Application Note Binding of Fc-engineered IgG antibodies to FcRn Published on December 5th, 2022 Authors: Matt Gothard¹, Alex Spice2, Hannah Turn Ey¹, Katharina Mahal¹, Sean Devenish2, Sebastian Fiedler2, And David Perez-Martinez¹ 1 – Biopharmaceutical Development, Biopharmaceuticals R&D, Astrazeneca, Cambridge, United Kingdom 2 – Fluidic Analytics, Cambridge, United Kingdom Abstract Fc engineering of IgGs to extend antibody serum half-life or to modulate immune-receptor binding is an essential part of antibody drug development. However, quantifying human IgG-Fc receptor binding using surface-based, kinetic biomolecular interaction analysis has proven difficult due to reagent heterogeneity resulting in complex binding kinetics with multiple KD values. Because of these challenges, scientists in many cases resort to reporting derived qualitative binding parameters which are difficult to compare across different labs or studies. In this application note, we present a straightforward equilibrium binding assay that yields easy to interpret and universally comparable KD values for IgG–Fc receptor binding affinities. The assay utilizes microfluidic diffusional sizing (MDS) as a robust and physically meaningful way to investigate biomolecular interactions. An experiment requires only 20 minutes of hands-on time per IgG–Fc receptor pair and uses nanogram and low microgram amounts of Fc receptor and IgG, respectively. DOWNLOAD THE APPLICATION NOTE Introduction Therapeutic monoclonal antibodies are widely used in many disease areas including cancer, auto-immune disease, and infectious disease. All FDA-approved therapeutic antibodies are members of the IgG class. IgG molecules are multifunctional and multivalent as they are composed of two identical fragment antigen binding (Fab) domains and a single fragment crystallizable (Fc) domain. The two Fab domains are responsible for antigen recognition while the Fc domain is involved in humoral and effector cell functions. Fc engineering1 is often required to modulate the function of therapeutic antibodies to either promote or abolish antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) or to modify serum half-life, for example. The neonatal Fc receptor (FcRn) is responsible for controlling serum IgG concentrations. The key is that IgG molecules only bind to FcRn at pH <6.5, whereas they have very low affinities at physiological pH of 7.4. IgGs circulating in the blood stream get internalized in acidic FcRn-containing endosomes by circulating endothelial cells and monocytes. In the acidic endosome, monomeric IgGs bind to FcRn and are recycled by being released back into circulation at physiological pH. Increasing the binding affinity to FcRn increases the recycled fraction of antibody and so extends the IgG half-life. Polymeric IgGs bound to antigens as part of immune complexes do not bind FcRn and so are degraded in lysosomes to clear the antigen. An extended serum half-life is often thought to be beneficial for therapeutic antibodies as it should increase efficacy by sustaining serum concentrations, decrease dosing frequency, and lower cost to the patient and health system. In consequence, the Fc-engineered triple mutant IgG-YTE (M252Y/S254T/T256E)2 was developed which showed increased binding affinity to FcRn and a 4-fold extended half-life in serum. For antibodies that target antigens on the surface of immune cells, for example PD-1 blockers, it is essential to abolish immune effector functions such as ADCC and/or ADCP. To eliminate these pathways, Fc-engineering was employed to create another triple mutant molecule named IgG-TM (L234F/L235E/P331S)3 which lacks immune receptor binding. Despite the high therapeutic relevance of IgG–Fc receptor binding, measuring these interactions often proves to be challenging. In this application note, we present a straightforward in-solution assay based on microfluidic diffusional sizing (MDS) to determine binding affinities of Fc-engineered IgGs to FcRn in comparison with wild type IgG. Key Advantage MDS does not suffer from challenges faced by surface-based, kinetic Fc receptor binding assays such as complex binding kinetics due to reagent heterogeneity4 or varying results depending on the assay setup used. MDS provides easy to interpret KD values that provide quantitative information on the binding properties of Fc engineered antibodies. Results To analyze FcRn binding to IgGs, binding curves were measured for fluorescently labeled FcRn with different variants of IgG (Figure 1). Specifically, we used IgG wild type, IgG-TM, which lacks immune receptor binding, IgG-YTE, which has an extended serum half-life, and YTE-TM which combines both properties. To obtain binding curves, fluorescently labeled FcRn was kept at a constant concentration of 10 nM and titrated with the four