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Characterization and Modeling of Reversible Antibody Self-Association Provide Insights into Behavior, Prediction, and Correction
Reversible antibody self-association, while having major developability and therapeutic implications, is not fully understood or readily predictable and correctable. For a strongly self-associating humanized mAb variant, resulting in unacceptable viscosity, the monovalent affinity of self-interactio...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7931086/ https://www.ncbi.nlm.nih.gov/pubmed/33671864 http://dx.doi.org/10.3390/antib10010008 |
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author | Mieczkowski, Carl Cheng, Alan Fischmann, Thierry Hsieh, Mark Baker, Jeanne Uchida, Makiko Raghunathan, Gopalan Strickland, Corey Fayadat-Dilman, Laurence |
author_facet | Mieczkowski, Carl Cheng, Alan Fischmann, Thierry Hsieh, Mark Baker, Jeanne Uchida, Makiko Raghunathan, Gopalan Strickland, Corey Fayadat-Dilman, Laurence |
author_sort | Mieczkowski, Carl |
collection | PubMed |
description | Reversible antibody self-association, while having major developability and therapeutic implications, is not fully understood or readily predictable and correctable. For a strongly self-associating humanized mAb variant, resulting in unacceptable viscosity, the monovalent affinity of self-interaction was measured in the low μM range, typical of many specific and biologically relevant protein–protein interactions. A face-to-face interaction model extending across both the heavy-chain (HC) and light-chain (LC) Complementary Determining Regions (CDRs) was apparent from biochemical and mutagenesis approaches as well as computational modeling. Light scattering experiments involving individual mAb, Fc, Fab, and Fab’2 domains revealed that Fabs self-interact to form dimers, while bivalent mAb/Fab’2 forms lead to significant oligomerization. Site-directed mutagenesis of aromatic residues identified by homology model patch analysis and self-docking dramatically affected self-association, demonstrating the utility of these predictive approaches, while revealing a highly specific and tunable nature of self-binding modulated by single point mutations. Mutagenesis at these same key HC/LC CDR positions that affect self-interaction also typically abolished target binding with notable exceptions, clearly demonstrating the difficulties yet possibility of correcting self-association through engineering. Clear correlations were also observed between different methods used to assess self-interaction, such as Dynamic Light Scattering (DLS) and Affinity-Capture Self-Interaction Nanoparticle Spectroscopy (AC-SINS). Our findings advance our understanding of therapeutic protein and antibody self-association and offer insights into its prediction, evaluation and corrective mitigation to aid therapeutic development. |
format | Online Article Text |
id | pubmed-7931086 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-79310862021-03-05 Characterization and Modeling of Reversible Antibody Self-Association Provide Insights into Behavior, Prediction, and Correction Mieczkowski, Carl Cheng, Alan Fischmann, Thierry Hsieh, Mark Baker, Jeanne Uchida, Makiko Raghunathan, Gopalan Strickland, Corey Fayadat-Dilman, Laurence Antibodies (Basel) Article Reversible antibody self-association, while having major developability and therapeutic implications, is not fully understood or readily predictable and correctable. For a strongly self-associating humanized mAb variant, resulting in unacceptable viscosity, the monovalent affinity of self-interaction was measured in the low μM range, typical of many specific and biologically relevant protein–protein interactions. A face-to-face interaction model extending across both the heavy-chain (HC) and light-chain (LC) Complementary Determining Regions (CDRs) was apparent from biochemical and mutagenesis approaches as well as computational modeling. Light scattering experiments involving individual mAb, Fc, Fab, and Fab’2 domains revealed that Fabs self-interact to form dimers, while bivalent mAb/Fab’2 forms lead to significant oligomerization. Site-directed mutagenesis of aromatic residues identified by homology model patch analysis and self-docking dramatically affected self-association, demonstrating the utility of these predictive approaches, while revealing a highly specific and tunable nature of self-binding modulated by single point mutations. Mutagenesis at these same key HC/LC CDR positions that affect self-interaction also typically abolished target binding with notable exceptions, clearly demonstrating the difficulties yet possibility of correcting self-association through engineering. Clear correlations were also observed between different methods used to assess self-interaction, such as Dynamic Light Scattering (DLS) and Affinity-Capture Self-Interaction Nanoparticle Spectroscopy (AC-SINS). Our findings advance our understanding of therapeutic protein and antibody self-association and offer insights into its prediction, evaluation and corrective mitigation to aid therapeutic development. MDPI 2021-02-15 /pmc/articles/PMC7931086/ /pubmed/33671864 http://dx.doi.org/10.3390/antib10010008 Text en © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Mieczkowski, Carl Cheng, Alan Fischmann, Thierry Hsieh, Mark Baker, Jeanne Uchida, Makiko Raghunathan, Gopalan Strickland, Corey Fayadat-Dilman, Laurence Characterization and Modeling of Reversible Antibody Self-Association Provide Insights into Behavior, Prediction, and Correction |
title | Characterization and Modeling of Reversible Antibody Self-Association Provide Insights into Behavior, Prediction, and Correction |
title_full | Characterization and Modeling of Reversible Antibody Self-Association Provide Insights into Behavior, Prediction, and Correction |
title_fullStr | Characterization and Modeling of Reversible Antibody Self-Association Provide Insights into Behavior, Prediction, and Correction |
title_full_unstemmed | Characterization and Modeling of Reversible Antibody Self-Association Provide Insights into Behavior, Prediction, and Correction |
title_short | Characterization and Modeling of Reversible Antibody Self-Association Provide Insights into Behavior, Prediction, and Correction |
title_sort | characterization and modeling of reversible antibody self-association provide insights into behavior, prediction, and correction |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7931086/ https://www.ncbi.nlm.nih.gov/pubmed/33671864 http://dx.doi.org/10.3390/antib10010008 |
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