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A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display

There is growing interest in the fast and robust engineering of protein pH-sensitivity that aims to reduce binding at acidic pH, compared to neutral pH. Here, we describe a novel strategy for the incorporation of pH-sensitive antigen binding functions into antibody variable domains using combinatori...

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Autores principales: Schröter, Christian, Günther, Ralf, Rhiel, Laura, Becker, Stefan, Toleikis, Lars, Doerner, Achim, Becker, Janine, Schönemann, Andreas, Nasu, Daichi, Neuteboom, Berend, Kolmar, Harald, Hock, Björn
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4622719/
https://www.ncbi.nlm.nih.gov/pubmed/25523975
http://dx.doi.org/10.4161/19420862.2014.985993
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author Schröter, Christian
Günther, Ralf
Rhiel, Laura
Becker, Stefan
Toleikis, Lars
Doerner, Achim
Becker, Janine
Schönemann, Andreas
Nasu, Daichi
Neuteboom, Berend
Kolmar, Harald
Hock, Björn
author_facet Schröter, Christian
Günther, Ralf
Rhiel, Laura
Becker, Stefan
Toleikis, Lars
Doerner, Achim
Becker, Janine
Schönemann, Andreas
Nasu, Daichi
Neuteboom, Berend
Kolmar, Harald
Hock, Björn
author_sort Schröter, Christian
collection PubMed
description There is growing interest in the fast and robust engineering of protein pH-sensitivity that aims to reduce binding at acidic pH, compared to neutral pH. Here, we describe a novel strategy for the incorporation of pH-sensitive antigen binding functions into antibody variable domains using combinatorial histidine scanning libraries and yeast surface display. The strategy allows simultaneous screening for both, high affinity binding at pH 7.4 and pH-sensitivity, and excludes conventional negative selection steps. As proof of concept, we applied this strategy to incorporate pH-dependent antigen binding into the complementary-determining regions of adalimumab. After 3 consecutive rounds of separate heavy and light chain library screening, pH-sensitive variants could be isolated. Heavy and light chain mutations were combined, resulting in 3 full-length antibody variants that revealed sharp, reversible pH-dependent binding profiles. Dissociation rate constants at pH 6.0 increased 230- to 780-fold, while high affinity binding at pH 7.4 in the sub-nanomolar range was retained. Furthermore, binding to huFcRn and thermal stability were not affected by histidine substitutions. Overall, this study emphasizes a generalizable strategy for engineering pH-switch functions potentially applicable to a variety of antibodies and further proteins-based therapeutics.
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spelling pubmed-46227192015-12-18 A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display Schröter, Christian Günther, Ralf Rhiel, Laura Becker, Stefan Toleikis, Lars Doerner, Achim Becker, Janine Schönemann, Andreas Nasu, Daichi Neuteboom, Berend Kolmar, Harald Hock, Björn MAbs Reports There is growing interest in the fast and robust engineering of protein pH-sensitivity that aims to reduce binding at acidic pH, compared to neutral pH. Here, we describe a novel strategy for the incorporation of pH-sensitive antigen binding functions into antibody variable domains using combinatorial histidine scanning libraries and yeast surface display. The strategy allows simultaneous screening for both, high affinity binding at pH 7.4 and pH-sensitivity, and excludes conventional negative selection steps. As proof of concept, we applied this strategy to incorporate pH-dependent antigen binding into the complementary-determining regions of adalimumab. After 3 consecutive rounds of separate heavy and light chain library screening, pH-sensitive variants could be isolated. Heavy and light chain mutations were combined, resulting in 3 full-length antibody variants that revealed sharp, reversible pH-dependent binding profiles. Dissociation rate constants at pH 6.0 increased 230- to 780-fold, while high affinity binding at pH 7.4 in the sub-nanomolar range was retained. Furthermore, binding to huFcRn and thermal stability were not affected by histidine substitutions. Overall, this study emphasizes a generalizable strategy for engineering pH-switch functions potentially applicable to a variety of antibodies and further proteins-based therapeutics. Taylor & Francis 2014-12-18 /pmc/articles/PMC4622719/ /pubmed/25523975 http://dx.doi.org/10.4161/19420862.2014.985993 Text en © 2015 The Author(s). Published with license by Taylor & Francis Group, LLC http://creativecommons.org/licenses/by-nc/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.
spellingShingle Reports
Schröter, Christian
Günther, Ralf
Rhiel, Laura
Becker, Stefan
Toleikis, Lars
Doerner, Achim
Becker, Janine
Schönemann, Andreas
Nasu, Daichi
Neuteboom, Berend
Kolmar, Harald
Hock, Björn
A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display
title A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display
title_full A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display
title_fullStr A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display
title_full_unstemmed A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display
title_short A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display
title_sort generic approach to engineer antibody ph-switches using combinatorial histidine scanning libraries and yeast display
topic Reports
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4622719/
https://www.ncbi.nlm.nih.gov/pubmed/25523975
http://dx.doi.org/10.4161/19420862.2014.985993
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