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Fusion gene RIL-7 combined CCL19 recombinant oncolytic vaccinia virus and application thereof in preparation of antitumor drugs

A VV-RIL-7-CCL19, RIL-7 technology, applied in the field of medicine, can solve the problems of cytokine side effects, blockage of anti-tumor immune circulation, limited direct promotion of anti-tumor immune cells, etc., to avoid cytokines Storm, the effect of promoting anti-tumor immune response

Pending Publication Date: 2021-12-24
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
[0013] 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
[0023] However, there are inevitably some defects in the above applications. For example, the cytokine IL-7 can enhance the anti-tumor immune effect, but it also has the potential risk of its toxic side effects as a cytokine.
The chemokine CCL19 plays a role in promoting the migration of immune cells to tumor tissue, but its direct promotion effect on anti-tumor immune cell function is limited
In addition, the anti-tumor immunity mediated by IL-7 and CCL19 is easily suppressed by negative immunity in patients, which is the bottleneck restricting the efficacy of their tumor immunotherapy
[0024] 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 combined CCL19 recombinant oncolytic vaccinia virus and application thereof in preparation of antitumor drugs
  • Fusion gene RIL-7 combined CCL19 recombinant oncolytic vaccinia virus and application thereof in preparation of antitumor drugs
  • Fusion gene RIL-7 combined CCL19 recombinant oncolytic vaccinia virus and application thereof in preparation of antitumor drugs

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] 1. Establishment of recombinant oncolytic vaccinia virus VV-RIL-7

[0100] 1) Construction of the fusion gene of IL-7 and the cell membrane anchor sequence: the gene sequence of the wild-type IL-7 with the stop codon deleted, the EA3K rigid peptide gene sequence [or (G4S)3 flexible linker peptide sequence] and human CD16b by PCR The GPI-anchored gene sequence was spliced ​​into a new type of fusion gene, which was named RIL-7.

[0101] 2) Restriction enzyme cutting sites are introduced into the two wings of the fusion gene RIL-7: SdaI and HpaI restriction enzyme cutting sites are introduced into the two wings of the fusion gene RIL-7 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 for later use.

[0102] 3) Preparation of the cohesive end of the VV homologous recombination vector: Use SdaI and HpaI to cut the VV homologous recombi...

Embodiment 2

[0128] Example 2 Test of recombinant oncolytic virus infection of tumor cells expressing immune factors RIL-7 and CCL19

[0129] figure 2 It is the result of detecting the expression of IL-7 in the cell membrane by flow cytometry after the fusion gene RIL-7 recombinant oncolytic virus infected MC-38 cells. figure 2 Shown in is to confirm whether the fusion protein encoded by the RIL-7 gene carried by the recombinant oncolytic viruses VV-RIL-7 and VV-RIL-7-CCL19 can be anchored to the cell membrane. After infecting MC-38 cells with VV-RIL-7 and VV-RIL-7-CCL19 recombinant oncolytic viruses, the expression of IL-7 in the cell membrane was detected by anti-IL-7 monoclonal antibody staining and flow cytometry. The results showed that the presence of membrane-anchored IL-7 was detected on the surface of intestinal cancer cell MC38 cell membranes in the VV-RIL-7 and VV-RIL-7-CCL19 infection groups after recombination into the RIL-7 gene, while VV-D (knockout VV except the TK gene...

Embodiment 3

[0138] Embodiment 3 biological activity test

[0139] 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 the recombinant vaccinia virus carrying the fusion gene RIL-7 could effectively stimulate the proliferation of resting and activated T cells, and this effect could be blocked by anti-IL-7 neutralizing antibody, whi...

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Abstract

The invention discloses a fusion gene RIL-7 combined CCL19 recombinant oncolytic vaccinia virus and application thereof in preparation of antitumor drugs. The fusion gene RIL-7 combined CCL19 recombinant oncolytic vaccinia virus VV-RIL-7-CCL19 is a gene sequence of inserting a fusion gene RIL-7, a chemotactic factor CCL19 and a regulatory element behind the 81001bp position of a vaccinia virus female parent genome (the genome sequence is based on GenBank: AY243312.1), wherein the gene sequence inserted into the vaccinia virus female parent genome is shown as S1 or S1'. The fusion gene RIL-7 combined CCL19 recombinant oncolytic vaccinia virus can avoid potential adverse reactions such as cytokine storm while promoting antitumor immune response, and can promote chemotaxis of T cells and other immune cells to tumor microenvironment, thus further enhancing the antitumor effect of the recombinant oncolytic virus.

Description

technical field [0001] The invention belongs to the field of medicine, and in particular relates to fusion gene RIL-7 combined with CCL19 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 affect...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/85C12N15/66C12N15/65C12N15/62C12N15/19A61K38/19A61K38/20A61K35/768A61K48/00A61P35/00C12R1/93
CPCC12N7/00C12N15/85C12N15/65C07K14/5418C07K14/523A61K38/195A61K38/2046A61K35/768A61K48/0008A61K48/005A61P35/00C12N2710/24121C12N2710/24152C12N2710/24132C12N2800/107C07K2319/035Y02A50/30
Inventor 居颂光葛彦
Owner 居颂光
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