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Characterization and Modeling of Reversible Antibody Self-Association Provide Insights into Behavior, Prediction, and Correction

Authors
  • Mieczkowski, Carl1
  • Cheng, Alan2
  • Fischmann, Thierry3
  • Hsieh, Mark1
  • Baker, Jeanne1
  • Uchida, Makiko1
  • Raghunathan, Gopalan1
  • Strickland, Corey3
  • Fayadat-Dilman, Laurence1
  • 1 (L.F.-D.)
  • 2 Discovery Chemistry, Modeling and Informatics, Merck & Co., Inc., South San Francisco, CA 94080, USA
  • 3 (C.S.)
Type
Published Article
Journal
Antibodies
Publisher
MDPI
Publication Date
Feb 15, 2021
Volume
10
Issue
1
Identifiers
DOI: 10.3390/antib10010008
PMID: 33671864
PMCID: PMC7931086
Source
PubMed Central
Keywords
Disciplines
  • Article
License
Green

Abstract

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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