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Engineering Bispecificity into a Single Albumin-Binding Domain

Bispecific antibodies as well as non-immunoglobulin based bispecific affinity proteins are considered to have a very high potential in future biotherapeutic applications. In this study, we report on a novel approach for generation of extremely small bispecific proteins comprised of only a single str...

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Autores principales: Nilvebrant, Johan, Alm, Tove, Hober, Sophia, Löfblom, John
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185003/
https://www.ncbi.nlm.nih.gov/pubmed/21991353
http://dx.doi.org/10.1371/journal.pone.0025791
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author Nilvebrant, Johan
Alm, Tove
Hober, Sophia
Löfblom, John
author_facet Nilvebrant, Johan
Alm, Tove
Hober, Sophia
Löfblom, John
author_sort Nilvebrant, Johan
collection PubMed
description Bispecific antibodies as well as non-immunoglobulin based bispecific affinity proteins are considered to have a very high potential in future biotherapeutic applications. In this study, we report on a novel approach for generation of extremely small bispecific proteins comprised of only a single structural domain. Binding to tumor necrosis factor-α (TNF-α) was engineered into an albumin-binding domain while still retaining the original affinity for albumin, resulting in a bispecific protein composed of merely 46 amino acids. By diversification of the non albumin-binding side of the three-helix bundle domain, followed by display of the resulting library on phage particles, bispecific single-domain proteins were isolated using selections with TNF-α as target. Moreover, based on the obtained sequences from the phage selection, a second-generation library was designed in order to further increase the affinity of the bispecific candidates. Staphylococcal surface display was employed for the affinity maturation, enabling efficient isolation of improved binders as well as multiparameter-based sortings with both TNF-α and albumin as targets in the same selection cycle. Isolated variants were sequenced and the binding to albumin and TNF-α was analyzed. This analysis revealed an affinity for TNF-α below 5 nM for the strongest binders. From the multiparameter sorting that simultaneously targeted TNF-α and albumin, several bispecific candidates were isolated with high affinity to both antigens, suggesting that cell display in combination with fluorescence activated cell sorting is a suitable technology for engineering of bispecificity. To our knowledge, the new binders represent the smallest engineered bispecific proteins reported so far. Possibilities and challenges as well as potential future applications of this novel strategy are discussed.
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spelling pubmed-31850032011-10-11 Engineering Bispecificity into a Single Albumin-Binding Domain Nilvebrant, Johan Alm, Tove Hober, Sophia Löfblom, John PLoS One Research Article Bispecific antibodies as well as non-immunoglobulin based bispecific affinity proteins are considered to have a very high potential in future biotherapeutic applications. In this study, we report on a novel approach for generation of extremely small bispecific proteins comprised of only a single structural domain. Binding to tumor necrosis factor-α (TNF-α) was engineered into an albumin-binding domain while still retaining the original affinity for albumin, resulting in a bispecific protein composed of merely 46 amino acids. By diversification of the non albumin-binding side of the three-helix bundle domain, followed by display of the resulting library on phage particles, bispecific single-domain proteins were isolated using selections with TNF-α as target. Moreover, based on the obtained sequences from the phage selection, a second-generation library was designed in order to further increase the affinity of the bispecific candidates. Staphylococcal surface display was employed for the affinity maturation, enabling efficient isolation of improved binders as well as multiparameter-based sortings with both TNF-α and albumin as targets in the same selection cycle. Isolated variants were sequenced and the binding to albumin and TNF-α was analyzed. This analysis revealed an affinity for TNF-α below 5 nM for the strongest binders. From the multiparameter sorting that simultaneously targeted TNF-α and albumin, several bispecific candidates were isolated with high affinity to both antigens, suggesting that cell display in combination with fluorescence activated cell sorting is a suitable technology for engineering of bispecificity. To our knowledge, the new binders represent the smallest engineered bispecific proteins reported so far. Possibilities and challenges as well as potential future applications of this novel strategy are discussed. Public Library of Science 2011-10-03 /pmc/articles/PMC3185003/ /pubmed/21991353 http://dx.doi.org/10.1371/journal.pone.0025791 Text en Nilvebrant 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, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Nilvebrant, Johan
Alm, Tove
Hober, Sophia
Löfblom, John
Engineering Bispecificity into a Single Albumin-Binding Domain
title Engineering Bispecificity into a Single Albumin-Binding Domain
title_full Engineering Bispecificity into a Single Albumin-Binding Domain
title_fullStr Engineering Bispecificity into a Single Albumin-Binding Domain
title_full_unstemmed Engineering Bispecificity into a Single Albumin-Binding Domain
title_short Engineering Bispecificity into a Single Albumin-Binding Domain
title_sort engineering bispecificity into a single albumin-binding domain
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185003/
https://www.ncbi.nlm.nih.gov/pubmed/21991353
http://dx.doi.org/10.1371/journal.pone.0025791
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