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Fusion gene RIL-7 recombinant vaccinia virus and application thereof in preparation of antitumor drugs

A RIL-7, anti-tumor drug technology, applied in the field of medicine, can solve the problems of restricting the effect of tumor immunotherapy, blocking the anti-tumor immune circulation, destroying tumor cells, etc. , the effect of avoiding cytokine storm

Pending Publication Date: 2021-12-28
居颂光
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 2. Blockage of all links in the tumor immune cycle is a bottleneck that seriously restricts the effect of tumor immunotherapy
[0008] 3. The concept, curative effect and disadvantages of oncolytic virus
[0014] But at the same time, the single use of vaccinia virus (VV) as an oncolytic virus also has disadvantages: ①VV will eventually be cleared by the body's antiviral immunity in the body; ②The anti-tumor immune response triggered by VV will also be suppressed by the negative immune mechanism in the patient ; ③ The systemic and memory advantages of VV-induced anti-tumor immunity need to be further enhanced
[0021] Oncolytic viruses can selectively infect and destroy tumor cells, and then induce anti-tumor immunity, making them a new field of tumor immunotherapy. The common problem that the anti-tumor immune cycle has been blocked

Method used

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  • Fusion gene RIL-7 recombinant vaccinia virus and application thereof in preparation of antitumor drugs
  • Fusion gene RIL-7 recombinant vaccinia virus and application thereof in preparation of antitumor drugs
  • Fusion gene RIL-7 recombinant vaccinia virus and application thereof in preparation of antitumor drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] 1. Establishment of recombinant oncolytic vaccinia virus VV-RIL-7

[0083] 1) Construction of the fusion gene RIL-7: the gene sequence of the wild-type IL-7 with the termination codon deleted, the EA3K rigid peptide gene sequence [or (G4S)3 flexible linker peptide sequence] and the GPI anchor gene of human CD16b are removed by PCR The sequence was spliced ​​into a new type of fusion gene, named RIL-7 fusion gene, [Note: In the subsequent related expressions, the RIL-7 linked by EA3K is referred to as RIL-7 / EA3K; the RIL-7 linked by (G4S)3 is referred to as RIL-7 / (G4S)3. ].

[0084] 2) Restriction enzyme cutting sites were introduced into the two wings of the fusion gene RIL-7: SdaI and HpaI restriction enzyme cutting sites were introduced into the two wings of the RIL-7 fusion gene by PCR. Enzyme digestion, gel electrophoresis to separate the gene fragments, cut out the fragments of expected size, and use the DNA gel electrophoresis kit to separate the gene fragments ...

Embodiment 2

[0098] Example 2 Test of expression of immune factor RIL-7 in tumor cells infected by recombinant oncolytic virus

[0099] figure 2 MC-38 cells are infected with the RIL-7 fusion gene recombinant oncolytic virus, and the results of detecting the expression of IL-7 in the cell membrane by flow cytometry are shown. figure 2 Shown in is to confirm whether the fusion protein encoded by the RIL-7 gene carried by the recombinant oncolytic virus VV-RIL-7 can be anchored to the cell membrane. After infecting MC-38 cells with VV-RIL-7 recombinant oncolytic virus, the expression of IL-7 in the cell membrane was detected by staining with anti-IL-7 monoclonal antibody and flow cytometry. The results showed that the presence of membrane-anchored IL-7 was detected on the surface of the intestinal cancer cell MC38 cell membrane of the VV-RIL-7 infection group after recombination into the RIL-7 gene, while VV-D (only the VV with the TK gene knocked out), There was no presence of membrane-...

Embodiment 3

[0106] Embodiment 3 biological activity test

[0107] In order to identify whether the expression product of the fusion gene RIL-7 carried by the recombinant vaccinia virus has biological activity, T cells were obtained from spleen cells in this experiment, and divided into a resting T cell group and a PHA-activated T cell group and seeded in 96-well plates respectively. After infecting MC-38 cells with different recombinant vaccinia viruses at MOI=1 for 24 hours, digest and prepare single cell suspension, after washing with PBS, add the resting T cell group and PHA activated T cell group in proportion, and after co-incubating for 48 hours, use the MTT method The OD (optical density) value of each group was detected. The results showed that tumor cells infected with recombinant vaccinia virus carrying the RIL-7 gene could effectively stimulate the proliferation of resting and activated T cells, and this effect could be blocked by an anti-IL-7 neutralizing antibody, further dem...

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Abstract

The invention discloses a fusion gene RIL-7 recombinant vaccinia virus and application thereof in preparation of antitumor drugs. The code name of the fusion gene RIL-7 recombinant vaccinia virus is VV-RIL-7, the recombinant vaccinia virus is obtained by inserting a fusion gene RIL-7 and a gene sequence of a regulatory element behind the 81001bp position of a vaccinia virus female parent genome (the genome sequence takes GenBank:AY243312.1 as the reference), wherein the gene sequence inserted into the vaccinia virus female parent genome is shown as S1 or S1'. By means of the fusion gene RIL-7 recombinant vaccinia virus, while the anti-tumor immune response is promoted, potential adverse reactions such as cytokine storms and the like can be avoided, local IL-7 high-concentration expression of tumor tissues can also be realized, and the stronger local anti-tumor efficiency can be exerted.

Description

technical field [0001] The invention belongs to the field of medicine, and in particular relates to fusion gene RIL-7 recombinant oncolytic vaccinia virus and its application in the preparation of antitumor drugs. Background technique [0002] 1. Immunotherapy is increasingly becoming an important means of tumor treatment [0003] Overall, the current cancer attack rate and mortality rate remain high. Existing tumor treatment methods include surgery, radiotherapy, chemotherapy, targeted therapy, traditional Chinese medicine and other traditional medical treatments, but their efficacy needs to be improved urgently. [0004] During the pathological process of tumors, 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 T cells, the main force of anti-tumor immunity) in the tumor microenvironment are one of the key factors affecting the effect of tu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/863C12N15/65A61K48/00A61K38/20A61K35/768A61P35/00
CPCC12N7/00C07K14/5418C12N15/86C12N15/65C12N9/1211C12Y207/01021A61K48/005A61K38/2046A61K35/768A61P35/00C12N2710/24121C07K2319/035C12N2710/24143C12N2800/107A61K2300/00Y02A50/30
Inventor 居颂光葛彦
Owner 居颂光
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