Structural and Functional Characterization of a Hole–Hole Homodimer Variant in a “Knob-Into-Hole” Bispecific Antibody

Zhang, Huimin; Li, Charlene; Lei, Ming; Lundin, Victor; Lee, Ho‐Young; Niñonuevo, Milady R.; Lin, Kevin K.; Han, Guanghui; Sandoval, Wendy; Lei, Dongsheng; Ren, Gang; Zhang, Jennifer; Liu, Hongbin

doi:10.1021/acs.analchem.7b03830

Cited by 32 publications

(40 citation statements)

References 34 publications

(66 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…KiH (H) was chosen as a recipient for the D84.4Q substitution over KiH "knob" (denoted K; T366W) because we found its characteristics to be the closest match to BEAT (B); both KiH (H) and BEAT (B) are the chains receiving the smaller residues, creating cavities to accommodate the larger residues introduced into KiH (K) and BEAT (A). Furthermore, some characterization data for KiH (H) homodimers had been published (13). Upon purification, we found that this antibody was not abrogated for PA binding (Fig.…”

Section: D844q Substitution Abrogates Pa and Pg Binding In The Fc Ofmentioning

confidence: 65%

“…The increase in Rh was less significant than for the BEAT (B) D84.4Q homodimers. Zhang et al ( 13 ) attributed the increase in Rh of the KiH (H) homodimer to a reduced compactness of the domains caused by their poor pairing capabilities. This also may explain the relatively mild reduction in FcRn and FcγR1a binding compared with the corresponding KiH heterodimer ( Fig.…”

Section: Discussionmentioning

confidence: 99%

“…10 B ). The mild increase in ANS fluorescence for KiH could be explained by the reported existence of multiple conformational isomers of varying folding compactness and varying hydrophobicity ( 13 ). Increased hydrophobicity may lead to some increase in ANS binding, which could also explain the mildly increased fluorescence emission observed for BEAT (A) homodimers, which may suffer from suboptimally paired CH3 domains.…”

Section: Discussionmentioning

confidence: 99%

“…Finally, our results shed structural insights on bsAb Hc hetero- versus homodimer assembly, and the nature of half-antibodies on which only little data could be found prior to this work ( 13 ). As in our previous studies ( 10 ), antibodies based on KiH were used as comparators.…”

mentioning

confidence: 81%

See 3 more Smart Citations

A single mutation increases heavy-chain heterodimer assembly of bispecific antibodies by inducing structural disorder in one homodimer species

Stutz¹,

Blein²

2020

Journal of Biological Chemistry

View full text Add to dashboard Cite

We previously reported efficient heavy-chain assembly of heterodimeric bispecific antibodies by exchanging the interdomain protein interface of the human IgG1 CH3 dimer with the protein interface of the constant α and β domains of the human T-cell receptor, a technology known as bispecific engagement by antibodies based on the T-cell receptor (BEAT). Efficient heterodimerization in mammalian cell transient transfections was observed, but levels were influenced by the nature of the binding arms, particularly in the Fab-scFv-Fc format. In this study, we report a single amino acid change that significantly and consistently improved the heterodimerization rate of this format (≥95%) by inducing partial disorder in one homodimer species without affecting the heterodimer. Correct folding and assembly of the heterodimer were confirmed by the high-resolution (1.88–1.98 Å) crystal structure presented here. Thermal stability and 1-anilinonaphthalene-8-sulfonic acid–binding experiments, comparing original BEAT, mutated BEAT, and “knobs-into-holes” interfaces, suggested a cooperative assembly process of heavy chains in heterodimers. The observed gain in stability of the interfaces could be classified in the following rank order: mutated BEAT > original BEAT > knobs-into-holes. We therefore propose that the superior cooperativity found in BEAT interfaces is the key driver of their greater performance. Furthermore, we show how the mutated BEAT interface can be exploited for the routine preparation of drug candidates, with minimal risk of homodimer contamination using a single Protein A chromatography step.

show abstract

Section: D844q Substitution Abrogates Pa and Pg Binding In The Fc Ofmentioning

confidence: 65%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 81%

See 2 more Smart Citations

A single mutation increases heavy-chain heterodimer assembly of bispecific antibodies by inducing structural disorder in one homodimer species

