cells can achieve expression of the 4-1BBL molecule on the tumor cell surface.

[0212] 4. Virus titer assay to detect the proliferation ability of VV-ΔTK-4-1BBL in different tumor cells:

[0213] To confirm the effect of gene recombination on the replication and proliferation ability of vaccinia 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 24 h, 48 h and 72 h after infection, and then repeatedly frozen and thawed and diluted to infect green monkey kidney CV-1 cells. The virus yield was determined by plaque lysis assay.

[0214] The experimental results ( FIG. 17 ) show that the recombinant introduction of the 4-1BBL gene into the vaccinia virus does not change the replication properties of the virus itself in tumor cells.

[0215] 5. Detection and comparison of the killing ability of VV-ΔTK-4-1BBL on tumor cells:

[0216] To confirm whether the gene recombination in this example affects the viral oncolytic effect, in this experiment, each recombinant virus was infected with LTPA cells, B16-F10 cells, Panc02 / Luc cells, and MC-38 cells at different MOIs (0, 0.1, 0.5, and 5), and the proportion of live cells was detected by MTT assay after 48 h.

[0217] The experimental results ( FIG. 18 ) showed that the oncolytic activity of the virus itself was not altered after the 4-1BBL gene was recombined into the vaccinia virus.

[0218] 6. Detection of synonymous mutations in 4-1BBL during tumor treatment with recombinant oncolytic poxvirus:

[0219] To verify whether a recombinant oncolytic poxvirus carrying the synonymous 4-1BBL mutation could be detected during tumor treatment, a mouse colorectal cancer model was established. Experimental treatment was performed using VV-ΔTK-4-1BBL, and tumor tissue was then collected for testing. The specific process is as follows.

[0220] Select 8-12 week old C57BL / 6 mice and sample them at 5×10 5 MC-38 colorectal cancer cells were inoculated into the abdominal cavity of mice. After 9 days, the mice were randomly divided into two groups. 8 PFU / mice were intraperitoneally injected with VV-ΔTK-4L, while another group was intraperitoneally injected with an equal volume of PBS. 48 hours later, the mice were sacrificed, and tumor tissue was removed. 10 mg of tumor tissue was dissolved in Trizol and total RNA was extracted. RNA was reverse transcribed into cDNA and then analyzed by PCR.

[0221] Primers were designed based on the CDS region of the mouse 4-1BBL gene (NCBI Reference Sequence ID: NM_009404.3). The primer sequences are as follows:

[0222] Pmf (SEQ ID No. 19):

[0223] 5'-TATAAAGCTTATGGACCAGCACACACTTGATG-3';

[0224] Pmr (SEQ ID No. 20):

[0225] 5'-TATATCTAGATCATTCCCATGGGTTGTCGG-3'.

[0226] Using the obtained cDNA as a template and primers Pmf and PmR, a full-length fragment encompassing the CDS of the mouse 4-1BBL gene was amplified by PCR. The PCR product was ligated with the pUMc-T vector and transformed into competent DH5α E. coli. The cells were then plated on ampicillin-resistant LB plates. After resistant colonies formed, individual colonies were randomly selected for unidirectional sequencing. The sequencing primer sequence was 5'-GTTGTAAAACGACGGCCAG-3' (SEQ ID No. 21).

[0227] The experimental results showed that in the PBS treatment group, 3 clones were randomly selected for sequencing, and the 4-1BBL fragments obtained were all wild-type 4-1BBL, that is, 100% matching the CDS region of the mouse 4-1BBL gene (NCBI Reference Sequence ID: NM_009404.3) (Figures 19 and 20). In Figure 19, the arrow indicates that there is no mutation at position C of the 309th base of the CDS of the mouse 4-1BBL gene. In the VV-△TK-4-1BBL group, 10 clones were randomly selected for sequencing, and the 4-1BBL sequence in one clone matched the mouse 4-1BBL gene (NCBI Reference Sequence ID: NM_009404.3). ID: NM_009404.3) The CDS region of the other 9 clones was 100% matched, while the 309th C base of CDS was detected to be a T base in all the other 9 clones, while there was no mutation in the other sites (Figures 21 and 22). In Figure 21, the arrow shows that the 309th C base of the mouse 4-1BBL gene CDS mutated to a T base. In Figure 22, the arrow shows that the 309th C base of the mouse 4-1BBL gene CDS mutated to a T base. The wild-type 4-1BBL gene comes from tumor-bearing mice, while a high proportion of synonymous mutations 4-1BBL gene expression is derived from the oncolytic virus VV-△TK-4-1BBL.

[0228] This result proves that tumor cells have a background level of 4-1BBL gene expression, and also confirms that during in vivo treatment with VV-△TK-4-1BBL, after infecting tumor cells, the virus rapidly replicates and expresses high levels of synonymous mutant 4-1BBL genes, which can be detected by molecular biological methods.

