Production and Characterization of Recombinant Antibody Variants Neutralizing SARS-CoV-2
| Autor: | Iakubovskaia, Alina |
|---|---|
| Přispěvatelé: | Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology, Tampere University |
| Jazyk: | angličtina |
| Rok vydání: | 2022 |
| Předmět: |
Bioteknologian ja biolääketieteen tekniikan maisteriohjelma - Master's Programme in Biotechnology and Biomedical Engineering
coronaviruses SARS-CoV-2 virus antibody fragment DNA tricistronic gene recombinant proteins proteins pseudovirus neutralization assay antigens antibodies viruses single chain fragment variable genes biotechnology |
| Popis: | Coronaviruses are ssRNA viruses, some of which can infect human lung epithelium via binding the ACE2 receptor. The outbreak of the severe acute respiratory syndrome corona-virus-2 (SARS-CoV-2) reported at the end of 2019 has forced the biomedical field to shift the focus to intensive studying of the novel virus, its diagnostics and treatment strategies. This thesis work aimed to produce recombinant IgG, Fab, scFv and scFv-Fc antibodies against receptor binding site (RBD) of SARS-CoV-2 and to perform their characterization regarding their specificity and overall functionality. The antibody constructs are based on two antibodies (REGN10933 and REGN10987) developed by Regeneron Pharmaceuticals, which have demonstrated high efficiency in neutralizing SARS-CoV-2 in animal testing and humans. The approach for recombinant antibody production includes the expression of antibodies and antibody fragments using mammalian expression vectors comprising single chain frag-ment variable (scFv) and IRES-mediated tricistronic genes as well as fusions with mFc and hFc regions of IgG. In particular, this approach allows beneficial outcomes regarding protein yields and stability. The DNA for the antibody constructs was cloned in E.coli and expressed in HEK293 cells. Proteins were purified with affinity chromatography techniques. The quality of the product was assessed with SDS-PAGE gel staining and Western blotting. Obtained results have demonstrated satisfactory purity and yields for most proteins as well as the expected formation of covalent bonds between antibody chains (di-, tetramerization). However, some of the antibody variants’ expression and functionality failed presumably due to the possible flaw in the sequence alignment. Analysis of binding properties of the antibody constructs was performed by pseudovirus neutralization assay against Wuhan, Beta, Delta and Omicron SARS-CoV-2 variants. The assay was carried out on ACE2 overexpressing HEK cells, which allowed to assess specific inhibition of viral entry by neutralizing anti-RBD antibodies and antibody fragments. The results demonstrated that SCF1-mFc and LIH2-mFc appear to be the most efficient and specific recombinant antibody constructs against RBD of certain SARS-CoV-2 variants, able to neutralize pseudovirus in a dose-dependent manner. However, Omicron variant fully resisted the neutralization. The results have also demonstrated that the presence of Fc region significantly improves the stability and neutralizing ability of recombinant antibodies. The production system of tricistronic, scFv and scFv-Fc constructs as well as their func-tionality was mainly successful. Most efficient recombinant antibody constructs will be poten-tially utilized for research and diagnostic purposes, e.g. for immunological assays detecting SARS-CoV-2. |
| Databáze: | OpenAIRE |
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