Increasing FcγR Characterization Throughput for Drug Candidates Using LSA^XT

Key Highlights

• Rigorous characterization of FcγR binding is a key step required in the development of Fc-formatted therapeutics.

• The historical throughput limitations of real-time, label-free biosensors have resulted in challenges with performing detailed FcγR binding characterization.

• This study highlights how LSA^XT and high quality FcγR reagents can produce data which is nearly identical to traditional, lower throughput biosensor platforms.

• The Carterra LSA^XT reduced the cost per measurement of FcγR binding from $89 down to $3.

Introduction

With around 175 antibody therapeutics approved globally, the US market is nearly $300 billion. Fc gamma receptors (FcγRs) are immune cell surface proteins that can produce activating or inhibitory signals when bound by Fc-containing molecules and are critical to the mediation of immune activities such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). As such, FcγR binding will remain an important characterization step for Fc-containing drug candidates.

FcγR binding assessment allows for the determination of wild-type FcγR binding, a step frequently required by regulatory agencies to demonstrate that the Fc-formatted therapeutic has an acceptable safety profile. Additionally, relevant species FcγR cross-reactivity and the impact of mutations further aid in understanding safety as well as overall mechanism of action (MOA). The impacts of these factors are potentially significant, as non-human primate (NHP) studies must consider both human and NHP FcγR binding, and critically, mutations may silence or enhance binding. A review by Liu et al. highlighted how virtually any change made to an antibody’s Fc region changed the binding affinity for multiple other FcγR receptors. This emphasizes that even a single Fc mutation affecting the modulation of effector function should be reviewed in depth, necessitating full characterization of FcγR binding. A challenge in the past has been identifying the fastest, most accurate process for this characterization.

The Carterra LSA^XT builds on the established capabilities of the LSA, providing unmatched throughput for real-time, label-free binding assays. The enhanced data quality of the LSA^XT allows for more rigorous characterization of systems that have rapid kinetics, which is the case for many FcyRs binding to Fc-containing molecules. Uniformity of responses for replicate ligands on the LSA^XT is typically < 15% which aids in accurate reporting of steady state affinities. Additionally, increased signal-to-noise (at least 2-fold better than LSA) allows for the use of lower ligand density surfaces and more accurate quantitation of lower binding signals. The experiments here were used to evaluate the performance of the LSA^XT for characterization of a well-studied commercial mAb against a panel of FcγRs and highlight opportunities to increase the depth of FcyR characterization while reducing resource requirements.

Posted by Noah T. Ditto