CN114606204A

CN114606204A - Oncolytic vaccinia virus and preparation method and application thereof

Info

Publication number: CN114606204A
Application number: CN202011405828.7A
Authority: CN
Inventors: 魏继武; 左曙光; 董杰
Original assignee: Nanjing Weiyade Biomedical Co ltd
Current assignee: Nanjing Weiyade Biomedical Co ltd
Priority date: 2020-12-04
Filing date: 2020-12-04
Publication date: 2022-06-10

Abstract

The invention discloses an oncolytic vaccinia virus and a preparation method and application thereof. According to the invention, the TIGIT single-chain antibody gene is inserted into the TK region of the vaccinia virus genome to inactivate the TK gene dependent on virus replication, so that the virus can only replicate in high-activity tumor cells of the TK, and the safety of oncolytic vaccinia virus is obviously enhanced.

Description

Oncolytic vaccinia virus and preparation method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering and biological medicine, and particularly relates to an oncolytic vaccinia virus containing a gene sequence of an anti-mouse/human TIGIT single-chain antibody and expressing the TIGIT single-chain antibody, and a preparation method and application thereof.

Background

In recent years, with the rapid development of cell biology, molecular biology and bioengineering technologies, the immunotherapy of cancer has made a major breakthrough. The major immunotherapeutic modalities currently include cytokines, tumor vaccines, cell therapy, immune checkpoint inhibitors and oncolytic viruses, of which oncolytic viruses have received much attention in recent years.

Oncolytic viruses are a class of natural or recombinant viruses that replicate selectively in tumor cells compared to normal cells, thereby causing tumor cell lysis. Types of oncolytic viruses include Herpes Simplex Virus (HSV), Vaccinia Virus (VV), adenovirus (AdV), Measles Virus (MV), Vesicular Stomatitis Virus (VSV), Newcastle Disease Virus (NDV), reovirus, and the like. Oncolytic viruses can not only directly lyse tumor cells, but also indirectly induce anti-tumor immune responses. Thus, oncolytic viruses are considered a new cancer immunotherapy approach. Oncolytic viruses can induce local infiltration of immune cells in tumors, converting "cold" tumors with few immune cells to "hot" tumors with many immune cells. The infiltrated immune cells can effectively kill virus-infected and non-infected tumor cells and form long-lasting immune memory. However, with the activation of T cells, their surface immunosuppressive checkpoint molecules [ e.g. programmed cell death 1(PD-1), CTL antigen 4(CTLA-4) and T cell immunoglobulin and ITIM domain (TIGIT) ] are also activated simultaneously, helping tumor cells to escape killing of immune cells.

To address the above problems, immune checkpoint inhibitors in combination with oncolytic viruses are used to relieve the immunosuppressive effects of immune checkpoints. Preclinical studies have shown that treatment of colon and ovarian cancer with oncolytic vaccinia virus in combination with PD-L1 antibody significantly reduces tumor burden and increases survival in mice bearing tumors. Recently, a phase Ib clinical study has shown that the combination of T-VEC [ oncolytic herpes simplex virus 1(HSV-1) encoding human granulocyte macrophage colony stimulating factor (GM-CSF) ] and pembrolizumab (Keytruda, the first FDA approved PD-1 inhibitor) has a better therapeutic effect in treating melanoma than either therapy alone. Another phase Ib/II clinical study showed that the combination of T-VEC and ipilimumab [ a cytotoxic T lymphocyte antigen 4(CTLA-4) inhibitor ] showed better efficacy than T-VEC or ipilimumab monotherapy. However, this combination therapy strategy has raised some concerns in terms of increased patient side effects and medical costs. Since oncolytic viruses can carry antibody genes, the search for engineered oncolytic viruses expressing immune checkpoint inhibitors is a new direction for tumor virus development.

TIGIT, also known as Vstm3, VSIG9 or WUCAM, is a new target for immune checkpoint therapy. The two primary ligands of TIGIT are the poliovirus receptor (PVR, CD155) and poliovirus receptor-associated 2(PVRL2, CD112, nectin-2), both expressed by tumor cells and myeloid cells. DNAX helper molecule 1(DNAM-1, CD226) and CD96 are alternative receptors for PVR, both expressed on naive T cells and on effector T cells. Interaction of TIGIT and CD96 with PVR induces an immunosuppressive signal, while DNAM-1 provides a costimulatory activation signal. Moreover, TIGIT is highly expressed on activated Natural Killer (NK), T cells and regulatory T (treg) cells, suggesting that blocking PVR/TIGIT signaling may be a promising approach to tumor immunotherapy. However, the engineered oncolytic virus carrying the TIGIT single-chain antibody gene is still lacking for research and development of antitumor drugs.

Disclosure of Invention

The invention aims to solve the problem that although oncolytic virus can induce T cells to effectively infiltrate in tumor tissues, the cold tumor with less T cells is converted into the hot tumor with more T cells. However, TIGIT molecules expressed on the surface of T cells can inhibit T cell activation, leading to depletion of activated T cells, thereby assisting tumor cells in evading the killing effect of T cells. At present, a new oncolytic virus is urgently needed to be developed to overcome the inhibiting effect of TIGIT molecules on anti-tumor immune response excited by the oncolytic virus, so that the curative effect of the existing oncolytic virus is improved.

In order to solve the above problems, the present invention provides the following technical solutions:

the Thymidine Kinase (TK) region of the genome of the oncolytic vaccinia virus comprises a gene sequence of an anti-mouse/human TIGIT single-chain antibody, and the oncolytic vaccinia virus can infect tumor cells to enable the tumor cells to express the anti-mouse/human TIGIT single-chain antibody.

Further, the nucleotide sequence of the anti-mouse/human TIGIT single-chain antibody is shown in SEQ ID NO. 1.

Furthermore, the anti-mouse/human TIGIT single-chain antibody is formed by connecting a signal sequence, a tag protein, an antibody heavy chain variable region, a connecting peptide and an antibody light chain variable region in series. .

Furthermore, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 2.

Furthermore, the amino acid sequence of the variable region of the antibody light chain is shown as SEQ ID NO. 3.

Further, the oncolytic vaccinia virus is capable of lysing tumor cells.

Further, the single-chain antibody expressed by the oncolytic vaccinia virus can block the immune suppression function of a TIGIT molecule.

