Glycosylation of Fcγ receptors influences their interaction with various IgG1 glycoforms.

Most of therapeutic monoclonal antibodies belong to the immunoglobulin G1 (IgG1) family; they interact with the Fcγ receptors (FcγRs) at the surface of immune cells to trigger effector functions. The IgG1-Fc N-glycans impact the interaction with FcγRs and are considered a critical quality attribute. Pioneer studies on FcγR N-glycans have unveiled an additional complexity in that the N-glycan linked on the Asn-162 of FcγRIIIa was shown to be directly involved in the strong affinity for afucosylated IgG1. The last few years have thus seen the emergence of many studies investigating the complex influence of FcγRIIIa N-glycans on the interaction with IgG1 through their glycosylation sites or their glycoprofiles. In this context, we performed site-directed mutagenesis along with glycoengineering on FcγRs (FcγRI, FcγRIIaH131/b and FcγRIIIaV158/F158) in an effort to elucidate the impact of FcγRs N-glycans on the interaction with IgG1. Furthermore, we assessed their binding to various trastuzumab glycoforms with an enhanced surface plasmon resonance assay. The FcγRIIIa N-glycans had the highest impact on the interaction with IgG1. More specifically, the N162 glycan positively influenced the affinity (15-fold) for afucosylated IgG1 while the N45 glycan presented a negative impact (2-fold) regardless of the IgG1 glycoforms. Interestingly, only the FcγRIIIa glycoprofile had an impact on the interaction with IgG1 with a 1.5-fold affinity increase when FcγRIIIa displays high-mannose glycans. These results provide invaluable insights into the complex and strong influence of N-glycosylation upon FcγRs/IgG1 binding and are instrumental to further understand the impact of FcγRs N-glycosylation in their natural forms.

Impact of IgG1 N-glycosylation on their interaction with Fc gamma receptors.

The effector functions of the IgGs are modulated by the N-glycosylation of their Fc region. Particularly, the absence of core fucosylation is known to increase the affinity of IgG1s for the Fcγ receptor IIIa expressed by immune cells, in turn translating in an improvement in the antibody-dependent cellular cytotoxicity. However, the impact of galactosylation and sialylation is still debated in the literature. In this study, we have investigated the influence of high and low levels of core fucosylation, terminal galactosylation and terminal α2,6-sialylation of the Fc N-glycans of trastuzumab on its affinity for the FcγRIIIa. A large panel of antibody glycoforms (i.e., highly α2,6-sialylated or galactosylated IgG1s, with high or low levels of core fucosylation) were generated and characterized, while their interactions with the FcγRs were analysed by a robust surface plasmon resonance-based assay as well as in a cell-based reporter bioassay. Overall, IgG1 glycoforms with reduced fucosylation display a stronger affinity for the FcγRIIIa. In addition, fucosylation, and the presence of terminal galactose and sialic acids are shown to increase the affinity for the FcγRIIIa as compared to the agalactosylated forms. These observations perfectly translate in the response observed in our reporter bioassay.

Surface Plasmon Resonance-Based Method for Rapid Product Sialylation Assessment in Cell Culture.

To streamline cell culture process development, surface plasmon resonance (SPR) biosensors offer a versatile platform for the rapid quantification and quality analysis of recombinant proteins. As a representative case study, the present chapter details a procedure employing a SPR biosensor for determining the differential sialylation levels of recombinant interferon α2b contained in cell culture samples, using immobilized Sambucus nigra lectin. Of interest, this semiquantitative approach can be adapted to work with other lectins with unique carbohydrate-binding specificities, enabling a wide range of product characterization analysis.

