CN111150748A

CN111150748A - Application of recombinant oncolytic virus in preparation of medicine for treating digestive tract cancer

Info

Publication number: CN111150748A
Application number: CN201911373616.2A
Authority: CN
Inventors: 钱文斌; 王世兵; 王俊杰; 黄筛
Original assignee: Hangzhou Ronggu Biotechnology Co Ltd
Current assignee: Hangzhou Ronggu Biotechnology Co Ltd
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2020-05-15

Abstract

The invention provides an application of a recombinant oncolytic vaccinia virus in preparation of a medicine for treating digestive tract cancer, belongs to gene engineering and oncology, and relates to an application of the recombinant oncolytic vaccinia virus carrying human IL-33 in treatment of cancer. The invention effectively combines gene therapy of malignant tumor with virus therapy to prepare the oncolytic vaccinia virus capable of efficiently expressing IL-33, and compared with simple gene therapy or virus therapy, the invention enhances the killing capacity to tumor. The invention adopts a tumor targeted therapy strategy, can effectively target tumor cells and specifically proliferate in the tumor cells. Thereby greatly enhancing the safety of oncolytic vaccinia virus vectors. The invention adopts a virus replication related gene deletion mode to ensure the intratumoral specific replication of the virus and greatly enhance the safety of the oncolytic vaccinia virus vector. The invention develops better effects of resisting gastric cancer, colon cancer and pancreatic cancer by constructing the recombinant oncolytic vaccinia virus carrying human IL-33.

Description

Application of recombinant oncolytic virus in preparation of medicine for treating digestive tract cancer

Technical Field

The invention belongs to gene engineering and oncology, and relates to application of a recombinant oncolytic vaccinia virus carrying an IL-33 encoding gene in preparation of a pharmaceutical composition, a medicament or a kit for treating gastric cancer, colon cancer and pancreatic cancer.

Background

According to the data of '2018 global cancer statistics', 1800 ten thousand new cancer patients are shown in the world. Among them, the patients with new gastric cancer, colon cancer and pancreatic cancer are 6.1%, 5.7% and 2.5% respectively, and the mortality rates are 5.8%, 8.2% and 4.5% respectively. The incidence and mortality of the disease are important characteristics of the digestive tract cancer, and the development of a novel safe and effective treatment method for improving the life cycle of digestive tract cancer patients has very important significance.

Oncolytic virus immunotherapy (oncolyticic Virotherapy) is a new approach to tumor therapy that has developed very rapidly in recent years and has achieved some encouraging results, and many products are undergoing phase I, phase II and phase III clinical studies both domestically and internationally. Among them, recombinant human GM-CSF oncolytic adenovirus (T-VEC) of American Advance corporation was excellent in clinical trials, and has been approved by the United states Food and Drug Administration (FDA) to be marketed in 2015 for local treatment of unresectable skin, subcutaneous and lymph node lesions in melanoma patients who recur after the first surgery. Recombinant human p53 adenovirus (now born) in China obtains new drug certificates issued by the State food and drug administration and is clinically used for treating patients with solid tumors. These results indicate that oncolytic viruses are a very promising new approach to cancer treatment. Oncolytic vaccinia virus (oncolyticic Poxvirus) is a novel vector for Oncolytic virus immunotherapy and has the advantages that: the virus has good stability, low pathogenicity, high gene transfection efficiency and good safety. Oncolytic vaccinia virus selectively infects tumor cells and replicates and kills tumor cells, while it has little infectivity on normal tissues and cells and thus little toxicity.

Human Interleukin-33 (Interleukin-33, IL-33) and IL-1 α, IL-1 β and IL-18 are members of the IL-1 cytokine family that initiate an inflammatory response after local stress IL-33 signals through the heterodimeric ST2 receptor encoded by the IL1rl1 gene, IL1rl1 gene is constitutively expressed on the surface of some innate immune cells including mast cells, type 2 innate lymphocytes (ILC2) and regulatory T cells (tregs). Akimoto et al report that in colorectal cancer, elevation of the soluble ST2 receptor in the tumor microenvironment can increase capture of soluble IL-33, thereby inhibiting growth and metastasis of the tumor and thus can be correlated well with the prognosis of colorectal cancer.