The preparation method of the oncolytic vaccinia virus comprises the following steps:

(1) artificially synthesizing a vaccinia virus shuttle plasmid pVV-Control, wherein the nucleotide sequence of the vaccinia virus shuttle plasmid pVV-Control is shown as SEQ ID NO. 4;

(2) adding EcoR1 and Xba1 enzyme cutting sites before and after the TIGIT single-chain antibody gene sequence SEQ ID NO.1 respectively, and artificially synthesizing a TIGIT single-chain antibody gene fragment;

(3) after the TIGIT single-chain antibody gene fragment is subjected to double enzyme digestion by EcoR1 and Xba1, the TIGIT single-chain antibody gene fragment is subcloned into a TK region of a shuttle plasmid pVV-Control, and a pVV-scFv-TIGIT plasmid is constructed, wherein the expression of the TIGIT single-chain antibody (scFv-TIGIT) is controlled by a vaccinia virus early and late promoter pSE/L;

(4) cells that had been previously infected with wild vaccinia virus (WR strain) were transfected with the pVV-scFv-TIGIT plasmid, and homologous recombination of the plasmid and wild vaccinia virus occurred to produce oncolytic vaccinia virus VV-scFv-TIGIT.

The invention also provides a recombinant plasmid, wherein the genome DNA of the recombinant plasmid comprises a thymidine kinase region of an oncolytic vaccinia virus genome, and the thymidine kinase region comprises a gene sequence of an anti-mouse/human TIGIT single-chain antibody.

Furthermore, the anti-mouse/human TIGIT single-chain antibody is formed by connecting a signal sequence, a tag protein, an antibody heavy chain variable region, a connecting peptide and an antibody light chain variable region in series.

The invention also protects the application of the oncolytic vaccinia virus or the recombinant plasmid in preparing a medicament for treating tumors.

Furthermore, the tumor is breast cancer, colorectal cancer, liver cancer, melanoma, lung cancer, gastric cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, glioma, cancerous ascites or cancerous pleural effusion.

Preferably, the tumor is breast cancer, colon cancer, melanoma or cancerous ascites.

Has the advantages that: according to the invention, the TK gene dependent on replication of the virus is inactivated by inserting the TIGIT single-chain antibody gene into the TK region of the vaccinia virus genome, so that the virus can only replicate in high-activity tumor cells of the TK, and the safety of the oncolytic vaccinia virus is obviously enhanced.

(1) The oncolytic effect of the oncolytic virus is effectively combined with the anti-tumor effect of an immune checkpoint inhibitor to prepare the oncolytic vaccinia virus capable of expressing TIGIT single-chain antibody; the virus can replicate in tumor cells and exert an oncolytic effect.

(2) The virus can effectively induce T cell infiltration, and the 'cold' tumor microenvironment is converted into a 'hot' tumor microenvironment. The virus can infect tumor cells and enable the tumor cells to express a TIGIT single-chain antibody, and the TIGIT single-chain antibody can effectively combine with TIGIT molecules on the surfaces of T cells, relieve the immunosuppressive action of the TIGIT molecules on the T cells, enhance the immune response of the T cells on the tumor cells, and inhibit the exhaustion of activated T cells, thereby exerting multiple antitumor effects.

(3) The TIGIT single-chain antibody secreted by the virus can effectively combine TIGIT molecules on the surface of local T cells of the tumor, relieve the immunosuppression effect of the TIGIT molecules on the T cells and enhance the immune response of the T cells on the tumor cells.

Drawings

The following will be further explained in conjunction with the attached drawings, in which:

FIG. 1 is a schematic diagram showing the construction of vaccinia virus VV-scFv-TIGIT according to the present invention.

FIG. 2 is a map of shuttle plasmid pVV-Control of vaccinia virus according to the invention.

FIG. 3 is a map of vaccinia virus shuttle plasmid pVV-scFv-TIGIT of the invention.

FIG. 4 is a schematic representation of viral plaques produced by infection of HELA-S3 cells with oncolytic vaccinia virus VV-scFv-TIGIT of the invention.

FIG. 5 shows a Western Blot method for detecting the expression of TIGIT single-chain antibody in the culture supernatant of HELA-S3 cells infected with vaccinia virus VV-scFv-TIGIT. As can be seen from the figure, the molecular weight of the TIGIT single-chain antibody is about 40 KD.

FIG. 6 is a graph showing the in vitro oncolytic capacity of the oncolytic vaccinia virus VV-scFv-TIGIT on the breast cancer cell line 4T1, the colon cancer cell line CT26 and the melanoma cell line B16/F10 by crystal violet staining. As can be seen from the figure, the Control viruses VV-Control and VV-scFv-TIGIT both had a gradually increasing tumor-dissolving ability with increasing multiplicity of infection (MOI).

FIG. 7 is a graph showing that the MTT assay of the present invention detects the in vitro oncolytic ability of oncolytic vaccinia virus VV-scFv-TIGIT on breast cancer cell line 4T1, colon cancer cell line CT26 and melanoma cell line B16/F10. As can be seen from the figure, the Control viruses VV-Control and VV-scFv-TIGIT both have gradually increased oncolytic abilities with the increase of multiplicity of infection (MOI), and compared with each other, there was no significant difference in oncolytic abilities.

FIG. 8 shows the therapeutic effect of intratumoral injection of oncolytic vaccinia virus VV-scFv-TIGIT on colon cancer CT26 subcutaneous tumor transplantation model. As can be seen from the figure, compared with PBS and a Control virus VV-Control, VV-scFv-TIGIT can obviously inhibit the growth of CT26 subcutaneous transplanted tumor and prolong the survival time of mice.

FIG. 9 shows the therapeutic effect of intratumoral injection of oncolytic vaccinia virus VV-scFv-TIGIT in model of subcutaneous tumor transplantation of colon cancer MC 38. As can be seen from the figure, compared with PBS and the Control virus VV-Control, VV-scFv-TIGIT can obviously inhibit the growth of MC38 subcutaneous transplantation tumor and prolong the survival time of mice.

FIG. 10 shows the therapeutic effect of intratumoral injection of oncolytic vaccinia virus VV-scFv-TIGIT in the 4T1 subcutaneous tumor engraftment model of breast cancer. As can be seen from the figure, compared with PBS and the Control virus VV-Control, VV-scFv-TIGIT can obviously inhibit the growth of 4T1 subcutaneous transplanted tumor and prolong the survival time of the mouse.

