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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...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2020
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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. |
format | Online Article Text |
id | pubmed-7075871 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
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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