![Sample our Bioscience journals, sign in here to start your access, Latest two full volumes FREE to you for 14 days]({"type":"image" , "src" : "/sda/5925/TOC-Subject-Sample-2021-Bioscience.jpg"})
ABSTRACT
A challenge when developing therapeutic antibodies is the identification of candidates with favorable pharmacokinetics (PK) early in development. A key determinant of immunoglobulin (IgG) serum half‑life in vivo is the efficiency of pH-dependent binding to the neonatal Fc receptor (FcRn). Numerous studies have proposed techniques to assess FcRn binding of IgG-based therapeutics in vitro, enabling prediction of serum half-life prior to clinical assessment. FcRn high-performance liquid chromatography (HPLC) assays FcRn binding of therapeutic IgGs across a pH gradient, allowing the correlation of IgG column retention time to the half‑life of a therapeutic IgG in vivo. However, as FcRn retention time cannot be directly compared to an in vivo parameter, modifications to FcRn-HPLC are required to enable interpretation of the data within a physiological context, to provide more accurate estimations of serum half-life. This study presents an important modification to this method, FcRn-pH-HPLC, which reproducibly measures FcRn dissociation pH, allowing correlation with previously established half-lives of therapeutic antibodies. Furthermore, the influence of incorporating various antibody modifications, binding modules, and their orientations within IgGs and bispecifics on FcRn dissociation pH was evaluated using antibodies from the redirected optimized cell killing (ROCK®) platform. Target and effector antigen-binding domain sequences, their presentation format and orientation within a bispecific antibody alter FcRn retention; tested Fc domain modifications and incorporating stabilizing disulfide bonds had minimal effect. This study may inform the generation of mono-, bi- and multi-specific antibodies with tailored half-lives based on FcRn binding properties in vitro, to differentiate antibody-based therapeutic candidates with optimal developability.
© 2023 Affimed GmbH. Published with license by Taylor&Francis Group, LLC
Abbreviations
| ADCC | = | antibody-dependent cellular cytotoxicity |
| bsAb | = | bispecific antibody |
| CV | = | column volumes |
| DSB | = | disulfide bridges |
| Fab | = | fragment antigen binding |
| Fc | = | fragment crystallizable |
| FcRn | = | neonatal Fc receptor |
| HPLC | = | high-performance liquid chromatography |
| ICE | = | innate cell engager |
| IgG | = | immunoglobulin G |
| KiH | = | knobs-into-holes |
| NIST | = | National Institute of Standards and Technology |
| NK | = | natural killer |
| pI | = | isoelectric point |
| PK | = | pharmacokinetic |
| ROCK® | = | redirected optimized cell killing |
| scDb | = | single-chain diabody |
| scFv | = | single-chain variable fragment |
| stgFab | = | staggered Fabs |
| WT | = | wild type |
Acknowledgments
The authors would like to thank Marvin Hofmann and Frederic Bethke for supporting the manuscript by conducting the experiments utilizing FcRn-pH-HPLC in the lab. The authors also thank Arndt Schottelius for providing scientific support for the manuscript.
Disclosure statement
All authors are employees of Affimed GmbH.
Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2245519.
Correction Statement
This article has been corrected with minor changes. These changes do not impact the academic content of the article.