FIG. 11 shows the therapeutic effect of intratumoral injection of oncolytic vaccinia virus VV-scFv-TIGIT on colon cancer CT26 subcutaneous transplantation tumor model and the induction of immune cell infiltration. As can be seen, compared with PBS and the Control virus VV-Control, VV-scFv-TIGIT can obviously inhibit the growth of CT26 subcutaneous transplanted tumor. Lymphocytes in subcutaneous tumor tissue, CD3, in VV-scFv-TIGIT treated mice compared to PBS and Control virus VV-Control 2 days after 2 nd virus treatment⁺T cell, CD4⁺T cells and CD8⁺The proportion of T cells was significantly increased. CD8 in mouse spleen cells⁺The proportion of T cells increases significantly.

FIG. 12 shows the therapeutic effect of intraabdominal injection of oncolytic vaccinia virus VV-scFv-TIGIT on H22 ascites tumor model of liver cancer. As can be seen from the figure, compared with PBS and the Control virus VV-Control, VV-scFv-TIGIT can obviously improve the cure rate of the mice.

FIG. 13 is a dynamic observation of tumor cells and immune cell components in ascites of H22 ascites model for the treatment of hepatoma H22 by intraperitoneal injection of vaccinia virus VV-scFv-TIGIT in accordance with the present invention.

FIG. 14 is a graph of the immune cell infiltration in ascites of H22 ascites model for the treatment of liver cancer by intraperitoneal injection of vaccinia virus VV-scFv-TIGIT in accordance with the invention.

FIG. 15 shows the intraabdominal injection of VV-scFv-TIGIT for H22 ascites tumor infiltration CD8 of hepatocarcinoma⁺T cell immunosuppressive and immunostimulatory moleculesThe influence of (c).

FIG. 16 shows NK cell and CD8 depletion⁺The effect of T cells on the curative effect of the vaccinia virus VV-scFv-TIGIT on treating the hepatoma H22 ascites tumor.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention, unless otherwise indicated.

Example 1

Construction of shuttle plasmid pVV-scFv-TIGIT

The total length of pVV-Control plasmid is synthesized by artificial synthesis, the gene sequence is shown in SEQ ID NO.4, and the plasmid map is shown in FIG. 2.

The amino acid sequences of heavy and light chain variable regions of the hamster anti-mouse/human TIGIT antibody are respectively referred to the sequences of SEQ ID nos. 21 and 22 in patent US20090258013a1, and the nucleotide sequences of the TIGIT antibody heavy and light chain variable regions of the present application are obtained through amino acid deduction and codon optimization. According to the design of the heavy chain variable region-connecting peptide-light chain variable region, a gene sequence for coding GGSGGGGSGGGGS connecting peptide is added between the heavy chain variable region and the light chain variable region, and then a gene sequence for coding human interleukin 2(hIL-2) signal peptide and a gene sequence for coding HA tag protein are added at the front end of the heavy chain variable region and the light chain variable region to form a single-chain antibody gene sequence shown as SEQ ID NO. 1. The recognition sequences GAATTC and Kozak GCCACC of endonuclease EcoR1 and recognition sequence TCTAGA of endonuclease Xba1 are respectively added at the front end of the strain and the tail end of the strain, and then the sequences are synthesized by adopting a manual synthesis method.

Artificially synthesized TIGIT single-chain antibody DNA is subjected to double enzyme digestion by EcoR1 and Xba1, meanwhile, the pVV-Control plasmid is also subjected to double enzyme digestion by EcoR1 and Xba1, agarose gel electrophoresis is carried out, a TIGIT single-chain antibody gene fragment and a pVV-Control plasmid fragment are respectively recovered by a DNA gel recovery kit (Kangshiji), the recovered target gene fragment and the plasmid fragment are connected for 30min at room temperature by T4DNA ligase (Takara company), then a connection product is transformed into DH5 alpha competent cells, the competent cells are coated on an LB plate containing 100 mu g/ml of ampicillin, and the culture is carried out overnight at 37 ℃. Selecting 5-10 single colonies the next day, and carrying out PCR amplification by using primers 5'-caggtgatctgtttttattgtggag-3' and 5'-gatctacttccttaccgtgc-3', wherein the amplification reaction is carried out under the conditions of 98 ℃ for 3 minutes, 98 ℃ for 10 seconds, 55 ℃ for 5 seconds, 72 ℃ for 20 seconds, 30 cycles and 72 ℃ for 5 minutes. Agarose gel electrophoresis of the PCR amplification product shows that the colonies with 1100BP positive bands are positive colonies. Then, the positive colonies were inoculated into 5mL of LB liquid medium containing 100. mu.g/mL of ampicillin, and cultured with shaking at 37 ℃ for 12 hours; after the culture is finished, extracting plasmids by using a plasmid extraction kit, carrying out double enzyme digestion identification on EcoR1 and Xba1, further sequencing the correctly sequenced plasmid and naming the plasmid as pVV-scFv-TIGIT, wherein the plasmid map is shown in figure 3.

Example 2

Oncolytic vaccinia virus packaging and screening

HEK293 cells were routinely grown in DMED high sugar medium containing 10% FBS, cells were digested with 0.5% trypsin, counted and counted at 3X 10⁵cells/well were seeded in 6-well plates at 37 ℃ with 5% CO₂Culturing in an incubator. When the cells grew to more than 90% confluence, wild vaccinia virus (WR strain, purchased from ATCC, accession number:

VR-1354) for 2 hours (MOI ═ 1). The plasmid pVV-scFv-TIGIT is adopted

Transfection reagent (Polyplus-transfection Co.) transfection of HEK293 cells previously infected with wild vaccinia virus, the transfected HEK293 cells were placed at 37 ℃ and 5% CO₂The plasmid and the wild vaccinia virus are subjected to homologous recombination in an incubator to generate the oncolytic vaccinia virus VV-scFv-TIGIT, and the homologous recombination mode of the oncolytic vaccinia virus is shown in figure 1. Inserting a guanine-hypoxanthine phosphoribosyl transferase (gpt) gene and a fluorescent protein reporter gene into a wild virus TK region of an oncolytic vaccinia virus VV-scFv-TIGIT, connecting the guanine-hypoxanthine phosphoribosyl transferase (gpt) gene and the fluorescent protein reporter gene by adopting 2A peptide, and controlling the expression of the guanine-hypoxanthine phosphoribosyl transferase (gpt) gene and the fluorescent protein reporter gene by a p7.5K promoter; expression of inserted TIGIT Single chain antibody (scFv) GeneIs controlled by a p-SE/L promoter, and the gene sequence of the promoter is shown as SEQ ID NO. 1. The packaging of VV-Control virus is similar to VV-scFv-TIGIT, and is obtained by homologous recombination of pVV-Control plasmid and wild vaccinia virus.