different IgG molecules. Binding was assessed through an increase in average size of the FcRn molecule as determined by MDS on the Fluidity One-M. Figure 1. Equilibrium binding curves to analyze FcRn binding of IgG with engineered Fc regions. FcRn labeled with Alexa FluorTM 488 was used at a constant concentration of 10 nM. Errors on data points are the standard deviation from at least three measurements per point, and uncertainty on KD values are standard errors from nonlinear least squares fitting. As shown in Figure 2, IgG molecules that contain the YTE triple mutation in their Fc domains exhibited the highest binding affinities to FcRn, consistent with the extended half-life reported for this IgG variant. IgG-TM showed a slightly reduced binding affinity to FcRn as compared with IgG wild type. This affinity reduction could however be overcome by combining the TM mutations with the YTE mutations such that IgG YTE-TM benefits from both Figure 2. Overview of dissociation constants, KD, measured for FcRn binding of wild type IgG and three different Fc engineered IgG molecules. IgG-TM exhibits no immune effector function, IgG-YTE has extended serum-half life, and IgG YTE-TM combines the properties of IgG-YTE and IgG-TM. Conclusion Here, we present an in-solution based equilibrium FcRn–IgG binding assay which does not suffer from the challenges faced by surface-based, kinetic biomolecular interaction analysis and provides KD values for quantitative assessment and comparison of binding affinities. The assay requires only 20 min of hands-on time per FcRn–IgG pair and uses only 70 ng of FcRn and 30 µg of IgG. Workflow and assay features Methods Fluorescent labeling of proteins FcRn labeling with Alexa FluorTM 488 NHS ester FcRn (P55899, R&D systems) was fluorescently labeled with Alexa FluorTM 488 NHS ester (Thermo Fisher). A solution containing 100 µg of FcRn was mixed with dye at a three-fold molar excess in the presence of NaHCO3 (Merck) buffer at pH 8.3 and incubated at 4 °C overnight. Unbound label was removed using a ZebaTM desalting chromatography cartridge (7K MWCO, 1 mL, Thermo Fisher). FcRn was stored at −80 °C in PBS pH 7.4 containing 10% (w/v) glycerol. FcRn–IgG binding affinity assay IgGs were diluted into PBS containing 0.05% (v/v) Tween 20 to achieve a five-fold serial dilution series. IgG dilutions were subsequently mixed with a solution of labeled FcRn to obtain a final FcRn concentration of 10 nM. Samples were incubated for 30 min at ambient temperature prior to measurement. For MDS measurements on a Fluidity One-M instrument (Fluidic Analytics, Cambridge, UK), 3.5 µL of the FcRn–IgG mixtures were loaded on the microfluidic chip having 3.5 µL of PBS with Tween 20 (0.05% (v/v)) as the auxiliary fluid. Measurements were performed in triplicate using the (2.0–9.3) nm size range and viscosity setting 1. The binding affinity was quantified in terms of the equilibrium dissociation constant KD, which was determined by non-linear least-squares fitting. To this end, the concentration of FcRn was set to 10 nM. Equations Equations fd fraction of labeled species that diffused into the auxiliary flow of the microfluidic chip [L] total concentration of labeled species [B] equilibrium concentration of bound ρf fraction of free labeled species ρb fraction of bound labeled species [U] total concentration of binding sites in the unlabeled species α α is the fractional concentration of unlabeled species in the binding measurement KD dissociation constant (affiniy) References (1) Kang, T. H.; Jung, S. T. Boosting Therapeutic Potency of Antibodies by Taming Fc Domain Functions. Exp. Mol. Med. 2019, 51 (11), 1–9. https://doi.org/10.1038/s12276-019-0345-9. (2) Dall’Acqua, W. F.; Kiener, P. A.; Wu, H. Properties of Human IgG1s Engineered for Enhanced Binding to the Neonatal Fc Receptor (FcRn). J. Biol. Chem. 2006, 281 (33), 23514–23524. https://doi.org/10.1074/JBC.M604292200. (3) Oganesyan, V.; Gao, C.; Shirinian, L.; Wu, H.; Dall’acqua, W. F. Structural Characterization of a Human Fc Fragment Engineered for Lack of Effector Functions. Acta Cryst. 2008, D64, 700–704. https://doi.org/10.1107/ S0907444908007877. (4) Hayes, J. M.; Frostell, A.; Cosgrave, E. F. J.; Struwe, W. B.; Potter, O.; Davey, G. P.; Karlsson, R.; Anneren, C.; Rudd, P. M. Fc Gamma Receptor Glycosylation Modulates the Binding of IgG Glycoforms: A Requirement for Stable Antibody Interactions. J. Proteome Res. 2014, 13 (12), 5471–5485. https://doi.org/10.1021/PR500414Q. For research use only. Document reference: AP-0022v1 Download application note Fill out the form below to download the application note "Binding of Fc-engineered IgG antibodies to FcRn". You can print it, share it with friends and family or just use it as light reading! We hope you enjoy this application note as much as we did creating it!