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 N-glycosylation profile of immunoglobulin G (IgG) is considered a critical quality attribute due to its impact on IgG-Fc gamma receptor (FcγR) interactions, which subsequently affect antibody-dependent cell-based immune responses. In this study, we investigated the impact of the FcγR capture method, as well as FcγR N-glycosylation, on the kinetics of interaction with various glycoforms of trastuzumab (TZM) in a surface plasmon resonance (SPR) biosensor assay. More specifically, we developed a novel strategy based on coiled-coil interactions for the stable and oriented capture of coil-tagged FcγRs at the biosensor surface. Coil-tagged FcγR capture outperformed all other capture strategies applied to the SPR study of IgG-FcγR interactions, as the robustness and reproducibility of the assay and the shelf life of the biosensor chip were excellent (> 1,000 IgG injections with the same biosensor surface). Coil-tagged FcγRs displaying different N-glycosylation profiles were generated either by different expression systems, in vitro glycoengineering or by size-exclusion chromatography, and roughly characterized by lectin blotting. Of salient interest, the overlay of their kinetics of interaction with several TZM glycoforms revealed key differences on both association and dissociation kinetics, confirming a complex influence of the FcγR N-glycosylation and its inherent heterogeneity upon receptor interaction with mAbs. This work is thus an important step towards better understanding of the impact of glycosylation upon binding of IgGs, either natural or engineered, to their receptors.

Process development for an inducible rituximab-expressing Chinese hamster ovary cell line.

Inducible mammalian expression systems are becoming increasingly available and are not only useful for the production of cytotoxic/cytostatic products, but also confer the unique ability to uncouple the growth and production phases. In this work, we have specifically investigated how the cell culture state at the time of induction influences the cumate-inducible expression of recombinant rituximab by a GS-CHO cell line. To this end, cells grown in batch and fed-batch cultures were induced at increasing cell densities (1 to 10 × 10 6 cells/mL). In batch, the cell specific productivity and the product yield were found to reduce with increasing cell density at induction. A dynamic feeding strategy using a concentrated nutrient solution applied prior and postinduction allowed to significantly increase the integral of viable cells and led to a 3-fold increase in the volumetric productivity (1.2 g/L). The highest product yields were achieved for intermediate cell densities at induction, as cultures induced during the late exponential phase (10 × 10 6 cells/mL) were associated with a shortened production phase. The final glycosylation patterns remained however similar, irrespective of the cell density at induction. The kinetics of growth and production in a 2 L bioreactor were largely comparable to shake flasks for a similar cell density at induction. The degree of galactosylation was found to decrease over time, but the final glycan distribution at harvest was consistent to that of the shake flasks cultures. Taken together, our results provide useful insights for the rational development of fed-batch cell culture processes involving inducible CHO cells. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2742, 2019.

Real-time QCM-D monitoring of cancer cell death early events in a dynamic context.

Since a few years, the acoustic sensing of whole cell is the focus of increasing interest for monitoring the cytoskeletal cellular response to morphological modulators. We aimed at illustrating the potentialities of the quartz crystal microbalance with dissipation (QCM-D) technique for the real-time detection of the earliest morphological changes that occur at the cell-substrate interface during programmed cell death. Human breast cancer cells (MCF-7) grown on serum protein-coated gold sensors were placed in dynamic conditions under a continuous medium flow. The mass and viscoelasticity changes of the cells were tracked by monitoring the frequency and dissipation shifts during the first 4h of cell exposure to staurosporine, a well-known apoptosis inducer. We have identified a QCM-D signature characteristic of morphological modifications and cell detachment from the sensing surface that are related to the pro-apoptotic treatment. In particular, for low staurosporine doses below 1 µM, we showed that recording the dissipation shift allows to detect an early cell response which is undetectable after the same duration by the classical analytical techniques in cell biology. Furthermore, this sensing method allows quantifying the efficiency of the drug effect in less than 4h without requiring labeling and without interfering in the system, thus preventing any loss of information. In the actual context of targeted cancer therapy development, we believe that these results bring new insights in favor of the use of the non invasive QCM-D technique for quickly probing the cancer cell sensitivity to death inducer drugs.