The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants

Abstract In vitro affinity-maturation potentially generates antibody fragments with enhanced antigen-binding affinities that allow for developing more sensitive diagnostic systems and more effective therapeutic agents. Site-directed mutagenesis targeting “hot regions,” i.e., amino acid substitutions...

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Autores principales:	Yuki Kiguchi, Hiroyuki Oyama, Izumi Morita, Yasuhiro Nagata, Naoko Umezawa, Norihiro Kobayashi
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/8bb6256cd6294d32b16c979a6b21bdcb
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spelling	oai:doaj.org-article:8bb6256cd6294d32b16c979a6b21bdcb2021-12-02T14:26:12ZThe VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants10.1038/s41598-021-87501-72045-2322https://doaj.org/article/8bb6256cd6294d32b16c979a6b21bdcb2021-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-87501-7https://doaj.org/toc/2045-2322Abstract In vitro affinity-maturation potentially generates antibody fragments with enhanced antigen-binding affinities that allow for developing more sensitive diagnostic systems and more effective therapeutic agents. Site-directed mutagenesis targeting “hot regions,” i.e., amino acid substitutions therein frequently increase the affinities, is desirable for straightforward discovery of valuable mutants. We here report two “designed” site-directed mutagenesis (A and B) targeted the N-terminal 1–10 positions of the VH framework region 1 that successfully improved an anti-cortisol single-chain Fv fragment (K a, 3.6 × 108 M−1). Mutagenesis A substituted the amino acids at the position 1–3, 5–7, 9 and 10 with a limited set of substitutions to generate only 1,536 different members, while mutagenesis B inserted 1–6 random residues between the positions 6 and 7. Screening the resulting bacterial libraries as scFv-phage clones with a clonal array profiling system provided 21 genetically unique scFv mutants showing 17–31-fold increased affinity with > 109 M−1 K a values. Among the mutants selected from the library A and B, scFv mA#18 (with five-residue substitutions) and mB1-3#130 (with a single residue insertion) showed the greatest K a value, 1.1 × 1010 M−1.Yuki KiguchiHiroyuki OyamaIzumi MoritaYasuhiro NagataNaoko UmezawaNorihiro KobayashiNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-11 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Yuki Kiguchi Hiroyuki Oyama Izumi Morita Yasuhiro Nagata Naoko Umezawa Norihiro Kobayashi The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants
description	Abstract In vitro affinity-maturation potentially generates antibody fragments with enhanced antigen-binding affinities that allow for developing more sensitive diagnostic systems and more effective therapeutic agents. Site-directed mutagenesis targeting “hot regions,” i.e., amino acid substitutions therein frequently increase the affinities, is desirable for straightforward discovery of valuable mutants. We here report two “designed” site-directed mutagenesis (A and B) targeted the N-terminal 1–10 positions of the VH framework region 1 that successfully improved an anti-cortisol single-chain Fv fragment (K a, 3.6 × 108 M−1). Mutagenesis A substituted the amino acids at the position 1–3, 5–7, 9 and 10 with a limited set of substitutions to generate only 1,536 different members, while mutagenesis B inserted 1–6 random residues between the positions 6 and 7. Screening the resulting bacterial libraries as scFv-phage clones with a clonal array profiling system provided 21 genetically unique scFv mutants showing 17–31-fold increased affinity with > 109 M−1 K a values. Among the mutants selected from the library A and B, scFv mA#18 (with five-residue substitutions) and mB1-3#130 (with a single residue insertion) showed the greatest K a value, 1.1 × 1010 M−1.
format	article
author	Yuki Kiguchi Hiroyuki Oyama Izumi Morita Yasuhiro Nagata Naoko Umezawa Norihiro Kobayashi
author_facet	Yuki Kiguchi Hiroyuki Oyama Izumi Morita Yasuhiro Nagata Naoko Umezawa Norihiro Kobayashi
author_sort	Yuki Kiguchi
title	The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants
title_short	The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants
title_full	The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants
title_fullStr	The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants
title_full_unstemmed	The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants
title_sort	vh framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scfv mutants
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/8bb6256cd6294d32b16c979a6b21bdcb
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The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants

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