Screening for oncolytic vaccinia virus was performed by plaque purification, and vaccinia virus VV-scFv-TIGIT without wild virus was obtained by plaque purification under conditions of medium containing 25. mu.g/ml mycophenolic acid (MPA), 250. mu.g/ml Xanthine (Xanthine), and 15. mu.g/ml Hypoxanthine (Hypoxanthine) for screening and removing wild virus 48 hours after transfection, after plaque was generated in oncolytic vaccinia virus (as shown in FIG. 4). Then, a virus genome extraction kit (kang century company) is adopted to extract the genome of the oncolytic vaccinia virus, the following primers are adopted to respectively amplify the target gene and the Thymidine Kinase (TK) gene of the wild virus, and the amplification conditions are the same as the above. The target gene: 5'-caggtgatctgtttttattgtggag-3' for F, 5'-gatctacttccttaccgtgc-3' for R; TK: F: 5'-tgtgaagacgataaattaatgatc-3', and R: 5'-gtttgccatacgctcacag-3'. The virus in which the target gene is successfully amplified by PCR but the wild virus TK gene is not simultaneously amplified is the successfully screened oncolytic vaccinia virus. The screened successful oncolytic vaccinia viruses were further expanded in cell culture plates using HELA-S3 cells. The titer of the virus was determined by the TCID50 method and was calculated as 0.7X 10^ (1+ S (D-0.5)) where S is log10 (dilution) and D is the sum of EGFP positive ratios at each dilution.

Example 3

Determination of oncolytic vaccinia virus expression and secretion of TIGIT single-chain antibody

HELA-S3 cells were cultured in DMED high sugar medium containing 10% FBS, the cells were digested with 0.5% trypsin, counted and counted at 5X 10⁵The cells/well were seeded in 6-well plates at 37 ℃ in 5% CO₂Culturing in an incubator. When the cells grew to more than 90% confluence, oncolytic vaccinia virus infection (MOI ═ 0.1) was added. After 24 hours, cell culture supernatants were collected, and Western Blot was performed to detect the expression of TIGIT single-chain antibody in the cell culture supernatants using anti-HA-tagged antibody as the primary antibody. As shown in FIG. 5, oncolytic vaccinia VV-scFv-TIGIT infected HELA-S3 thin linesThe cells can secrete TIGIT single-chain antibody with the molecular weight of about 40KD in cell culture supernatant.

Example 4

Determination of the in vitro oncolytic Capacity of oncolytic vaccinia Virus on tumor cells

Breast cancer cells 4T1, colon cancer cells CT26 and melanoma cells B16/F10 were cultured in DMED high sugar medium containing 10% FBS, cells were digested with 0.5% trypsin and counted at 5X 10³Cells/well were seeded in 96-well plates at 37 ℃ in 5% CO₂Culturing in an incubator. When the cells grow to a fusion degree of more than 90%, oncolytic vaccinia viruses with different multiplicity of infection (MOI of 0, 0.1, 1, 5 and 10) are added respectively. After 72 hours of culture, the supernatant was removed, the cells were stained with 0.5% crystal violet stain, and the oncolytic capacity of the oncolytic vaccinia virus was observed.

The oncolytic capacity of oncolytic vaccinia virus on tumor cells is quantitatively analyzed by adopting an MTT test. Cell culture and viral infection were tested with crystal violet staining. After 72 hours of viral infection, 20. mu.l of thiazole blue (MTT) was added to each well to continue the culture. After 4 hours, the supernatant was removed and 150. mu.l of isopropanol was added to dissolve Formazan (Formazan), and the OD value was measured at 450nm using a microplate reader, and the cell viability was calculated by the following formula (experimental well-blank control well)/(control well-blank control group). times.100%).

As shown in FIGS. 6 and 7, the oncolytic ability of both VV-Control and VV-scFv-TIGIT gradually increased with the increase of the multiplicity of infection (MOI); compared with the oncolytic capacity of VV-scFv-TIGIT, the VV-Control and the VV-scFv-TIGIT have no significant difference.

Example 5

Therapeutic effect of oncolytic vaccinia virus on CT26 colon cancer subcutaneous transplantation tumor

Establishing colon cancer subcutaneous tumor model with CT26 cells, selecting 6-8 week old male BALB/c mice (SPF grade, purchased from Nanjing university model animal institute), and inoculating 5 × 10 subcutaneous tissue to each mouse⁵And CT26 cells. At 7 days after modeling, when the tumor diameter had grown to approximately 5mm, the mice were randomly assigned to 3 groups, PBS Control group, VV-Control group and VV-scFv-TIGIT treatment group, respectivelyThe treatment was performed by intratumoral multiple injections of PBS, VV-Control and VV-scFv-TIGIT. The dose of virus injection is 1X 10⁷PFU was treated 1 time every 1 day for 3 times per mouse. After the first virus treatment, tumor length and length were measured every 1 day, and the weight of the mice was weighed. The tumor volume is given by the formula [ long meridian (mm) × short meridian (mm)²]The tumor volume size was calculated 2. When the tumor volume is larger than 2000mm³When the mouse died, the mouse was judged to be dead. As shown in FIG. 8, VV-scFv-TIGIT significantly inhibited the growth of CT26 subcutaneous transplantable tumor on day 11 after the first viral injection, compared to PBS and VV-Control. Meanwhile, VV-scFv-TIGIT treatment significantly prolonged the survival time of CT26 tumor-bearing mice. P<0.01；****P<0.0001. Compared among the 3 groups, the weight of the mice has no significant difference, which indicates that the treatment of intratumoral injection of oncolytic vaccinia virus VV-scFv-TIGIT has no obvious toxic or side effect on BALB/c mice.

