Biotinylation of the Fcγ receptor ectodomains by mammalian cell co-transfection: application to the development of a surface plasmon resonance-based assay

Abstract

We here report the production of four biotinylated Fcγ receptor (FcγR) ectodomains and their subsequent stable capture on streptavidin-biosensor surfaces. For receptor biotinylation, we first describe an in-cell protocol based on the co-transfection of two plasmids corresponding to one of the FcγR ectodomains and the BirA enzyme in mammalian cells. This strategy is compared with a standard sequential in vitro enzymatic biotinylation with respect to biotinylation level and yield. Biotinylated FcγR ectodomains that have been prepared with both strategies are then compared by analytical ultracentrifugation and surface plasmon resonance (SPR) analyses. Overall, we demonstrate that in-cell biotinylation is an interesting alternative to standard biotinylation protocol, as it requires less purification steps while yielding higher titers. Finally, biotin-tagged FcγRs produced with the in-cell approach are successfully applied to the development of SPR-based assays to evaluate the impact of the glycosylation pattern of monoclonal antibodies on their interaction with CD16a and CD64. In that endeavor, we unambiguously observe that highly galactosylated trastuzumab (TZM-gal), non-glycosylated trastuzumab (TZM-NG), and reference trastuzumab are characterized by different kinetic profiles upon binding to CD16a and CD64 that had been captured at the biosensor surface via their biotin tag. More precisely, while TZM-NG binding to CD16a was not detected, TZM-gal formed a more stable complex with CD16a than our reference TZM. In contrast, both glycosylated TZM bound to captured CD64 in a stable and similar fashion, whereas the interaction of their non-glycosylated form with CD64 was characterized by a higher dissociation rate.

Impact of N-glycosylation on Fcγ receptor / IgG interactions: unravelling differences with an enhanced surface plasmon resonance biosensor assay based on coiled-coil interactions

“…The FcγRIIIa/TZM dissociation occurred within less than 180 s (F158) and 480 s (V158) and were biphasic, in excellent agreement with other studies. 5,22 Steady-state analysis (insets in Figure 3(a,b)) of the TZM/ FcγRIIIa interactions (three independent repetitions for both F158 and V158 variants) was performed, assuming a one-toone interaction for the sake of comparison with values reported in the literature. The apparent K D values (4,015 ± 214 nM for FcγRIIIa F158 and 717 ± 27 nM for FcγRIIIa V158 ) were within the same order of magnitude as those previously reported (Table 1).…”

“…The apparent K D values (4,015 ± 214 nM for FcγRIIIa F158 and 717 ± 27 nM for FcγRIIIa V158 ) were within the same order of magnitude as those previously reported (Table 1). 5,21,25,26,38 Differences in binding kinetics between captured E5-tagged FcγRs and several TZM glycoform preparations were then assessed. To this end, our reference TZM (a TZM preparation with low galactosylation level), TZM-gal (a TZM preparation with high galactosylation level), TZM-afuc (a TZM preparation with low fucosylation level) and TZM-ng (an aglycosylated N297Q TZM mutant) were injected in triplicate at 1,000 nM over captured E5-tagged FcγRs.…”

“…4 In contrast, the type II (FcγRIIa-c or CD32a-c) and type III (FcγRIIIa/b or CD16a/b) receptors display lower affinities for IgGs (apparent K D s of ≈ 10 -6 -10 -7 M). 5 Depending on the immune cell type and the receptors engaged, interactions between IgGs and FcγRs trigger effector functions such as phagocytosis, antigen presentation, secretion of soluble factors (e.g. cytokines) and antibodydependent cell-mediated cytotoxicity.…”

“…23 Surface plasmon resonance (SPR)-based assays have been extensively applied to characterize FcγR-IgG interactions and assess the effect of glycosylation on binding. 5,[10][11][12][20][21][22][23][24][25][26] However, up to now, the use of diverse SPR assays has led to the recording of kinetic and affinity constants that are hard to compare and interpret from one report to another. 25,27 Indeed, these differences may be attributed to variations in IgG and FcγR glycosylation profiles, IgG aggregation state, as well as to biases emanating from biosensor surface preparation.…”

“…[22][23][24]26 We demonstrated that a capture approach relying on the recruitment of biotinylated FcγRs on streptavidin-decorated biosensor surfaces eliminated this artifact, but the numerous sequential injections required for the assay negatively impacted reproducibility. 5 Altogether, these studies have highlighted the critical importance of an appropriate FcγR tethering strategy for the development of a standardized SPRbased biosensor assay for routine and robust analysis.…”