[0229] VII. Detection of the effects and mechanisms of the synonymous mutation 4-1BBL recombinant oncolytic poxvirus on peripheral immune organs:

[0230] 1. In order to comprehensively investigate the in vivo anti-tumor effect and mechanism of action of VV-ΔTK-4-1BBL and verify the effect of VV-ΔTK-4-1BBL on peripheral immune tissues and organs, 8-12 week old C57BL / 6 mice were selected and 5×10 5 MC-38 colorectal cancer cells were inoculated into the abdominal cavity of mice. 9 days later, the mice were randomly divided into groups and the number of 8 VV-ΔTK-4L, VV-ΔTK, and an equal volume of PBS were injected intraperitoneally with 100 PFU / mouse. Mice were sacrificed 5 days later, and spleens were removed, weighed, and single-cell suspensions were prepared and counted.

[0231] The experimental results ( FIG23 ) showed that VV-ΔTK-4-1BBL treatment of MC-38 tumor-bearing mice resulted in an increase in spleen weight ( FIG23A ) and spleen cell number ( FIG23B ), thereby promoting the proliferation of peripheral immune cells and the proliferation of peripheral immune tissues and organs.

[0232] The experimental results (Figure 24) show that after VV-ΔTK-4-1BBL treatment of MC-38 tumor-bearing mice, the proportion of CD3+T cells (Figure 24A), CD4+T cells (Figure 24B) and CD8+T cells (Figure 24C) in spleen cells can be promoted. This confirms that VV-ΔTK-4-1BBL can promote the proliferation of T cells and their subsets and NK cells in peripheral immune tissues and organs through the combined action of oncolytic viruses and the 4-1BBL molecules they express.

[0233] 2. In order to comprehensively investigate the in vivo anti-tumor effect and mechanism of action of VV-ΔTK-4-1BBL and verify the effect of VV-ΔTK-4-1BBL on the expression of PD-1 and other immune checkpoint molecules in CD4+T and CD8+T cells, which are key cells of anti-tumor immunity, 8-12 week old C57BL / 6 mice were selected and cultured at 5×10 5 MC-38 colorectal cancer cells were inoculated into the abdominal cavity of mice. 9 days later, the mice were randomly divided into groups and the number of 8 VV-ΔTK-4L, VV-ΔTK, and an equal volume of PBS were injected intraperitoneally with 100 PFU / mouse. Mice were sacrificed 5 days later, and spleens were harvested to prepare single-cell suspensions, which were then analyzed by immunofluorescence staining and flow cytometry.

[0234] Experimental results (Figure 25) show that VV-ΔTK-4-1BBL treatment generates an anti-tumor immune response, which can feedback-regulate PD-1 expression on the surfaces of immune cells such as CD4+ T cells and CD8+ T cells. This result demonstrates the necessary foundation for the concept of combining VV-ΔTK-4-1BBL with PD-1 / PD-L1 pathway blockers to enhance anti-tumor efficacy.

[0235] 8. Detection of the effects and mechanisms of the synonymous mutation 4-1BBL recombinant oncolytic poxvirus on the tumor microenvironment:

[0236] 1. In order to verify the effect of VV-ΔTK-4-1BBL on the anti-tumor immune status in the tumor microenvironment, 8-12 week old C57BL / 6 mice were selected and 5×10 5 MC-38 colorectal cancer cells were inoculated into the abdominal cavity of mice. 9 days later, the mice were randomly divided into groups and the number of 8 VV-ΔTK-4L, VV-ΔTK, and an equal volume of PBS were injected intraperitoneally with 100 PFU / mouse. Mice were sacrificed 5 days later, and tumor tissues were harvested to prepare single-cell suspensions for analysis by immunofluorescence staining and flow cytometry.

[0237] The experimental results (Figure 26) show that after VV-ΔTK-4-1BBL treatment of MC-38 tumor-bearing mice, the proportions of CD3+T, CD8+T, and CD4+T cells in tumor tissue increased significantly. This suggests that VV-ΔTK-4-1BBL can promote the increase in the number of T cells and their subsets in tumor tissue through the combined action of the oncolytic virus and the 4-1BBL molecule it expresses, thereby altering the anti-tumor immune landscape.

[0238] 2. In order to verify the effect of VV-ΔTK-4-1BBL on the anti-tumor immune status in the tumor microenvironment, 8-12 week old C57BL / 6 mice were selected and 5×10 5 MC-38 colorectal cancer cells were inoculated into the abdominal cavity of mice. 9 days later, the mice were randomly divided into groups and the number of 8 VV-ΔTK-4L, VV-ΔTK, and an equal volume of PBS were injected intraperitoneally with 100 PFU / mouse. Mice were sacrificed 5 days later, and 50 mg of each tumor tissue was dissolved in Trizol. Total RNA was extracted, reverse-transcribed into cDNA, and analyzed using real-time quantitative PCR.