Example 6

Therapeutic effect of oncolytic vaccinia virus on MC38 colon cancer subcutaneous transplantation tumor

Establishing colon cancer subcutaneous tumor model with MC38 cells, selecting 6-8 week old male C57BL/6 mice (SPF grade, purchased from Nanjing university model animal institute), and injecting 2 × 10 subcutaneous injection per mouse⁶MC38 cells. At 5 days after modeling, when tumors grew approximately to 5mm in diameter, the mice were randomized into 3 groups, PBS Control, VV-Control and VV-scFv-TIGIT treatment. The treatment dosage, treatment mode and detection index were the same as those in example 5. As shown in FIG. 9, after 15 days of the first injection of the virus, VV-scFv-TIGIT significantly inhibited the growth of MC38 subcutaneous transplants compared to PBS and the Control virus VV-Control. Meanwhile, VV-scFv-TIGIT treatment significantly prolongs the survival time of MC38 tumor-bearing mice. P<0.05；**P<0.01；***P<0.001. Compared with the PBS, VV-Control and VV-scFv-TIGIT treatment groups, the weight of the mice has no significant difference, which indicates that the intratumoral injection oncolytic vaccinia virus VV-scFv-TIGIT treatment has no obvious toxic or side effect on the C57bl/6 mice.

Example 7

Therapeutic effect of oncolytic vaccinia virus on 4T1 breast cancer subcutaneous transplantation tumor

Using 4T1 cellsEstablishing breast cancer subcutaneous tumor model, selecting 6-8 week female BALB/c mouse (SPF grade, purchased from Nanjing university model animal institute), injecting 2 × 10 subcutaneous injection into each mouse⁵4T1 cells. At 7 days after modeling, when tumors grew approximately to 5mm in diameter, the mice were randomly assigned to 3 groups, PBS Control group, VV-Control and VV-scFv-TIGIT treatment group, respectively. The treatment dosage, treatment mode and detection index were the same as in example 5. As shown in FIG. 10, compared with PBS and the Control virus VV-Control, VV-scFv-TIGIT can significantly inhibit the growth of 4T1 subcutaneous transplanted tumor and prolong the survival time of mice. P<0.05；**P<0.01； ***P<0.001. Compared among the 3 groups, the weight of the mice has no significant difference, which shows that the treatment of intratumoral injection of the oncolytic vaccinia virus VV-scFv-TIGIT has no obvious toxic or side effect on BALB/c mice.

Example 8

Effects of oncolytic vaccinia virus on treating CT26 colon carcinoma subcutaneous tumor and inducing lymphocyte infiltration

Establishing colon cancer subcutaneous tumor model with CT26 cells, selecting 6-8 week old male BALB/c mice (SPF grade, purchased from Nanjing university model animal institute), injecting 5 × 10 subcutaneously into each mouse⁵And CT26 cells. At 7 days after modeling, when tumors grew approximately to 5mm in diameter, the mice were randomly assigned to 3 groups, PBS Control group, VV-Control and VV-scFv-TIGIT treatment group, respectively. The treatment dosage, treatment mode and detection index were the same as those in example 5. As shown in FIGS. 11A-D, VV-scFv-TIGIT significantly inhibited the growth of CT26 subcutaneous transplantable tumors compared to PBS and VV-Control at day 7 after the first viral injection. P<0.05； **P<0.01. There was no significant difference in mouse body weight compared between the 3 groups.

On day 7 after the first viral injection, mice were anesthetized and sacrificed, tumors were denuded, tumor tissue was cut into pieces of about 1mm with scissors, then digested with 0.2% collagenase solution IV for 2 hours, filtered with a 200 mesh screen, and prepared into single cell suspensions. T cells and NK cells were labeled with APC-CD45, FITC-CD3, PerCP-Cy5.5-CD8 and PE-Cy5.5-CD49b monoclonal antibodies (BD Co., Ltd.), respectively. Incubate for 15 minutes at room temperature in the dark, and fix the cells by adding 4% paraformaldehyde. Flow with FACS CaliburThe detection was performed by a cytometer (BD Co., Ltd.) and data analysis was performed by using FlowJo software. As shown in FIGS. 11E-G, lymphocytes in tumor tissue, CD3, from the VV-scFv-TIGIT treated mice compared to PBS and the Control virus VV-Control⁺T，CD4⁺T and CD8⁺T cells were significantly increased, while NK and NKT cells were not significantly different compared between the 3 treatment groups. CD8 in splenocytes from mice treated with VV-scFv-TIGIT as compared to PBS treatment⁺The proportion of T cells is increased, but has no significant difference compared with the VV-Control treatment group mice. NK, CD3 in splenocytes⁺T and CD4⁺The proportion of T cells was not significantly different in comparison between the three groups. The above results indicate that oncolytic vaccinia virus can efficiently induce T cell infiltration in tumor tissues. P<0.05；**P<0.01；***P<0.001；****P<0.0001。

Example 9

Effect of oncolytic vaccinia virus in treating H22 hepatoma ascites tumor and inducing lymphocyte infiltration

Establishing a hepatoma ascites tumor model by using H22, selecting 6-8 week-old male C57BL/6 mice (SPF grade, purchased from Nanjing university model animal institute), injecting 1 × 10 per mouse into abdominal cavity⁷And H22 cells. Ascites were produced in all mice on day 5 after tumor cell injection, and then the mice were randomly divided into 3 groups, which were a PBS Control group, a VV-Control group and a VV-scFv-TIGIT treatment group, respectively. Respectively treating by intraperitoneal injection of PBS, VV-Control and VV-scFv-TIGIT at a dose of 1 × 10⁷PFU, 1 treatment every 1 day for a total of 3 treatments.

As shown in FIG. 12, the VV-Control and VV-scFv-TIGIT treated groups significantly prolonged the survival time of tumor-bearing mice compared to the PBS treated group. The cure rate of the mice in the VV-scFv-TIGIT treatment group is 93.75 percent (15/16), the cure rate of the mice in the VV-Control treatment group is 25.00 percent (4/16), and the cumulative survival rates of the two groups have significant difference (P < 0.001).

