Towards an optimized in-vitro SPR assay for antibody Fcg receptor binding kinetics Åsa Frostell 1, Robert Karlsson 1, Jerrard Hayes 2, Matilda Lindgren 1, Pauline Rudd 2, and Cecilia Annerén 1 1 GE Healthcare Bio-Sciences AB, Rapsgatan 23, SE-751 84 Uppsala, Sweden 2 National Institute for Bioprocessing, Research and Training, Dublin, Ireland First published in Sep 2012 at BEBPA, Lisbon, Portugal
Introduction Therapeutic antibodies are now approved for a number of different indications. One important antibody (Ab) attribute is its Fc effector function, and Abs are now being engineered with the aim of obtaining desired binding of the Ab Fc region to Fc receptors (FcR) on immune effector cells. Optimized and standardized FcR assays may impact the understanding of the function FcR-Ab interactions. They can be used through all stages of antibody development, process development and QC; they may also play an important role in comparability studies and for the evaluation of biosimilars. 2
Introduction The use of Biacore as a biochemical assay for studies of FcR Ab Fc binding is already established in this area; however there is a lack of consensus regarding assay set up and evaluation of the often complex binding kinetics. Examples of published assay setups include amine coupling of FcR (1), anti-his capture of His tagged FcR (2), amine coupling of Ab (3), Protein A capture of Ab (4), anti-kappa F(ab )2 capture of Ab (5), anti-fab capture of Ab (6). In the present study, assay setups and kinetic evaluations using Biacore T200 are discussed. Results using commercially available, recombinant extracellular FcgRs from two different expression systems, HEK293 (Sino Biological) and NS0 cells (R&D Systems), are presented. 3
Kinetics depend on assay setup Rituximab binding to Fcg receptors After feasibility studies of most of the published Biacore assay setups, we focused on and compared two formats, with the aim of finding a setup suitable for screening of Ab - FcgR binding. Protein A capture of Ab was compared more closely with anti-his mab capture of His-tagged FcR. 4
Kinetics depend on assay setup Significant differences in kinetic data were found upon visual comparison of sensorgrams, depending on whether the FcR or the Ab was immobilized. 5
His-tag FcγR capture method Therap. Ab FcR Anti-His Assay condition details: Sensor chip CM5 with 6000 to 10000 RU amine coupled anti-his mab (from His Capture Kit) in active and ref. flow cell Running buffer: PBS-P+ Capture: 0.5 1 μg/ml His-tagged FcR, 60 s injection, 5 μl/min in active flow cell Capture stabilization time, 1 3 min, added for some FcRs. Capture levels for these FcRs: 40 200 RU Single cycle kinetics (2-5 injections) over active and reference flow cell. 1-300 nm Ab conc. series for FcγRI, 25-2000 nm for FcγRIIIa. 60 s injection at 30 μl/min. Regeneration: 10 mm Glycine, ph 1.5, 30 s Blank cycles (FcR capture + buffer injections) performed first, last and upon change of FcR + The product (Ab) is in solution, retaining full flexibility + Avoiding Protein A and possible risk of Ab binding also with Fab part + Ab is more often than FcR available in the high concentrations needed to measure week interactions + Oriented capture of His tagged FcR. Neutral buffer for immobilization. + Single cycle kinetics consumes low amounts of FcR + His capture kit adds convenience. Easy switch of different FcRs and Abs. - Need to know Ab concentration 6
Specific capture of FcR His-tagged Fcγ receptors binding to immobilized anti-his Ab (blue bar) compared to binding to dextran and to immobilized irrelevant Abs. All IgG1. Specific binding of FcγRs to the anti-his Ab was obtained. Binding to irrelevant Abs were negligible (< 7% of binding to anti-his). 7
FcgRIIIa Two state model 8
FcgRI Heterogeneous ligand model 9
Antibody binding to Fcγ receptors from different cell lines 10
IgG3, with different glycosylation patterns, binding to FcγRIIIa Binding kinetics for IgG3 binding to FcγRIIIa F158 (HEK) Glycosylation analysis of IgG3 by UHPLC. Significant kinetic differences betwee the IgG3 preparations IgG3, Sigma: heterogenous collection N-glycans; minor sialylation (11.5%) and extensive core fucosylation (91% IgG3, The Binding Site and Millipore: very different glycan profiles, albeit identical to each other. Higher levels of sialylation, monosialylated structures in 19.5% of glycans, di-sialylated in 1.5%. Core fucosylatio in only 23% of glycans. Differences in e.g. core fucosylation could possibly be used to explain the binding differences, a phenomenon previously reported for IgG1 binding to FcγRIIIa*. *Lifely et al. Glycobiol, 1995. Shields et al, J Biol Chem, 2002. Shinkawa et al, J Biol Chem 2003, and others. 11
Conclusions An His FcR capture assay was found suitable for antibody-fcgr binding studies. Antibodies exhibited complex binding to both FcgRI and FcgRIIIa receptors. Binding data indicated different binding mechanisms to FcgRI and FcgRIIIa respectively. Binding to FcgRI could be modeled as two independent reactions, while binding to FcgRIIIa could be modeled by assuming two states of the reaction. Significant differences in binding to FcgRIIIa were observed for IgG3 preparations with differences in glycosylation pattern. 12
References 1. Bruhns P., et al. Blood, 2009, 113, 3716-25. 2. Shibata-Koyama M., et al. Glycobiology, 2009, 19, 126-134. 3. Hatayama K., et al. Appl Microbiol Biotechnol, 2012, 94, 1051-59. 4. Heider K., et al. Blood, 2011, 118, 4159-68. 5. Ha S., et al. Glycobiology, 2011, 21, 1087-96. 6. Ferrara C., et al. PNAS, 2011, 108, 12669-74. 7. Starovasnik M., et al. Protein Science, 1999, 8, 1423-31. 8. Royle L., et al. Methods Mol Biol. 2006, 347, 125-43. 9. Ferrara C., et al. Biotech Bioeng, 2006, 93, 51-61. 13
Acknowledgment 2013 General Electric Company - All rights reserved. GE, imagination at work, and GE monogram are trademarks of General Electric Company. Biacore is a trademark of GE Healthcare companies. All goods and services are sold subjects to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. First published Sep 2012. 14