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Seeking high-priority mutations enabling successful antibody-breeding: systematic analysis of a mutant that gained over 100-fold enhanced affinity.
Sci Rep. 2020 Mar 16; 10(1):4807.SR

Abstract

"Antibody-breeding" has provided therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Typically, random point mutations are introduced into the VH and VL 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 (Ka = 1.19 × 1010 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 Kas indicated that a revertant with four substitutions (VH-L100gQ, VL-I29V, -L36M, -S77G) exhibited somewhat higher affinity (Ka = 1.46 × 1010 M-1). Finally, the VH-L100gQ substitution, occurring in VH complementarity-determining region (CDR) 3, was found to be the highest-priority for improving the affinity, and VL-I29V and/or VL-L36M cooperated significantly. These findings encouraged us to reconsider the potential of VH-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.

Authors+Show Affiliations

Kobe Pharmaceutical University, 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe, 658-8558, Japan.Kobe Pharmaceutical University, 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe, 658-8558, Japan.Kobe Pharmaceutical University, 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe, 658-8558, Japan.Kobe Pharmaceutical University, 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe, 658-8558, Japan.Kobe Pharmaceutical University, 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe, 658-8558, Japan.Kobe Pharmaceutical University, 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe, 658-8558, Japan.Kobe Pharmaceutical University, 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe, 658-8558, Japan.Kobe Pharmaceutical University, 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe, 658-8558, Japan.Kobe Pharmaceutical University, 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe, 658-8558, Japan.Kobe Pharmaceutical University, 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe, 658-8558, Japan.Kobe Pharmaceutical University, 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe, 658-8558, Japan.Kobe Pharmaceutical University, 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe, 658-8558, Japan. no-kobay@kobepharma-u.ac.jp.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32179767

Citation

Oyama, Hiroyuki, et al. "Seeking High-priority Mutations Enabling Successful Antibody-breeding: Systematic Analysis of a Mutant That Gained Over 100-fold Enhanced Affinity." Scientific Reports, vol. 10, no. 1, 2020, p. 4807.
Oyama H, Kiguchi Y, Morita I, et al. Seeking high-priority mutations enabling successful antibody-breeding: systematic analysis of a mutant that gained over 100-fold enhanced affinity. Sci Rep. 2020;10(1):4807.
Oyama, H., Kiguchi, Y., Morita, I., Yamamoto, C., Higashi, Y., Taguchi, M., Tagawa, T., Enami, Y., Takamine, Y., Hasegawa, H., Takeuchi, A., & Kobayashi, N. (2020). Seeking high-priority mutations enabling successful antibody-breeding: systematic analysis of a mutant that gained over 100-fold enhanced affinity. Scientific Reports, 10(1), 4807. https://doi.org/10.1038/s41598-020-61529-7
Oyama H, et al. Seeking High-priority Mutations Enabling Successful Antibody-breeding: Systematic Analysis of a Mutant That Gained Over 100-fold Enhanced Affinity. Sci Rep. 2020 Mar 16;10(1):4807. PubMed PMID: 32179767.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Seeking high-priority mutations enabling successful antibody-breeding: systematic analysis of a mutant that gained over 100-fold enhanced affinity. AU - Oyama,Hiroyuki, AU - Kiguchi,Yuki, AU - Morita,Izumi, AU - Yamamoto,Chika, AU - Higashi,Yuka, AU - Taguchi,Miku, AU - Tagawa,Tatsuya, AU - Enami,Yuri, AU - Takamine,Yuriko, AU - Hasegawa,Hanako, AU - Takeuchi,Atsuko, AU - Kobayashi,Norihiro, Y1 - 2020/03/16/ PY - 2019/10/30/received PY - 2020/02/27/accepted PY - 2020/3/18/entrez PY - 2020/3/18/pubmed PY - 2020/3/18/medline SP - 4807 EP - 4807 JF - Scientific reports JO - Sci Rep VL - 10 IS - 1 N2 - "Antibody-breeding" has provided therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Typically, random point mutations are introduced into the VH and VL 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 (Ka = 1.19 × 1010 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 Kas indicated that a revertant with four substitutions (VH-L100gQ, VL-I29V, -L36M, -S77G) exhibited somewhat higher affinity (Ka = 1.46 × 1010 M-1). Finally, the VH-L100gQ substitution, occurring in VH complementarity-determining region (CDR) 3, was found to be the highest-priority for improving the affinity, and VL-I29V and/or VL-L36M cooperated significantly. These findings encouraged us to reconsider the potential of VH-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. SN - 2045-2322 UR - https://www.unboundmedicine.com/medline/citation/32179767/Seeking_high-priority_mutations_enabling_successful_antibody-breeding:_systematic_analysis_of_a_mutant_that_gained_over_100-fold_enhanced_affinity L2 - http://dx.doi.org/10.1038/s41598-020-61529-7 DB - PRIME DP - Unbound Medicine ER -
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