Ascites were extracted on day 5 (before virus treatment), 7 (2 days after 1 virus treatment), 9 (2 days after 2 virus treatment) and 11 (2 days after 3 virus treatment) after the ascites tumor model was established, respectively, counted and adjusted for each sampleThe number of the cells is 2X 10⁷And (4) respectively. 100. mu.l of the cell suspension was labeled with APC-CD45, FITC-NK1.1, FE-CD3, and PerCP-Cy5.5-CD8 (Biolegend), incubated at room temperature for 15 minutes in the dark, and the cells were fixed by adding 4% paraformaldehyde. The detection was performed by a FACS Calibur flow cytometer (BD Co.), and data analysis was performed by using FlowJo software. As shown in fig. 13, there was no significant change in tumor cell, lymphocyte and lymphocyte/tumor cell ratio in ascites of PBS group mice on

days

5, 7, 9 and 11 after ascites tumor model establishment. The tumor cells in the ascites cells of the mice in the two treatment groups of VV-Control and VV-scFv-TIGIT are gradually reduced, and the ratios of the lymphocytes and the lymphocytes/tumor cells are gradually increased. On the 11 th day (2 nd day after 3 rd virus treatment) of model establishment, tumor cells in ascites cells of mice in the VV-scFv-TIGIT treatment group almost disappeared, and the ratios of lymphocytes and lymphocytes/tumor cells were all significantly higher than those in the VV-Control treatment group.

As shown in FIG. 14, at day 9 after the ascites tumor model was established, the proportion of tumor cells in ascites of mice in the VV-Control-treated group and the VV-scFv-TIGIT-treated group was significantly decreased, as compared with the PBS-treated group. And the proportion of tumor cells in ascites of mice in the VV-scFv-TIGIT treatment group is obviously lower than that in the VV-Control treatment group. Lymphocyte, lymphocyte/tumor cell ratio, NK cell, CD3 in ascites of mice in VV-scFv-TIGIT treatment group compared with PBS and VV-Control group⁺T、CD4⁺T and CD8⁺The proportion of T cells was significantly increased.

As shown in FIG. 15, TIGIT in ascites of VV-scFv-TIGIT-treated mice as compared with PBS-treated group⁺CD8⁺T、PD1⁺CD8⁺T、TIM3⁺CD8⁺T、LAG3⁺CD8⁺T、CD107A⁺CD8⁺T、 IFN-γ⁺CD8⁺T、TNF-α⁺CD8⁺T and GzB⁺CD8⁺T cell occupancy CD8⁺The proportion in T cells was significantly increased; TIM3 in ascites of mice in VV-scFv-TIGIT treatment group compared with VV-Control treatment group⁺CD8⁺T、LAG3⁺CD8⁺T、CD107A⁺CD8⁺T、IFN-γ⁺CD8⁺T、TNF-α⁺CD8⁺T and GzB⁺CD8⁺T cells were all significantly increased; TIGIT in mouse ascites⁺CD8⁺T and PD1⁺CD8⁺T accounts for CD8⁺The proportion of T cells was not significantly different between the two treatment groups VV-Control and VV-scFv-TIGIT.

The results show that VV-scFv-TIGIT has higher cure rate on H22 hepatoma ascites tumor, and can recruit and activate CD8⁺T cells.

Example 10

CD8⁺Effect of T and NK cell depletion on the efficacy of VV-scFv-TIGIT in the treatment of H22 hepatoma ascites

H22 model of ascites carcinoma of liver cancer was established as in example 9, and the treatment regimen is shown in FIG. 16A. On day 3 after the mice were inoculated with tumor cells intraperitoneally, Anti-CD8 and Anti-NK1.1 monoclonal antibodies were intraperitoneally injected to eliminate CD8⁺T and NK cells. After 1 day of antibody injection, peripheral blood was taken from the tail vein and CD8 was detected by flow cytometry⁺T and NK cell ratios, cell fluorescent staining protocol as in example 9. On the 6 th day after tumor cell inoculation, PBS, VV-Control and VV-scFv-TIGIT are injected into the abdominal cavity respectively for treatment, and the dose of virus injection is 1 × 10⁷PFU, 1 treatment every 1 day for a total of 3 treatments. Ascites was extracted and the proportions of tumor cells, lymphocytes and subpopulations thereof in the ascites were examined 2 days before and 2 nd virus treatment, respectively. The survival time of the mice was observed.

As shown in FIGS. 16B-C, mice peripheral blood CD8 was injected with Anti-CD8 and Anti-NK1.1 monoclonal antibody for 1 day⁺T cells and NK cells were depleted, respectively. No CD8 could be detected in the ascites of mice 3 days after Anti-CD8 and Anti-NK1.1 monoclonal antibody injection (day 6 after tumor cell intraperitoneal inoculation)⁺T and NK cells.

As shown in FIGS. 16D-E, the tumor cell ratio in ascites of the mice of the VV-scFv-TIGIT treatment group was significantly lower than that of the PBS treatment group, and lymphocytes, NK, NKT, CD3 were present on day 2 after the 2 nd oncolytic virus treatment⁺T、CD4⁺T and CD8⁺The proportion of T cells is obviously higher than that of PBS treatmentAnd (4) grouping. Clearing CD8⁺T and NK cells can partially eliminate VV-scFv-TIGIT mediated anti-tumor effect, and increase tumor cell proportion in ascites, while lymphocyte, NK, NKT, CD3⁺T、CD4⁺T and CD8⁺The proportion of T cells was significantly reduced. Injection of Anti-CD8 resulted in CD8 in ascites in VV-scFv-TIGIT treated mice⁺T cell depletion and NK, NKT, CD3⁺T、CD4⁺T cells were significantly reduced, making these cells below or at the same level as the PBS treated group. Similarly, injection of Anti-NK1.1 resulted in NK cell depletion in ascites and NKT, CD3 in VV-scFv-TIGIT treated mice⁺T、CD4⁺T and CD8⁺T cells were significantly decreased. However, the proportion of these cells in ascites was significantly higher than in the PBS treated group.

As shown in FIG. 16F, the VV-Control and VV-scFv-TIGIT treated groups significantly prolonged the survival time of tumor-bearing mice compared to the PBS treated group. The cure rate of VV-scFv-TIGIT treated mice is 100% (8/8), the cure rate of anti-NK1.1+ VV-scFv-TIGIT treated mice is 87.50% (7/8), and none of PBS and anti-CD8+ VV-scFv-TIGIT treated mice is cured.

The above results indicate that the in vivo antitumor effect of VV-scFv-TIGIT is dependent on CD8⁺T cells.

It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but various changes and modifications may be made without departing from the spirit and scope of the invention, the scope of which is defined by the appended claims, the description and the equivalents thereof.

