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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...

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Autores principales: Mieczkowski, Carl, Cheng, Alan, Fischmann, Thierry, Hsieh, Mark, Baker, Jeanne, Uchida, Makiko, Raghunathan, Gopalan, Strickland, Corey, Fayadat-Dilman, Laurence
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
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.
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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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