Oncolytic virus construction body, oncolytic virus and application thereof

A technology of oncolytic virus and construct, applied in the direction of virus/bacteriophage, application, virus, etc.

Inactive Publication Date: 2018-11-02
LIFESEQ LTDRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Based on the above research results, it can be found that oncolytic virus is indeed a powerful tool for treating tumors, b

Method used

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  • Oncolytic virus construction body, oncolytic virus and application thereof
  • Oncolytic virus construction body, oncolytic virus and application thereof
  • Oncolytic virus construction body, oncolytic virus and application thereof

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0046] Embodiment 1 Materials and methods

[0047] Cell line African green monkey kidney fibroblasts (CV-1) were obtained from the American Germplasm Collection. CV-1 cells were cultured in DMEM supplemented with antibiotic-antimycotic solution (100 U / mL penicillin G, 250 ng / mL amphotericin B, 100 units / mL streptomycin) and 10% fetal bovine serum (FBS; Invitrogen Corporation) Base. The culture environment is 37°C, 5% CO 2 . Human 143TK-cells (ATCC) were cultured in basal medium supplemented with 0.015 mg / ml of 5-bromo-2'-deoxyuridine (BUdR), 10% fetal bovine serum and Earle's BSS.

[0048] Human melanoma cell A375, human pancreatic cancer cell Panc1, human brain tumor cell U87, human lung cancer cell H226, and human head and neck cancer cell ATCC TCP-1012 were obtained from ATCC. Normal cell lines, including human normal cells PDF (human primary dermal fibroblasts), MRC5 (normal human lung fibroblasts), IMR-90 (ATCC CCL-186, normal human lung fibroblasts) and BJ (ATCC CRL-...

Embodiment 2

[0055] Example 2 Construction of recombinant vaccinia oncolytic virus (VV-iPD1 / GMCSF) co-expressing human PD1 inhibitor and GM-CSF

[0056] The nucleotide sequence encoding human fusion protein (iPD1) (comprising human PD1 signal peptide sequence, PD1 extracellular region and human IgG Fc fragment) is connected with restriction enzyme cutting sites (Not1 and SalI). After synthesis, it was cloned into the vaccinia virus shuttle vector (pSEL-DsRed) (RFP: red fluorescent protein red fluorescent protein), thereby constructing the pVV-iPD1 shuttle vector (such as figure 1 shown). After the human GM-CSF gene was synthesized with restriction enzyme sites (XhoI and EcoRV), it was cloned into the pVV-iPD1 shuttle vector to construct the shuttle vector pVV-iPD1 / GMCSF. In the obtained shuttle vector pVV-iPD1 / GMCSF, the iPD1 fusion gene is under the control of the pSEL promoter, and the GM-CSF gene is under the regulation of the p7.5 promoter. The human GM-CSF gene can also be cloned in...

Embodiment 3

[0058] Example 3 Construction of recombinant vaccinia oncolytic virus VV-imPD1 / mGMCSF co-expressing mouse PD1 inhibitor and GM-CSF

[0059] Human GM-CSF is not functional in mice. Human PD1-Fc fusion protein also does not efficiently bind mouse PD-L1. In order to detect the anti-tumor immune response mediated by fusion proteins of GM-CSF and PD1-Fc (iPD1), the inventors constructed a series of recombinant TK-VGF-vaccinia viruses to express the corresponding mouse proteins. The mouse fusion protein gene includes mouse PD1 signal peptide gene, mouse PD1 extracellular region gene and mouse IgG2a Fc fragment gene. The mouse fusion protein gene is linked to a restriction enzyme site. After synthesis, it was cloned into the vaccinia virus shuttle vector pSEL-DsRed to construct pVV-imPDL1 / mGMCSF (results such as figure 2 shown). Mouse pVV-imPD1 or pVV-mGMCSF shuttle vectors were also constructed. Next, recombinant double-deleted VGF expressing mPD1-Fc(imPD1) fusion protein gene...

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Abstract

The invention provides an oncolytic virus construction body, an oncolytic virus and application thereof. The oncolytic virus construction body comprises a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule encodes a secreted fusion protein molecule; the secreted fusion protein molecule is used for inhibiting immune checkpoints; and the second nucleic acid molecule encodes an immunostimulant molecule. According to the embodiment of the invention, the construction body encodes fusion protein and immunostimulant molecules for inhibiting the immune checkpoints and can inhibit immunosuppression mechanism mediated by the immune checkpoints and immunoreactions which causes individual tumor specificity so as to realize generalized and effectivetumor cell killing.

Description

technical field [0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to oncolytic virus constructs, oncolytic viruses, recombinant cells, and pharmaceutical compositions. Further, the present invention relates to oncolytic virus constructs, oncolytic viruses, and recombinant cells. The use in the preparation of medicaments, the medicaments are used for treating or preventing cancer and inhibiting the proliferation of malignant cells in mammals. Background technique [0002] Oncolytic virus refers to a class of viruses that have the ability to selectively replicate and package in tumor cells to achieve selective killing of tumor cells. At present, most studies have transformed some naturally occurring viruses with weak pathogenicity, so that they can be specifically expressed and packaged in tumor cells, and then achieve oncolysis. There are two main principles for oncolytic viruses to selectively kill tumor cells: fir...

Claims

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Application Information

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IPC IPC(8): C12N15/863C12N7/01C12N5/10A61P35/00
CPCC12N15/86A61K35/768C07K14/535C07K14/70521C07K14/70532C07K2319/30C12N7/00C12N2710/24121C12N2710/24143
Inventor 黄雪芬陈思毅
Owner LIFESEQ LTDRP
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