Disclosure of Invention

The invention aims to provide application of recombinant oncolytic vaccinia virus in preparing a medicament for treating digestive tract cancer aiming at weak effect of a method for treating cancer in the prior art, and particularly aims to prepare a medicament of recombinant oncolytic vaccinia virus (OVV-IL-33) carrying human interleukin 33(IL-33) and apply the medicament to a subject.

In one aspect, the invention provides a use of a recombinant oncolytic vaccinia virus in the preparation of a medicament for treating cancer, wherein the recombinant oncolytic vaccinia virus OVV-IL-33 has an exogenous gene encoding human interleukin 33.

Further, the recombinant oncolytic vaccinia virus carrying an exogenous gene encoding human interleukin 33 is realized by the following method: inserting an exogenous gene encoding human IL-33 into said oncolytic vaccinia virus; the exogenous gene for coding the human IL-33 is a polynucleotide sequence which is hybridized with the nucleotide sequence of the human IL-33 under strict conditions and codes the human IL-33 with the activity of inhibiting the proliferation of digestive tract cancer cells, a polynucleotide sequence with 60 percent of identity or more of the exogenous gene or a complementary sequence thereof;

furthermore, the sequence of the exogenous gene for coding the human IL-33 is a sequence shown in SEQ ID NO. 1.

Further, the recombinant oncolytic vaccinia virus of the invention may be further modified to carry a protein active ingredient for expression in the treatment of digestive tract cancer. Illustratively, the active ingredient of the protein for expression in the present invention for the treatment of cancer is p 53.

Further, the medicament also comprises a pharmaceutically acceptable carrier and other active ingredients for treating cancer. Illustratively, the other active ingredient is paclitaxel. Pharmaceutically acceptable carriers include excipients and adjuvants that facilitate processing of the recombinant oncolytic vaccinia virus into formulations.

Further, the medicament is in the form of a solid or an injection. Preferably, the medicament is packaged in a kit; more preferably, the kit further comprises instructions for use of the pharmaceutical composition.

Further, the digestive tract cancer includes stomach cancer, colon cancer and pancreatic cancer.

The invention has the beneficial effects that:

1. the invention effectively combines gene therapy of malignant tumor with virus therapy to prepare the oncolytic vaccinia virus capable of efficiently expressing IL-33, and compared with simple gene therapy or virus therapy, the invention enhances the killing capacity to tumor.

2. The invention adopts a tumor targeted therapy strategy, can effectively target tumor cells and specifically proliferate in the tumor cells. Thereby greatly enhancing the safety of oncolytic vaccinia virus vectors.

3. The invention adopts the mode of virus replication related gene deletion, ensures the specific replication of the virus in the tumor, and greatly enhances the safety of the oncolytic vaccinia virus vector.

4. The invention develops better effects of resisting gastric cancer, colon cancer and pancreatic cancer by constructing the recombinant oncolytic vaccinia virus carrying human IL-33.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a graph showing the level of expression of GFP in recombinant oncolytic vaccinia virus carrying GFP infected HGC-27, SW-480 and ASPC-1 cells using a microplate reader.

FIG. 2 shows the expression level of IL-33 in HGC-27, SW-480 and ASPC-1 cells infected with recombinant vaccinia virus carrying IL-33 by RT-PCR.

FIGS. 3-1 and 3-2 are graphs of MTT assay for cell viability, data expressed as percentage of dose gradient relative to PBS group.

FIG. 4 is a graph of OVV and OVV-IL-33 versus time for tumor size in HGC-27, SW-480, and ASPC-1 cell tumor-bearing mice.

Detailed Description

The invention provides an application of recombinant oncolytic vaccinia virus OVV-IL-33 in preparing a medicine for treating digestive tract cancer, wherein the recombinant oncolytic vaccinia virus carries an exogenous gene for coding human interleukin 33. Obtaining a recombinant oncolytic vaccinia virus carrying an exogenous gene encoding human interleukin 33 by inserting an exogenous gene encoding human IL-33 into the oncolytic vaccinia virus;

specifically, the coding sequence of human IL-33 of the present invention is any DNA sequence capable of encoding human IL-33, preferably, the sequence is SEQ ID NO.1 or its complementary sequence. On the other hand, the coding sequence of human IL-33 of the invention can be a polynucleotide which hybridizes with the nucleotide sequence of human IL-33 under strict conditions and codes human IL-33 with the activity of inhibiting the proliferation of gastric cancer, colon cancer and pancreatic cancer cells or a complementary sequence thereof;

