Binding Kinetics of Anti-CD20 Antibodies

The binding kinetics of chimeric anti-CD20 mutant antibodies for each shFcγR (shFcγRI, shFcγRIIa, shFcγRIIIa-158V, shFcγRIIIa-158F, and shFcγRIIIb) were analyzed by surface plasmon resonance (SPR) measurement using a T100 biosensor instrument and CM5 sensor chips (BIAcore; GE Healthcare, Pittsburgh, PA), as described previously [24 (link)]. Briefly, assays were performed with anti-tetra-His antibody-immobilized CM5 sensor chips using an Amine Coupling Kit (BIAcore). The individual hexa-His-tagged shFcγRs were captured by the immobilized anti-tetra-His antibodies at a flow rate of 5 μL/min. Antibodies were diluted in HBS-EP+ Buffer (BIAcore) at various concentrations (for shFcγRI and shFcγRIIIa-158V: from 4 to 267 nM; for shFcγRIIa, shFcγRIIIa-158F, and shFcγRIIIb: from 8 to 534 nM), and each diluted antibody was injected into the shFcγRs-coated sensor chip at a flow rate of 30 μL/min. The experiments were performed with HBS-EP+ as the running buffer at 25°C. The shFcγRs and antibodies bound to the sensor surface were removed by injecting 10 mM HCl. The data obtained by the injection of antibodies were corrected for the blank control prior to data analysis. The dissociation constant (K_D) for each shFcγR was calculated by steady-state analysis using BIAcore T100 kinetic evaluation software (BIAcore).