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Recombinant oncolytic vaccinia virus, preparation method and application thereof

A vaccinia virus and oncolytic technology, applied in the fields of biomedicine and genetic engineering, can solve the problem of lack of oncolytic virus and achieve the effect of enhancing safety

Pending Publication Date: 2020-10-13
NANJING VIROTHER BIOPHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, oncolytic viruses modified with TIGIT inhibitors for antitumor drug development are still lacking

Method used

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  • Recombinant oncolytic vaccinia virus, preparation method and application thereof
  • Recombinant oncolytic vaccinia virus, preparation method and application thereof
  • Recombinant oncolytic vaccinia virus, preparation method and application thereof

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Effect test

Embodiment 1

[0042] A recombinant oncolytic vaccinia virus of the present invention, the thymidine kinase TK region of the genome of the recombinant oncolytic vaccinia virus contains an anti-mouse / human TIGIT antibody gene sequence, and the thymidine kinase TK region of the genome of the recombinant oncolytic vaccinia virus Glycoside kinase TK domain can express anti-mouse / human TIGIT antibody.

[0043] The anti-mouse / human TIGIT antibody gene sequence is composed of antibody heavy chain gene, 2A peptide and antibody light chain gene in series, and the nucleotide sequence of the anti-mouse / human TIGIT antibody gene is as shown in SEQ ID NO.1 shown.

[0044] The TIGIT antibody heavy chain gene comprises the TIGIT antibody heavy chain variable region gene, and the amino acid sequence of the protein encoded by the TIGIT antibody heavy chain variable region gene is shown in SEQ ID NO.2.

[0045] The TIGIT antibody light chain gene comprises the TIGIT antibody light chain variable region gene,...

Embodiment 2

[0055] Construction of Shuttle Plasmid pVV-TIGIT

[0056] First, the full-length pVV-Control plasmid was synthesized by artificial synthesis, its gene sequence is shown in SEQ ID NO.4, and its plasmid map is shown in figure 1 shown.

[0057] Similarly, an anti-mouse / human TIGIT antibody gene carrying a luciferase reporter gene was synthesized by artificial synthesis, and the sequence is shown in SEQ ID NO.5. The antibody sequence uses secreted luciferase (Lucia)-antibody heavy chain variable region-antibody heavy chain constant region-E2A peptide-human IL-2 signal peptide-antibody light chain variable region-antibody light chain constant region in series made. The secreted luciferase Lucia gene fragment refers to the Lucia sequence in the plasmid pFUSE-Lucia-CHIg-mG2a (InvivoGen). The heavy chain and light chain variable region sequences of the hamster anti-mouse / human TIGIT antibody refer to the sequence of SEQ ID NO.21 and 22 in the patent US20090258013A1, respectively. A...

Embodiment 3

[0067] Expression and secretion of TIGIT antibody by recombinant oncolytic vaccinia virus and functional assay of the antibody.

[0068] Since the antibody protein encoded by the virus carries the luciferase reporter group Lucia, the antibody can be quantified by detecting the luciferase activity. Routinely culture HEK293 cells with DMED high-glucose medium containing 10% FBS, digest the cells with 0.5% trypsin, count, inoculate 5×105 cells / well in a 6-well plate, and place in a 37°C, 5% CO2 incubator cultivated in. When the cells grew to more than 90% confluence, they were infected with recombinant oncolytic vaccinia virus (MOI=1). Collect the cell culture supernatant after 24 hours, take 100 μl of the cell culture supernatant and add 10 μl of QUANTI-Luc TM Substrate (InvivoGen Company), the luciferase activity was detected with a microplate reader. Such as Image 6 As shown, HEK293 cells infected with recombinant oncolytic vaccinia VV-TIGIT can express TIGIT antibody, an...

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Abstract

The invention discloses a recombinant oncolytic vaccinia virus, a preparation method and application thereof. A thymidine kinase TK region of a genome of a recombinant oncolytic vaccinia virus comprises an anti-mouse / human TIGIT antibody genetic sequence, and the thymidine kinase TK region of the genome of the recombinant oncolytic vaccinia virus can express an anti-mouse / human TIGIT antibody. Theanti-mouse / human TIGIT antibody genetic sequence is formed by connecting an antibody heavy chain gene, 2A peptide and an antibody light chain gene in series, and the nucleotide sequence of the anti-mouse / human TIGIT antibody gene is shown as SEQ ID NO. 1. The recombinant oncolytic vaccinia virus oncolytic virus kills tumors through direct oncolysis and activation of a human immune system. The oncolytic vaccinia virus can effectively activate the immune response of T cells to tumor cells so as to exert multiple anti-tumor effects.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and biomedicine, and in particular relates to a recombinant oncolytic vaccinia virus containing anti-mouse / human TIGIT full antibody gene sequence and expressing TIGIT antibody, as well as its preparation method and application. Background technique [0002] Cancer is one of the important diseases that seriously affect human health and threaten human life. Current cancer treatment methods mainly include surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy and traditional Chinese medicine treatment. Since some cancer patients are already in the middle and advanced stage when they are diagnosed, they lose the chance of surgery, so surgery is only suitable for some tumor patients. Although radiotherapy and chemotherapy can radically cure sensitive tumors, most tumors will be resistant to radiotherapy and chemotherapy, and radiotherapy and chemotherapy can seriously damag...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/863C12N15/90A61K35/768A61K39/395A61P35/00A61P7/10
CPCC12N7/00C12N15/86C12N15/907C07K16/2803A61K35/768A61K39/39558A61P35/00A61P7/10C12N2710/24021C12N2710/24043A61K2300/00
Inventor 魏继武陈安仙左曙光魏敏王世兵董杰
Owner NANJING VIROTHER BIOPHARMACEUTICAL CO LTD
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