the "stringent conditions" as used herein may be any of low stringency conditions, medium stringency conditions or high stringency conditions, and preferably high stringency conditions. Illustratively, "low stringency conditions" can be conditions of 30 ℃, 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 52% formamide; "Medium stringency conditions" can be 40 ℃, 5 XSSC, 5 XDenhardt solution, 0.5% SDS, 52% formamide; the "high stringency conditions" may be 50 ℃ in 5 XSSC, 5 XDenhardt's solution, 0.5% SDS, 52% formamide. It will be appreciated by those skilled in the art that higher temperatures will result in polynucleotides with high homology. In addition, one skilled in the art can select the temperature, probe concentration, probe length, ionic strength, time, salt concentration, etc., which affect the stringency of hybridization to form a composite result to achieve the corresponding stringency.

The polynucleotides that can hybridize to each other may have about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1 or more, 99.2 or more, or the like, as calculated by FASTA, BLAST equivalent homology search software using default parameters set by the system, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more, identical polynucleotides.

The identity of nucleotide sequences can be determined using the algorithm rules BLAST of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-. The programs BLASTN, BLASTX based on the rules of the BLAST algorithm have been developed (Altschul SF, et al: JMol Biol215:403,1990). When a nucleotide sequence is analyzed using BLASTN, the parameters are preferably score 100 and wordlength 12; when an amino acid sequence is analyzed using BLASTX, the parameters are preferably score 50 and wordlength 3; when BLAST and Gapped BLAST programs are used, default parameter values can be set for the system using each program.

Optionally, the recombinant viruses of the invention express other proteins known to inhibit proliferation of gastric, colon, and pancreatic cancer cells, such as p 53. The pharmaceutical composition of the present invention may also contain other active ingredients for the treatment of gastric cancer, colon cancer and pancreatic cancer, such as paclitaxel.

The recombinant oncolytic vaccinia virus carrying an IL-33 encoding gene of the present invention as an active ingredient may be used together with a pharmaceutically acceptable carrier. In addition to the active ingredients, the methods, uses and products of the invention may also comprise suitable pharmaceutically acceptable carriers, including excipients and auxiliaries that facilitate processing of the recombinant viruses of the invention into preparations.

For example, formulations suitable for injection or infusion include aqueous and non-aqueous sterile injection solutions, which may optionally contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

The active ingredients of the invention may optionally be combined with solid excipients and, if desired, after addition of suitable auxiliaries, the mixture of granules is processed to give the desired dosage form. Suitable excipients are in particular fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose or starch preparations, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents, such as cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate, may be added.

An effective amount of the active ingredient of the present invention can be any amount that treats gastric, colon, and pancreatic cancers, or relieves the symptoms of or inhibits the cells of the three cancers, which can be about 1x10¹¹-1x10¹²v.p recombinant virus. Determination of an effective amount is within the ability of those skilled in the art, particularly in light of the disclosure provided herein.

According to the present invention, the pharmaceutical product (drug, medicament) or pharmaceutical composition of the present invention may be administered to a subject in any effective number of doses. Preferably, the pharmaceutical product (drug, medicament) or pharmaceutical composition of the invention may be administered in multiple doses, for example from about 2 to about 15 doses, more preferably from about 4-10 doses, most preferably about 6 doses. In a particularly preferred embodiment, the pharmaceutical product (drug, medicament) or pharmaceutical composition of the invention is administered to the subject during the course of administration, e.g. injection, infusion or oral administration, at a frequency of about once every three weeks. In a particularly preferred embodiment, the administration is by injection to the tumor-bearing site.

It will be appreciated that the pharmaceutical product (drug, medicament) or pharmaceutical composition of the invention may be formulated in any suitable manner for administration by any suitable route.

Dosage units of the pharmaceutical products (drugs, medicaments) or pharmaceutical compositions of the invention are based on conventional administration to a subject. For example, a dosage unit may be administered more than once daily, once weekly, once monthly, etc. Dosage units may be administered on a twice/week basis, i.e., twice weekly, e.g., once every three days.

As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional unrecited elements or method steps. The term "comprising" in any of the expressions herein, particularly in describing the method, use or product of the invention, is to be understood as including those products, methods and uses which consist essentially of and consist of the recited components or elements or steps. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

The instructions contained in the pharmaceutical product of the invention relating to the pharmaceutical product may contain the following: indications (e.g., gastric, colon, pancreatic cancer), dosages (e.g., as exemplified above), and possible side effects, among others.