[0239] The experimental results (Figure 27) show that the anti-tumor immune molecules such as perforin, granzyme B, IFN-γ, IL-1β, and TNF-α in the tumor tissue of the VV-ΔTK-4-1BBL treatment group were significantly increased, while negative immune molecules such as TGF-β were significantly reduced compared with the blank oncolytic virus control (VV-ΔTK) group. These results further prove that VV-ΔTK-4-1BBL can work together through oncolytic viruses and the 4-1BBL molecules they express to change the immune situation in tumor tissues and promote anti-tumor immune effects.

[0240] IX. Verification of the in vivo anti-tumor effect of the recombinant oncolytic poxvirus carrying the synonymous mutation 4-1BBL:

[0241] In this example, the 4-1BBL gene was recombined into the vaccinia virus genome to construct a novel recombinant vaccinia virus. To compare its in vivo anti-tumor activity, this experiment injected MC-38 mouse colon cancer cells into the peritoneal cavity of C57 / BL6 mice to establish a colon cancer peritoneal tumor model. Nine days later, the mice were randomly divided into groups and injected intraperitoneally with each recombinant virus. The survival of the mice was then observed. The results showed that compared with the phosphate buffer saline (PBS) treatment group, the survival time of tumor-bearing mice in the VV-ΔTK (VV with the TK gene knocked out) and VV-ΔTK-4-1BBL (VV with the TK gene knocked out and the 4-1BBL gene recombined) treatment groups was prolonged compared with the PBS treatment group, and the survival time of tumor-bearing mice in the VV-ΔTK-4-1BBL treatment group was prolonged compared with the VV-ΔTK treatment group.

[0242] The experimental results ( FIG. 28 ) show that VV-ΔTK-4-1BBL can produce significant anti-tumor effects when used to treat tumors and prolong the survival time of the treated subjects.

[0243] 10. Verification of the in vivo anti-tumor effect of the synonymous mutation 4-1BBL recombinant oncolytic poxvirus combined with PD-1 monoclonal antibody:

[0244] To verify the combined efficacy of VV-ΔTK-4-1BBL combined with immune checkpoint blockers, this experiment injected mouse colorectal cancer cell MC-38 into the peritoneal cavity of C57 / BL6 mice to establish a colorectal cancer peritoneal tumor model. Nine days later, the mice were randomly divided into four groups: PBS treatment group, VV-ΔTK-4-1BBL, PD-1 blocking monoclonal antibody treatment group, and VV-ΔTK-4-1BBL combined with PD-1 blocking monoclonal antibody treatment group. Each group was treated with intraperitoneal injection, and the survival time of the mice was observed.

[0245] The experimental results (Figure 29) show that the survival time of tumor-bearing mice in the VV-ΔTK-4-1BBL and PD-1 blocking mAb treatment groups was prolonged compared with the PBS treatment group, while the VV-ΔTK-4-1BBL and PD-1 blocking mAb treatment groups had comparable therapeutic effects. The VV-ΔTK-4-1BBL combined with PD-1 blocking mAb treatment group had a significantly better therapeutic effect than either the VV-ΔTK-4-1BBL or PD-1 blocking mAb treatment groups.

[0246] This result demonstrates that VV-ΔTK-4-1BBL may provide a powerful tool for enhancing the therapeutic efficacy of immune checkpoint blockade agents, such as PD-1 monoclonal antibodies. Combining VV-ΔTK-4-1BBL with immune checkpoint blockade can produce a more pronounced anti-tumor effect and improve the survival rate and duration of treated subjects.

[0247] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A recombinant oncolytic vaccinia virus, characterized in that: The recombinant oncolytic vaccinia virus can be operably inserted with a synonymous mutated foreign gene capable of expressing 4-1BBL.

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

3. The recombinant oncolytic vaccinia virus according to claim 1, characterized in that The DNA sequences of the exogenous genes are shown in SEQ ID No.1-SEQ ID No.

3.

4. The recombinant oncolytic vaccinia 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 vaccinia virus according to claim 1, characterized in that The foreign gene is connected via an IRES sequence.

6. The recombinant oncolytic vaccinia virus according to claim 5, characterized in that The IRES sequence is shown as SEQ ID No.

5.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the recombinant oncolytic vaccinia 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, characterized in that The pharmaceutical composition is administered by direct injection and / or combined with endoscopic injection; the endoscope is preferably a laparoscope, a choledochoscope, a thoracoscope, a colonoscope and a neuroendoscope.

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

10. The use according to claim 9, characterized in that: The tumor and / or cancer is a solid tumor, preferably a cold tumor and / or a hot tumor, and its histological type includes but is not limited to pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, gastric cancer, esophageal cancer, brain glioma, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, lung cancer, breast cancer, multiple myeloma, lymphoma, and melanoma.

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