Sequence listing

<110> Nanjing Security articles medicine science and technology Co., Ltd

<120> oncolytic vaccinia virus and preparation method and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

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<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgtacagga tgcagctgct gtcttgcatc gccctgagcc tggctctggt gaccaactct 60

tacccctacg acgtgcctga ttacgctgag gtgcagctgg tggagagcgg aggaggactg 120

acacagccag gcaagagcct gaagctgtcc tgtgaggctt ctggcttcac cttcagctcc 180

tttaccatgc actgggtgcg gcagtcccct ggaaagggcc tggagtgggt ggccttcatc 240

cgctctggaa gcggcatcgt gttttacgct gacgctgtgc ggggccgctt caccatctcc 300

agggataacg ctaagaacct gctgtttctg cagatgaacg acctgaagtc tgaggataca 360

gccatgtact actgcgctag gagacccctg ggccacaaca ccttcgacag ctggggacag 420

ggcacactgg tgaccgtgtc tagcggagga ggaggatccg gaggaggagg atctggaggc 480

ggaggcagcg atatcgtgat gacacagtcc ccttcctctc tggccgtgtc tccaggcgag 540

aaggtgacca tgacctgtaa gagctcccag agcctgtact actccggcgt gaaggagaac 600

ctgctggcct ggtaccagca gaagccaggc cagagcccca agctgctgat ctactacgct 660

tccatcaggt tcaccggcgt gccagacagg ttcaccggat ccggctctgg aaccgattac 720

accctgacaa tcacctccgt gcaggctgag gacatgggcc agtacttttg ccagcaggga 780

atcaacaacc ccctgacatt cggcgatgga accaagctgg agatcaagag ataa 834

<210> 2

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Gly Val Gly Leu Val Gly Ser Gly Gly Gly Leu Thr Gly Pro Gly Leu

1 5 10 15

Ser Leu Leu Leu Ser Cys Gly Ala Ser Gly Pro Thr Pro Ser Ser Pro

20 25 30

Thr Met His Thr Val Ala Gly Ser Pro Gly Leu Gly Leu Gly Thr Val

35 40 45

Ala Pro Ile Ala Ser Gly Ser Gly Ile Val Pro Thr Ala Ala Ala Val

50 55 60

Ala Gly Ala Pro Thr Ile Ser Ala Ala Ala Ala Leu Ala Leu Leu Pro

65 70 75 80

Leu Gly Met Ala Ala Leu Leu Ser Gly Ala Thr Ala Met Thr Thr Cys

85 90 95

Ala Ala Ala Pro Leu Gly His Ala Thr Pro Ala Ser Thr Gly Gly Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 3

<211> 113

<212> PRT

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Ala Ile Val Met Thr Gly Ser Pro Ser Ser Leu Ala Val Ser Pro Gly

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Gly Leu Val Thr Met Thr Cys Leu Ser Ser Gly Ser Leu Thr Thr Ser

20 25 30

Gly Val Leu Gly Ala Leu Leu Ala Thr Thr Gly Gly Leu Pro Gly Gly

35 40 45

Ser Pro Leu Leu Leu Ile Thr Thr Ala Ser Ile Ala Pro Thr Gly Val

50 55 60

Pro Ala Ala Pro Thr Gly Ser Gly Ser Gly Thr Ala Thr Thr Leu Thr

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Ile Thr Ser Val Gly Ala Gly Ala Met Gly Gly Thr Pro Cys Gly Gly

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Gly Ile Ala Ala Pro Leu Thr Pro Gly Ala Gly Thr Leu Leu Gly Ile