The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

For a clearer explanation of the present invention, reference will now be made in detail to the following examples, which are merely illustrative of the present invention and are not to be construed as limiting the present application; the sources of the cells, the medium and the like used herein are indicated in parentheses after the corresponding cells and medium, but are not limited thereto. The present invention may employ, unless otherwise indicated, conventional techniques in the art.

Example 1, a method of preparing a recombinant oncolytic vaccinia virus, sequentially comprising the steps of:

1) obtaining a DNA sequence (SEQ ID NO.1) for coding the IL-33 by a gene synthesis method;

2) the sequence encoding IL-33 was loaded onto a shuttle plasmid pCB prepared according to the following: construction of a recombinant vaccinia virus vector with double screening markers of Zeocin and GFP, International epidemiology infectious disease journal, No. 39, No. 3 in 6 months of 2012; it should be noted that other pCBs, such as shuttle plasmids pCBs without selectable markers, may also be used in the present invention.

3) Transferring the plasmid pCB-IL-33 and a wild type vaccinia virus (purchased from ATCC company in America) into HEK293 cells, carrying out homologous recombination on the plasmid pCB-IL-33 and the wild type vaccinia virus in the HEK293 cells (a human embryonic kidney cell line purchased from cell banks of Chinese academy of sciences), and inserting a DNA sequence coding the IL-33 into a Thymidine Kinase (TK) gene of the wild type vaccinia virus;

4) and screening the mycophenolic acid drug to obtain the purified recombinant oncolytic vaccinia virus OVV-IL-33.

Example 2: the expression level of GFP in human HGC-27, SW-480 and ASPC-1 cells (human gastric cancer, colon cancer and pancreatic cancer cell lines, respectively) infected by recombinant oncolytic vaccinia virus OVV-IL-33-GFP carrying GFP (green fluorescent protein) is detected by a flow cytometer.

Six-well plate was inoculated with 2X 10 cells⁵Each mL of HGC-27, SW-480, and ASPC-1 cells (purchased from cell banks of Chinese academy of sciences) were added with recombinant oncolytic vaccinia virus carrying GFP (OVV-IL-33-GFP) at 0.1MOI, 0.5MOI, 1MOI, 5MOI, and 10MOI, respectively, and the control group was added with an equal amount of PBS,37℃、5％CO₂and (5) culturing. After 24h, cells were harvested and the samples of each group were analyzed for GFP expression using a flow cytometer (BioRad). The results are shown in FIG. 1, where the expression level of GFP increased with increasing OVV-IL-33 dose, indicating that the recombinant oncolytic vaccinia virus efficiently invaded gastric, colon and pancreatic cancer cells.

Example 3: PCR detects the expression level of human cell factor IL-33 in human HGC-27, SW-480 and ASPC-1 cells infected by recombinant oncolytic vaccinia virus carrying human cell factor IL-33 coding gene.

Recombinant oncolytic vaccinia virus carrying human cytokine IL-33 encoding gene (OVV-IL-33) infected HGC-27, SW-480, ASPC-1 cells at a dose of 4MOI, 37 ℃, 5% CO₂Incubated under conditions for 24 hours. Total cellular RNA was extracted by the TRIZOL method, and reverse-transcribed into cDNA using the extracted total RNA as a template, and primers (SEQ ID NO: 3) were designed using Primer 5.0 software. The cDNA was subjected to fluorescent quantitative PCR by SYBR qPCR Mix in an amplification system of 20. mu.L.

The amplification parameters were: 1min at 95 ℃, 15s at 95 ℃, 1min at 60 ℃ and 40 cycles. The results were analyzed by applied biosystems7300 real-time PCR instrument software. The results are shown in FIG. 2, and the expression level of IL-33 is remarkably increased on mRNA level after HGC-27, SW-480 and ASPC-1 cells are treated by OVV-IL-33, which indicates that OVV-IL-33 can stably express IL-33 in gastric cancer, colon cancer and pancreatic cancer cells.

Example 4: effect of recombinant oncolytic vaccinia virus carrying IL-33 on the proliferative Activity of gastric, colon and pancreatic cancer cells HGC-27, SW-480 and ASPC-1.