Stutz¹,

Blein²

2020

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Native MS utilizes 100% aqueous solutions of low-to-moderate concentrations of volatile salt (e.g., 50 mM ammonium acetate) buffered at neutral pH. 3,4 This type of analysis proceeds commonly after removal of non-volatile salts by buffer exchange followed by nanospray infusion MS. 5–7 Native MS-based intact mass analysis is required for certain classes of biotherapeutics that require preservation of non-covalent associations of protein-protein or protein-ligand complexes. 8 Native MS also affords a fundamental benefit to intact mass analysis of all types of heterogeneous samples where proteins acquire fewer charges and yield spectra at high m/z relative to denatured conditions.…”

Section: Introductionmentioning

confidence: 99%

Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis

Bailey

Han²,

Phung³

et al. 2018

mAbs

Self Cite

View full text Add to dashboard Cite

The preponderance and diversity of charge variants in therapeutic monoclonal antibodies has implications for antibody efficacy and degradation. Understanding the extent and impact of minor antibody variants is of great interest, and it is also a critical regulatory requirement. Traditionally, a combination of approaches is used to characterize antibody charge heterogeneity, including ion exchange chromatography and independent mass spectrometric variant site mapping after proteolytic digestion. Here, we describe charge variant native mass spectrometry (CVMS), an integrated native ion exchange mass spectrometry-based charge variant analytical approach that delivers detailed molecular information in a single, semi-automated analysis. We utilized pure volatile salt mobile phases over a pH gradient that effectively separated variants based on minimal differences in isoelectric point. Characterization of variants such as deamidation, which are traditionally unattainable by intact mass due to their minimal molecular weight differences, were measured unambiguously by mass and retention time to allow confident MS1 identification. We demonstrate that efficient chromatographic separation allows introduction of the purified forms of the charge variant isoforms into the Orbitrap mass spectrometer. Our CVMS method allows confident assignment of intact monoclonal antibody isoforms of similar mass and relative abundance measurements across three orders of magnitude dynamic range.

show abstract

Optimized Negative-Staining Protocol for Lipid–Protein Interactions Investigated by Electron Microscopy

Liu

Huang

et al. 2019

Methods in Molecular Biology

Self Cite

View full text Add to dashboard Cite

A large number of proteins are capable of inserting themselves into lipids, and interacting with membranes, such as transmembrane proteins and apolipoproteins. Insights into the lipid-protein interactions are important in understanding biological processes, and the structure of proteins at the lipid binding stage can help identify their roles and critical functions. Previously, such structural determination was challenging to obtain because the traditional methods, such as X-ray crystallography, are unable to capture the conformational and compositional heterogeneity of protein–lipid complexes. Electron microscopy (EM) is an alternative approach to determining protein structures and visualizing lipid–protein interactions directly, and negative-staining (OpNS), a subset of EM techniques, is a rapid, frequently used qualitative approach. The concern, however, is that current NS protocols often generate artifacts with lipid-related proteins, such as rouleaux formation from lipoproteins. To overcome this artifact formation, Ren and his colleagues have refined early NS protocols, and developed an optimized NS protocol that validated by comparing images of lipoproteins from cryo-electron microscopy (cryo-EM). This optimized NS protocol produces “near native-state” particle images and high contrast images of the protein in its native lipid-binding state, which can be used to create higher-quality three-dimensional (3D) reconstruction by single-particle analysis and electron tomography (e.g. IPET). This optimized protocol is thus a promising hands-on approach for examining the structure of proteins at their lipid-binding status.

show abstract

Structural and Functional Characterization of a Hole–Hole Homodimer Variant in a “Knob-Into-Hole” Bispecific Antibody

Cited by 32 publications

References 34 publications

A single mutation increases heavy-chain heterodimer assembly of bispecific antibodies by inducing structural disorder in one homodimer species

A single mutation increases heavy-chain heterodimer assembly of bispecific antibodies by inducing structural disorder in one homodimer species

Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis

Optimized Negative-Staining Protocol for Lipid–Protein Interactions Investigated by Electron Microscopy

Contact Info

Product

Resources

About