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Seeking high-priority mutations enabling successful antibody-breeding: systematic analysis of a mutant that gained over 100-fold enhanced affinity

“Antibody-breeding” has provided therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Typically, random point mutations are introduced into the V(H) and V(L) domains of parent antibodies to generate diverse libraries of single-chain Fv fragments (scFvs), from whic...

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Autores principales: Oyama, Hiroyuki, Kiguchi, Yuki, Morita, Izumi, Yamamoto, Chika, Higashi, Yuka, Taguchi, Miku, Tagawa, Tatsuya, Enami, Yuri, Takamine, Yuriko, Hasegawa, Hanako, Takeuchi, Atsuko, Kobayashi, Norihiro
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075871/
https://www.ncbi.nlm.nih.gov/pubmed/32179767
http://dx.doi.org/10.1038/s41598-020-61529-7
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author Oyama, Hiroyuki
Kiguchi, Yuki
Morita, Izumi
Yamamoto, Chika
Higashi, Yuka
Taguchi, Miku
Tagawa, Tatsuya
Enami, Yuri
Takamine, Yuriko
Hasegawa, Hanako
Takeuchi, Atsuko
Kobayashi, Norihiro
author_facet Oyama, Hiroyuki
Kiguchi, Yuki
Morita, Izumi
Yamamoto, Chika
Higashi, Yuka
Taguchi, Miku
Tagawa, Tatsuya
Enami, Yuri
Takamine, Yuriko
Hasegawa, Hanako
Takeuchi, Atsuko
Kobayashi, Norihiro
author_sort Oyama, Hiroyuki
collection PubMed
description “Antibody-breeding” has provided therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Typically, random point mutations are introduced into the V(H) and V(L) domains of parent antibodies to generate diverse libraries of single-chain Fv fragments (scFvs), from which evolved mutants are selected. We produced an scFv against estradiol-17β with 11 amino acid substitutions and a >100-fold improved affinity constant (K(a) = 1.19 × 10(10) M(−1)) over the parent scFv, enabling immunoassays with >30-fold higher sensitivity. We systematically analyzed contributions of these substitutions to the affinity enhancement. Comparing various partial scFv revertants based on their K(a)s indicated that a revertant with four substitutions (V(H)-L100gQ, V(L)-I29V, -L36M, -S77G) exhibited somewhat higher affinity (K(a) = 1.46 × 10(10) M(−1)). Finally, the V(H)-L100gQ substitution, occurring in V(H) complementarity-determining region (CDR) 3, was found to be the highest-priority for improving the affinity, and V(L)-I29V and/or V(L)-L36M cooperated significantly. These findings encouraged us to reconsider the potential of V(H)-CDR3-targeting mutagenesis, which has been frequently attempted. The substitution(s) wherein might enable a “high rate of return” in terms of selecting mutants with dramatically enhanced affinities. The “high risk” of generating a tremendous excess of “junk mutants” can be overcome with the efficient selection systems that we developed.
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spelling pubmed-70758712020-03-22 Seeking high-priority mutations enabling successful antibody-breeding: systematic analysis of a mutant that gained over 100-fold enhanced affinity Oyama, Hiroyuki Kiguchi, Yuki Morita, Izumi Yamamoto, Chika Higashi, Yuka Taguchi, Miku Tagawa, Tatsuya Enami, Yuri Takamine, Yuriko Hasegawa, Hanako Takeuchi, Atsuko Kobayashi, Norihiro Sci Rep Article “Antibody-breeding” has provided therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Typically, random point mutations are introduced into the V(H) and V(L) domains of parent antibodies to generate diverse libraries of single-chain Fv fragments (scFvs), from which evolved mutants are selected. We produced an scFv against estradiol-17β with 11 amino acid substitutions and a >100-fold improved affinity constant (K(a) = 1.19 × 10(10) M(−1)) over the parent scFv, enabling immunoassays with >30-fold higher sensitivity. We systematically analyzed contributions of these substitutions to the affinity enhancement. Comparing various partial scFv revertants based on their K(a)s indicated that a revertant with four substitutions (V(H)-L100gQ, V(L)-I29V, -L36M, -S77G) exhibited somewhat higher affinity (K(a) = 1.46 × 10(10) M(−1)). Finally, the V(H)-L100gQ substitution, occurring in V(H) complementarity-determining region (CDR) 3, was found to be the highest-priority for improving the affinity, and V(L)-I29V and/or V(L)-L36M cooperated significantly. These findings encouraged us to reconsider the potential of V(H)-CDR3-targeting mutagenesis, which has been frequently attempted. The substitution(s) wherein might enable a “high rate of return” in terms of selecting mutants with dramatically enhanced affinities. The “high risk” of generating a tremendous excess of “junk mutants” can be overcome with the efficient selection systems that we developed. Nature Publishing Group UK 2020-03-16 /pmc/articles/PMC7075871/ /pubmed/32179767 http://dx.doi.org/10.1038/s41598-020-61529-7 Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Oyama, Hiroyuki
Kiguchi, Yuki
Morita, Izumi
Yamamoto, Chika
Higashi, Yuka
Taguchi, Miku
Tagawa, Tatsuya
Enami, Yuri
Takamine, Yuriko
Hasegawa, Hanako
Takeuchi, Atsuko
Kobayashi, Norihiro
Seeking high-priority mutations enabling successful antibody-breeding: systematic analysis of a mutant that gained over 100-fold enhanced affinity
title Seeking high-priority mutations enabling successful antibody-breeding: systematic analysis of a mutant that gained over 100-fold enhanced affinity
title_full Seeking high-priority mutations enabling successful antibody-breeding: systematic analysis of a mutant that gained over 100-fold enhanced affinity
title_fullStr Seeking high-priority mutations enabling successful antibody-breeding: systematic analysis of a mutant that gained over 100-fold enhanced affinity
title_full_unstemmed Seeking high-priority mutations enabling successful antibody-breeding: systematic analysis of a mutant that gained over 100-fold enhanced affinity
title_short Seeking high-priority mutations enabling successful antibody-breeding: systematic analysis of a mutant that gained over 100-fold enhanced affinity
title_sort seeking high-priority mutations enabling successful antibody-breeding: systematic analysis of a mutant that gained over 100-fold enhanced affinity
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075871/
https://www.ncbi.nlm.nih.gov/pubmed/32179767
http://dx.doi.org/10.1038/s41598-020-61529-7
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