HGC-27, SW-480, ASPC-1 cells were cultured in RPMI-1640 containing 10% Fetal Bovine Serum (FBS) or DMEM (purchased from Gibco) (37 ℃, 5% CO), respectively₂Saturated humidity), the fourth generation of cells was taken at 1X10⁴Mu.l of the suspension was inoculated into a 96-well plate at a concentration of 100 ml. An oncolytic vaccinia virus group (OVV) and a recombinant oncolytic vaccinia virus group carrying an IL-33 encoding gene (OVV-IL-33) were set, and 3 replicates of each group were prepared. When the cell growth density reaches 60-70%, OVV (0.1MOI, 0.5MOI, 1MOI, 5MOI, 10MOI) and OVV-IL-33(0.1MOI, 0.5MOI, 1MOI, 5MOI, 10MOI) are respectively added, after culturing for 72 hours, 5mg/ml of tetramethyl azoazolate (MT) is addedT) 20. mu.l of the solution, after further incubation for 4 hours, the incubation was terminated, the culture was centrifuged off, 150. mu.l of DMSO was added to each well, after l0 min of shaking, the absorbance (A value) of each well was determined on a Thermo Varioskan Flash full-automatic enzyme scale at a wavelength of 490nm, and the above experiment was repeated 3 times. And calculating the cell inhibition rate according to the value A, wherein the calculation formula is as follows: the cell inhibition ratio (%) was (negative control group a value-addition group a value)/negative control group a value × 100%.

The results are shown in FIG. 3-1, with increasing doses, OVV and OVV-IL-33 have increasingly significant inhibitory effects on HGC-27, SW-480 and ASPC-1 proliferation of cells of stomach, colon and pancreas cancers, and OVV-IL-33 has a stronger inhibitory effect on HGC-27, SW-480 and ASPC-1 proliferation than OVV.

Example 5: effect of recombinant oncolytic vaccinia virus carrying the gene encoding IL-33 on the proliferative activity of human normal cells.

Human liver Normal cells QSG-7701 (purchased from cell Bank of Chinese academy of sciences) were cultured (37 ℃, 5% CO) in RPMI-1640 containing 10% Fetal Bovine Serum (FBS) (purchased from Gibco)₂Saturated humidity), the fourth generation of cells was taken at 1X10⁴Mu.l of the suspension was inoculated into a 96-well plate at a concentration of 100 ml. A control group, an oncolytic vaccinia virus group (OVV), and a recombinant oncolytic vaccinia virus group carrying a gene encoding IL-33 (OVV-IL-33) were set, and each group was plated in 3 replicates. When the cell growth density reaches 60-70%, OVV (0.1MOI, 0.5MOI, 1MOI, 5MOI, 10MOI) and OVV-IL-33(0.1MOI, 0.5MOI, 1MOI, 5MOI, 10MOI) are respectively added, after the cell is continuously cultured for 72 hours, 20 mu l of 5mg/ml tetramethyl azozolium (MTT) solution is added, after the cell is continuously cultured for 4 hours, the culture is stopped, the culture solution is removed by centrifugation, 150 mu l of DMSO is added into each well, after shaking for l0 min, the absorbance value (A value) of each well is determined by selecting the wavelength of 490nm on a Thermo Varioskan Flash full-automatic enzyme standard instrument, and the experiment is repeated for 3 times. And calculating the cell inhibition rate according to the value A, wherein the calculation formula is as follows: the cell inhibition ratio (%) was (negative control group a value-addition group a value)/negative control group a value × 100%.

The results are shown in FIG. 3-2, and OVV and OVV-IL-33 have no obvious inhibitory effect on human normal liver cells QSG-7701 with increasing dosage.

Example 6: recombinant oncolytic vaccinia viruses carrying IL-33 (OVV-IL-33) and OVV demonstrated tumor sizes in tumor-bearing model animals of gastric, colon and pancreatic cancer cells HGC-27, SW-480 and ASPC-1.

4-6w nude mice were used and inoculated subcutaneously with 5 x10, respectively⁶HGC-27, SW-480, ASPC-1 cells, when the tumor volume grows to 150mm³Left and right, respectively, injection of 1x10 into tumor⁷pfu OVV and OVV-IL-33 and equal volume of PBS, and at 0, 3, 7, 14, 21 days using vernier caliper measurement of tumor size and volume. The volume calculation formula is V ═ length x width²) And/2, wherein Length is the tumor major diameter, and Width is the tumor minor diameter. The results are shown in figure 4, OVV-IL-33 and OVV treatment groups can strongly inhibit tumor growth, and the tumor can not grow any more after OVV-IL-33 treatment, and the tumor growth inhibition effect is more obvious.

