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Expression vector of anti-sars-cov-2 neutralizing antibody

A technology for SARS-COV-2 and expression vectors, which is applied in the direction of vectors, antibodies, nucleic acid vectors, etc., and can solve problems such as inability to produce on a large scale and treatment limitations of convalescent plasma

Active Publication Date: 2022-04-15
NEWSOARA BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, convalescent plasma therapy is limited because plasma is donated by people who have recovered from COVID-19 and cannot be mass-produced

Method used

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  • Expression vector of anti-sars-cov-2 neutralizing antibody
  • Expression vector of anti-sars-cov-2 neutralizing antibody
  • Expression vector of anti-sars-cov-2 neutralizing antibody

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0279] Example 1: Anti-SARS-CoV-2 neutralizing antibodies

[0280] Materials and methods

[0281] human sample

[0282] Peripheral blood mononuclear cells (PBMC) from healthy control donors were collected from the Center Hospitalier Universitaire of Université Laval (Centre Hospitalier Universitaire of Université Laval; CHU-Université Laval), while those from COVID-19 survivors PBMCs were obtained from SunnyBrook Hospital, Toronto. Ethical approval from ethics committees was obtained from both institutions prior to sample collection, and all participants signed individual informed consent.

[0283] fluorescent cell sorting

[0284] SARS-CoV-2 virus-like particles ( VLP) (Medicago, Quebec, Canada) was biotinylated.

[0285] PBMCs from COVID-19 survivors were thawed and allowed to stand for 30 min before staining with 1 μg of biotinylated SARS-CoV-2 VLPs. Samples were then stained with viability dye (fixable viability dye eFluor, Thermo Fisher), A488-conjugated strepta...

example 2

[0294] Example 2: Characterization of Antibodies

[0295] After screening all obtained antibodies by a SARS-CoV-2 specific binding assay (data not shown), the three best antibodies (i.e., 37G2, 31C2 and 15A7) were obtained, which were among the highest among the numerous antibodies we screened. showed the best binding affinity. figure 1 The SARS-CoV-2 specific binding activities of the three antibodies are shown in . The CDR regions of the antibodies were sequenced and listed in Table 1. As far as the applicant knows, the SARS-CoV-2 antibody containing the CDRs listed in Table 1 has not been reported before. Therefore, this application provides a new SARS-CoV-2 antibody to fight against the SARS-CoV-2 epidemic. Provides more options.

[0296] These 3 antibodies were recombinantly expressed and in vitro neutralization assays were performed in Vero-E6 cells using live virus. Such as figure 2 As shown in , all antibodies exhibited significant neutralizing capacity against S...

example 3

[0298] Example 3: AAV-mediated antibody gene delivery

[0299] The experiment was designed to test the effect of AAV-mediated antibody gene transfer in preventing SARS-CoV-2 viral challenge in mice.

[0300] AAV-mAb vector construction

[0301] Preparation of antibody expression cassette: Monoclonal antibody expression cassette was synthesized by Genscript and contained signal peptide, variable heavy chain domain, human IgG1 constant domain, followed by 2A self-cleavage peptide sequence, signal peptide, variable light chain domain and the human lambda constant domain. Antibody heavy chain and light chain nucleic acid sequences were codon optimized by GenScript. Each antibody expression cassette was amplified by PCR using Taq polymerase, and the PCR products were gel purified, and the band of interest was excised and purified using a gel extraction kit.

[0302] AAV vector preparation: The pAVA-00200 plasmid backbone contains the CASI promoter (Alejandro B. Balazs et al., ...

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Abstract

The present disclosure provides recombinant expression vectors expressing novel neutralizing antibodies or antigen-binding fragments thereof against SARS-COV-2. Pharmaceutical compositions and kits comprising the same and uses thereof are also provided.

Description

technical field [0001] The present disclosure generally relates to expression vectors expressing anti-SARS-COV-2 neutralizing antibodies. Background technique [0002] The global pandemic of coronavirus disease 2019 (COVID-19) caused by the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has severely deteriorated public health and economic . While most of the infected population shows mild symptoms, some progress to acute respiratory distress syndrome and more than 2,000,000 deaths have been reported. [0003] SARS-CoV-2 infection of host cells is mediated by a glycoprotein expressed on the viral envelope called the spike (S) glycoprotein, which contains the S1 subunit and the S2 subunit. The S1 subunit contains a receptor-binding domain (RBD) that directly binds to the human angiotensin converting enzyme 2 (ACE2) receptor on the host cell, while the S2 subunit mediates viral The envelope fuses with the host cell membrane to facilitate vir...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/864C07K16/10C12N5/10A61K39/42A61K45/06A61P31/14
CPCC12N15/86C07K16/10A61K39/42A61K45/06A61P31/14C07K2317/565C07K2317/56C07K2317/52C07K2317/92C07K2317/41C12N2750/14143C12N2800/107C12N2830/50C12N2830/48A61K2039/505A61K2039/5256A61K2039/53A61K2039/5156A61K2039/523
Inventor 李元念王文义
Owner NEWSOARA BIOPHARMA CO LTD
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