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Rituximab-specific DNA aptamers are able to selectively recognize heat-treated antibodies

The monoclonal anti-CD20 IgG1 antibody rituximab is used as a first-line treatment for B cell lymphoma. Like all therapeutic antibodies, it is a complex protein for which both safety and efficacy heavily depend on the integrity of its three-dimensional structure. Aptamers, short oligonucleotides wit...

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Autores principales: Kohlberger, Michael, Wildner, Sabrina, Regl, Christof, Huber, Christian G., Gadermaier, Gabriele
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644011/
https://www.ncbi.nlm.nih.gov/pubmed/33151990
http://dx.doi.org/10.1371/journal.pone.0241560
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author Kohlberger, Michael
Wildner, Sabrina
Regl, Christof
Huber, Christian G.
Gadermaier, Gabriele
author_facet Kohlberger, Michael
Wildner, Sabrina
Regl, Christof
Huber, Christian G.
Gadermaier, Gabriele
author_sort Kohlberger, Michael
collection PubMed
description The monoclonal anti-CD20 IgG1 antibody rituximab is used as a first-line treatment for B cell lymphoma. Like all therapeutic antibodies, it is a complex protein for which both safety and efficacy heavily depend on the integrity of its three-dimensional structure. Aptamers, short oligonucleotides with a distinct fold, can be used to detect minor modifications or structural variations of a molecule or protein. To detect antibody molecules in a fold state occurring prior to protein precipitation, we generated DNA aptamers that were selected for extensively heat-treated rituximab. Using the magnetic bead-based systematic evolution of ligands by exponential enrichment (SELEX), we obtained six DNA aptamer sequences (40-mers) specific for 80°C heat-treated rituximab. In silico fold prediction and circular dichroism analysis revealed a G-quadruplex structure for one aptamer, while all others exhibited a B-DNA helix. Binding affinities ranging from 8.8–86.7 nM were determined by an enzyme-linked apta-sorbent assay (ELASA). Aptamers additionally detected structural changes in rituximab treated for 5 min at 70°C, although with lower binding activity. Notably, none of the aptamers recognized rituximab in its native state nor did they detect the antibody after it was exposed to lower temperatures or different physical stressors. Aptamers also reacted with the therapeutic antibody adalimumab incubated at 80°C suggesting similar aptamer binding motifs located on extensively heat-treated IgG1 antibodies. Within this work, we obtained the first aptamer panel, which is specific for an antibody fold state specifically present prior to protein aggregation. This study demonstrates the potential of aptamer selection for specific stress-based protein variants, which has potential impact for quality control of biopharmaceuticals.
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spelling pubmed-76440112020-11-16 Rituximab-specific DNA aptamers are able to selectively recognize heat-treated antibodies Kohlberger, Michael Wildner, Sabrina Regl, Christof Huber, Christian G. Gadermaier, Gabriele PLoS One Research Article The monoclonal anti-CD20 IgG1 antibody rituximab is used as a first-line treatment for B cell lymphoma. Like all therapeutic antibodies, it is a complex protein for which both safety and efficacy heavily depend on the integrity of its three-dimensional structure. Aptamers, short oligonucleotides with a distinct fold, can be used to detect minor modifications or structural variations of a molecule or protein. To detect antibody molecules in a fold state occurring prior to protein precipitation, we generated DNA aptamers that were selected for extensively heat-treated rituximab. Using the magnetic bead-based systematic evolution of ligands by exponential enrichment (SELEX), we obtained six DNA aptamer sequences (40-mers) specific for 80°C heat-treated rituximab. In silico fold prediction and circular dichroism analysis revealed a G-quadruplex structure for one aptamer, while all others exhibited a B-DNA helix. Binding affinities ranging from 8.8–86.7 nM were determined by an enzyme-linked apta-sorbent assay (ELASA). Aptamers additionally detected structural changes in rituximab treated for 5 min at 70°C, although with lower binding activity. Notably, none of the aptamers recognized rituximab in its native state nor did they detect the antibody after it was exposed to lower temperatures or different physical stressors. Aptamers also reacted with the therapeutic antibody adalimumab incubated at 80°C suggesting similar aptamer binding motifs located on extensively heat-treated IgG1 antibodies. Within this work, we obtained the first aptamer panel, which is specific for an antibody fold state specifically present prior to protein aggregation. This study demonstrates the potential of aptamer selection for specific stress-based protein variants, which has potential impact for quality control of biopharmaceuticals. Public Library of Science 2020-11-05 /pmc/articles/PMC7644011/ /pubmed/33151990 http://dx.doi.org/10.1371/journal.pone.0241560 Text en © 2020 Kohlberger et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Kohlberger, Michael
Wildner, Sabrina
Regl, Christof
Huber, Christian G.
Gadermaier, Gabriele
Rituximab-specific DNA aptamers are able to selectively recognize heat-treated antibodies
title Rituximab-specific DNA aptamers are able to selectively recognize heat-treated antibodies
title_full Rituximab-specific DNA aptamers are able to selectively recognize heat-treated antibodies
title_fullStr Rituximab-specific DNA aptamers are able to selectively recognize heat-treated antibodies
title_full_unstemmed Rituximab-specific DNA aptamers are able to selectively recognize heat-treated antibodies
title_short Rituximab-specific DNA aptamers are able to selectively recognize heat-treated antibodies
title_sort rituximab-specific dna aptamers are able to selectively recognize heat-treated antibodies
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644011/
https://www.ncbi.nlm.nih.gov/pubmed/33151990
http://dx.doi.org/10.1371/journal.pone.0241560
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