Although the present invention has been described in the above-mentioned embodiments, it is to be understood that the present invention may be further modified and changed without departing from the spirit of the present invention, and that such modifications and changes are within the scope of the present invention.

Sequence listing

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agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 2940

gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 3000

aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 3060

ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 3120

gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 3180

taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 3240

tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 3300

tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 3360

gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 3420

gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 3480

ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 3540

cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 3600

tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 3660

cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 3720

agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 3780

cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 3840

aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 3900

aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 3960

gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 4020

gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca 4080

tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggttccgcgcacat 4140

ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata 4200

aaaataggcg tatcacgagg ccctttcgtc ttcgaataaa tacctgtgac ggaagatcac 4260

ttcgcagaat aaataaatcc tggtgtccct gttgataccg ggaagccctg ggccaacttt 4320

tggcgaaaat gagacgttga tcggcacgta agaggttcca actttcacca taatgaaata 4380

agatcactac cgggcgtatt ttttgagtta tcgagatttt caggagctaa ggaagctaaa 4440

atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa 4500

cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt tcagagcttt 4560

tgggatcaat aaatggatca caaccagtat ctcttaacga tgttcttcgc agatgatgat 4620

tcatttttta agtatttggc tagtcaagat gatgaatctt cattatctga tatattgcaa 4680

atcactcaat atgtagctag actttctgtt attattattg atccaatcaa aaaataaatt 4740

agaagccgtg ggtcattgtt atgaatctct ttcagaggaa tacagacaat tgacaaaatt 4800

cacagacttt caagatttta aaaaactgtt taacaaggtc cctattgaca gatggaaggg 4860

tcaaacttaa taaaggatat ttgttcgact ttgtgattag tttgatgcga ttcaaaaaag 4920

aatcctctct agctaccacc gcaatagatc ctgttagata catagatcct cgtcgcaata 4980

tcgcattttc taacgtgatg gatatattaa agtcgaataa agtgaacaat aattaattct 5040

ttattgtcat catgaacggc ggacatattc agttgataat cggccccatg ttttcaggta 5100

aaagtacaga attaattaga cgagttagac gttatcaaat agctcaatat aaatgcgtga 5160

ctataaaata ttctaacgat aatagatacg gaacgggact atggacgcat gataagaata 5220

attttgaagc attggaagca actaaactat gtgatctctt ggaatcaatt acagatttct 5280

ccgtgatagg 5290

<210>3

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

aaccaccaaa aggccttcac 20

<210>4

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

aaaggcaaag cactccacag 20

Claims

1. Use of a recombinant oncolytic vaccinia virus in the manufacture of a medicament for the treatment of a cancer of the digestive tract, wherein the recombinant oncolytic vaccinia virus carries an exogenous gene encoding human interleukin 33.

2. The use according to claim 1, wherein the recombinant oncolytic vaccinia virus harboring an exogenous gene encoding human interleukin 33 is produced by: inserting an exogenous gene encoding human IL-33 into an oncolytic vaccinia virus; the exogenous gene encoding human IL-33 may be a polynucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of human IL-33 and encodes human IL-33 having activity of inhibiting proliferation of digestive tract cancer cells, a polynucleotide sequence that is 60% or more identical thereto, or a complementary sequence thereof.

3. The use according to claim 2, wherein the foreign gene encoding human IL-33 is the sequence shown in SEQ id No. 1.

4. The use according to any one of claims 1 to 3, wherein the recombinant oncolytic vaccinia virus further comprises an active ingredient for expressing a protein for treating a cancer of the digestive tract.

5. The use according to claim 4, wherein the protein active ingredient is p 53.

6. The use according to claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier, an additional active ingredient for the treatment of cancer.

7. The use according to claim 6, wherein the other active ingredient is paclitaxel.

8. The use of claim 6, wherein the pharmaceutically acceptable carrier comprises excipients and adjuvants that facilitate processing of the recombinant oncolytic vaccinia virus into a formulation.

9. The use according to claim 1, wherein the medicament is in the form of a solid or an injection solution.

10. The use according to claim 1, wherein the digestive tract cancer comprises gastric cancer, colon cancer and pancreatic cancer.