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Leu

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt 60

aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 120

ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 180

ttttttctgc gtgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 240

tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 300

cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct 360

gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 420

gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 480

tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 540

ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 600

gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 660

ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 720

tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggcccga 780

gtcagtctca tgttctcacc ggtataaata cttaataatc tcatttcagc tgaatatgaa 840

ggagcaaaag gttgtaacat tttattaccg tgtgggatat aaaagtcctt gatccattga 900

tctggaaacg ggcatctcca tttaagacta gacgccacgg ggtttaaaat actaatcatg 960

acattttgta gagcgtaatt acttagtaaa tccgccgtac taggttcatt tcctcctcgt 1020

ttggatctca catcagaaat taaaataatc ttagaaggat gcagttgttt tttgatggat 1080

cgtagatatt cctcatcaac gaaccgagtc actagagtca catcacgcaa tccatttaaa 1140

ataggatcat gatggcggcc gtcaattagc atccatttga tgatcactcc taaattatag 1200

aaatgatctc tcaaataacg tatatgtgta ccgggagcag atcctatata cactacggtg 1260

gcaccatcta atataccgtg tcgctgtaac ttactaagaa aaaataattc tcctagtaat 1320

agttttaact gtccttgata cggcagtttt tttgcgacct catttgcact ttctggttcg 1380

taatctaact cattatcaat ttcctcaaaa tacataaacg gtttatctaa cgacacaaca 1440

tccattttta agtattatat taaaatttaa tcaatgttta tttttagttt tttagataaa 1500

aaatataata ttatgagtcg atgtaacact ttctacacac cgattgatac atatcattac 1560

ctcctattat ttctatctcg gtttcctcac ccaatcgttt agaaaaggaa gcctccttaa 1620

agcatttcat acacacagca gttagtttta ccaccatttc agataatgga ataagattca 1680

aaatattatt aaacggttta cgttgaaatg tcccatcgag tgcggctact ataactgcgc 1740

gcaattaacc ctcactaaag ggaacaaaag ctgggatccg caggtttgcc tgtgtcatgg 1800

atgcagcctc cagaatactt actggaaact attgtaaccc gcctgaagtt aaaaagaaca 1860

acgcccggca gtgccaggcg ttgaaaagat tagcgaccgg agattggcgg gacgaatacg 1920

acgcccatat cccacggctg ttcaatccag gtatcttgcg ggatatcaac aacatagtca 1980

tcaaccagcg gacgaccagc cggttttgcg aagatggtga caaagtgcgc ttttggatac 2040

atttcacgaa tcgcaaccgc agtaccaccg gtatccacca ggtcatcaat aacgatgaag 2100

ccttcgccat cgccttctgc gcgtttcagc actttaagct cgcgctggtt gtcgtgatcg 2160

tagctggaaa tacaaacggt atcgacatga cgaataccca gttcacgcgc cagtaacgca 2220

cccggtacca gaccgccacg gcttacggca ataatgcctt tccattgttc agaaggcatc 2280

agtcggcttg cgagtttacg tgcatggatc tgcaacatgt cccaggtgac gatgtatttt 2340

tcgctcatag ggccgggatt ctcctccacg tcaccgcatg ttagaagact tcctctgccc 2400

tcgctagcct tgtacagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc 2460

agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctgggtgctc 2520

aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag 2580

tggtcggcga gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg 2640

atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc 2700

agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg 2760

ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc 2820

ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg 2880

tgctgcttca tgtggtcggg gtagcggctg aagcactgca cgccgtaggt cagggtggtc 2940

acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt cagggtcagc 3000

ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg 3060

tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc 3120

accatggtgg cgtgaagtgt cccagcctgt ttatctacgg cttaaaaagt gttcgagggg 3180

aaaataggtt gcgcgagatt atagagatcc ccaattcctc gagttatgat ctacttcctt 3240

accgtgcaat aaattagaat atattttcta cttttacgag aaattaatta ttgtatttat 3300

tatttatggg tgaaaaactt actataaaaa gcgggtgggt ttggaattag tgaaagctta 3360

aaaattgaaa ttttattttt tttttttgga atataaataa gctcgaagtc gacagatcta 3420

ggcctgagct tgatatcgaa ttcctgcagc ccgggggatc cactagttct agactccaca 3480

ataaaaacag atcacctgat ggataaaaag gcggttaacc gcgcagataa aaagagctcc 3540

aattcgccct atagtgagtc gtattacgcg cgcctatcac ggagaaatct gtaattgatt 3600

ccaagacatc acatagttta gttgcttcca atgcttcaaa attattctta tcatgcgtcc 3660

atagtcccgt tccgtatcta ttatcgttag aatattttat agtcacgcat ttatattgag 3720

ctatttgata acgtctaact cgtctaatta attctgtact tttacctgaa aacatggggc 3780

cgattatcaa ctgaatatgt ccgccgttca tgatgacaat aaagaattaa ttattgttca 3840

ctttattcga ctttaatata tccatcacgt tagaaaatgc gatattgcga cgaggatcta 3900

tgtatctaac aggatctatt gcggtggtag ctagagagga ttcttttttg aatcgcatca 3960

aactaatcac aaagtcgaac aaatatcctt tattaagttt gacccttcca tctgtaacaa 4020

tagggacctt gttaaacagt tttttaaaat cttgaaagtc tgtgaatttt gtcaattgtc 4080

tgtattcctc tgaaagagat tcataacaat gacccacggc ttctaattta ttttttgatt 4140

ggatcaataa taataacaga aagtctagct agatattgag tgatttgcaa tatatcagat 4200

aatgaagatt catcatcttg actagccaaa tacttaaaaa atgaatcatc atctgcgaag 4260

aacatcgtta agagatactg gttgtgatcc atttattgat cgcaaaagct ctgaacggtc 4320

tggttatagg tacattgagc aactgactga aatgcctcaa aatgttcttt acgatgccat 4380

tgggatatat caacggtggt atatccagtg atttttttct ccattttagc ttccttagct 4440

cctgaaaatc tcgataactc aaaaaatacg cccggtagtg atcttatttc attatggtga 4500

aagttggaac ctcttacgtg ccgatcaacg tctcattttc gccaaaagtt ggcccagggc 4560

ttcccggtat caacagggac accaggattt atttattctg cgaagtgatc ttccgtcaca 4620

ggtatttatt cgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat 4680

gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 4740

tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 4800

ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 4860

ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 4920

gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 4980

caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 5040

ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact 5100

cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 5160

gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 5220

taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 5280

tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 5340

agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg 5400

caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 5460

ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 5520

tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 5580

agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 5640

tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaa 5695

Claims

1. An oncolytic vaccinia virus comprising: the thymidine kinase region of the genome of the oncolytic vaccinia virus comprises a gene sequence of a mouse/human TIGIT single-chain antibody; and the oncolytic virus can infect tumor cells so that the tumor cells express anti-mouse/human TIGIT single-chain antibodies.

2. The oncolytic vaccinia virus of claim 1, wherein: the nucleotide sequence of the anti-mouse/human TIGIT single-chain antibody is shown in SEQ ID NO. 1.

3. The oncolytic vaccinia virus of claim 1, wherein: the anti-mouse/human TIGIT single-chain antibody is formed by connecting a signal sequence, a tag protein, an antibody heavy chain variable region, a connecting peptide and an antibody light chain variable region in series.

4. The oncolytic vaccinia virus of claim 3, wherein: the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 2.

5. The oncolytic vaccinia virus of claim 3, wherein: the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO. 3.

6. The oncolytic vaccinia virus of claim 1, wherein: the oncolytic vaccinia virus can lyse tumor cells; preferably, the single-chain antibody expressed by the oncolytic vaccinia virus can block the immunosuppressive function of a TIGIT molecule.

7. A method of producing an oncolytic vaccinia virus according to claim 1, comprising: the method comprises the following steps:

(2) EcoR1 and Xba1 enzyme cutting sites are respectively added before and after the TIGIT single-chain antibody gene sequence SEQ ID NO.1, and TIGIT single-chain antibody gene fragments are artificially synthesized;

(3) after the TIGIT single-chain antibody gene fragment is subjected to double enzyme digestion by EcoR1 and Xba1, the TIGIT single-chain antibody gene fragment is subcloned into a thymidine kinase region of a shuttle plasmid pVV-Control to construct pVV-scFv-TIGIT plasmid, wherein the expression of the TIGIT single-chain antibody is controlled by a vaccinia virus early and late promoter pSE/L;

(4) cells that had previously been infected with wild vaccinia virus were transfected with the pVV-scFv-TIGIT plasmid and homologous recombination of the plasmid and wild vaccinia virus occurred to produce oncolytic vaccinia virus VV-scFv-TIGIT.

8. A recombinant plasmid whose genomic DNA comprises the thymidine kinase region of the oncolytic vaccinia virus genome, said thymidine kinase region comprising anti-mouse/human TIGIT single-chain antibody gene sequence.

9. Use of an oncolytic vaccinia virus according to any one of claims 1-7 or recombinant plasmid according to claim 8 for the preparation of a medicament for the treatment of a tumor.

10. The use of claim 9, wherein: the tumor is breast cancer, carcinoma of large intestine, colon cancer, hepatocarcinoma, melanoma, lung cancer, gastric cancer, pancreatic cancer, gallbladder cancer, renal cancer, bladder cancer, prostatic cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, glioma, cancerous ascites or cancerous hydrothorax; preferably, the tumor is breast cancer, colon cancer, melanoma or cancerous ascites.