Recombinant oncolytic adenovirus and application thereof

An oncolytic adenovirus and adenovirus technology, applied in the field of biomedicine, can solve the problems of limited effect, high toxicity, short half-life, etc., and achieve the effect of enhancing targeting, strengthening killing effect, and avoiding adverse reactions

Pending Publication Date: 2022-01-25
SHANDONG UNIV QILU HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, BiTE has a short serum half-life, requires long-term continuous intravenous administration, and has high toxicity
In addition, the application of BiTEs in solid tumors has limited efficacy due to penetration issues in the tumor microenvironment and dose-limiting toxicity due to off-target effects.

Method used

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  • Recombinant oncolytic adenovirus and application thereof
  • Recombinant oncolytic adenovirus and application thereof
  • Recombinant oncolytic adenovirus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Construction of MUC16-BiTE

[0046] DNA sequences encoding two single-chain antibody fragments (scFvs) that recognize human MUC16 and CD3ε were combined with those encoding G 4 The DNA sequences of the S-linker were ligated to generate a MUC16-targeting BiTE (MUC16-BiTE), in which the anti-MUC16 scFv was directed against the human MUC16 cytoplasmic retention domain (MUC-CD). A human IgK signal peptide for mammalian secretion is added at the N-terminus and a 6×His tag for detection is added at the C-terminus.

[0047] The general structural formula of MUC16-BiTE is:

[0048] Signal peptide-VL(MUC16)-Linker1-VH(MUC16)-Linker2-VH(CD3)-Linker3-VL(CD3)-His.

[0049] The gene CDS region sequence and amino acid sequence of MUC16-BiTE are shown below, wherein, in the following amino acid or nucleotide sequences, the signal peptide sequence is indicated by a dotted line underline, the VL (MUC16) sequence is indicated by a single straight line underline, and the VH ...

Embodiment 2

[0055] Example 2 Construction of recombinant adenovirus OAd-MUC16-BiTE

[0056] 1. Adenovirus Packaging

[0057] 1.1 Construction of plasmid

[0058] Synthesis The above DNA sequence encoding MUC16-BiTE was inserted into the shuttle plasmid using Gibson assembly technique. Correct plasmid construction was confirmed by DNA sequencing.

[0059] 1.2 Plasmid transfection process

[0060] 1) 24 hours before transfection, HEK293 cells in the logarithmic growth phase were digested with 0.25% trypsin, the cell density was adjusted to 30%-40% with DMEM medium containing 10% FBS, and re-seeded in cell culture flasks, 37 °C, 5% CO 2 Cultured in an incubator. After about 24 hours, when the cell density reaches 50%-60%, it can be used for transfection. Cell state is critical for virus packaging, so good cell state and low passage times need to be guaranteed.

[0061] 2) 2 hours before transfection, the cell culture medium was replaced with serum-free DMEM medium.

[0062] 3) Add ...

Embodiment 3

[0096] Example 3 Detection of OAd-MUC16-BiTE expression of MUC16-BiTE

[0097] Infect human ovarian cancer cells HEY with MOI=100OAd (MOI is the multiplicity of infection) and OAd-MUC16-BiTE, respectively, extract HEY empty cells and virus-infected HEY cell proteins after 24 hours, and use anti-His antibody for western blot electrophoresis detection :

[0098] Primary antibody: Purified anti-His Tag Antibody (biolegend #362601) with a dilution ratio of 1:1000; the molecular weight of the target protein is about 58kDa.

[0099] Primary antibody: Anti-Beta-Actin antibody (Proteintech 66009-1-Ig) dilution ratio 1:2000; β-actin molecular weight is 42kDa.

[0100] Secondary antibody: Goat Anti-Rabbit IgG H&L (HRP) (abcam ab6721) dilution ratio 1:20000.

[0101] Result analysis: the result is as follows image 3 As shown, it can be seen from the results that HEY cells infected with adenovirus OAd-MUC16-BiTE have an obvious target band at the 58kDa position, while control HEY em...

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Abstract

The invention provides a recombinant oncolytic adenovirus and application thereof, the recombinant oncolytic adenovirus is constructed based on a type-5 adenovirus, the genome of the recombinant oncolytic adenovirus comprises a nucleotide sequence for coding a BiTE antibody or an antibody fragment, and the nucleotide sequence for coding the BiTE antibody or the antibody fragment is located between a virus element mCMV and a virus element MCS-3Flag. According to the recombinant oncolytic adenovirus, the targeting of T cells to tumor cells is improved, adverse reactions caused by systemic administration of BiTE are avoided, and the killing effect of the oncolytic adenovirus on solid tumors is improved through the activation effect of BiTE on an immune microenvironment.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a recombinant oncolytic adenovirus and its application. Background technique [0002] The information disclosed in the Background of the Invention is intended to enhance the understanding of the general background of the invention, and the disclosure should not necessarily be construed as an acknowledgment or any form of suggestion that the information has become the prior art that is already known to those skilled in the art. [0003] Ovarian cancer is the most lethal female reproductive system tumor. Thanks to cutting-edge research, advances in screening, diagnosis, surgery, and other treatments, survival rates for many cancers have improved dramatically, but survival rates for ovarian cancer have changed little over the decades. The standard treatment for ovarian cancer is surgery and platinum-paclitaxel combined chemotherapy. Although some progress has been made, the vas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01A61K35/761A61K39/395A61P35/00A61P15/08
CPCC12N7/00C07K16/2809C07K16/3092A61K35/761A61K39/3955A61P35/00A61P15/08C12N2710/10321C12N2710/10332C07K2317/31C07K2317/73C07K2317/622C07K2319/02C07K2319/21A61K2039/5256A61K2039/53A61K2300/00
Inventor 孔北华王秋曼苑存忠吴焕宋坤张青
Owner SHANDONG UNIV QILU HOSPITAL
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