FcγR-binding affinity of monoclonal murine IgG1s carrying different N-linked Fc oligosaccharides.

Glycosylation is one of the most common post-translational modifications which diversifies the structure and function of glycoproteins like immunoglobulin G (IgG). The effector function of IgG depends on N-glycan patterns located in the crystalline fragment (Fc). Fc gamma receptor (FcγR)-binding affinity is one of the most important effector functions in IgG, and it varies with different IgG isotypes. Murine IgG1 (mIgG1) triggers various immune effector functions via FcγRs, however, how N-glycans of mIgG1 impact interactions between mIgG1s and murine FcγRs remains largely unknown. Here, we generated mIgG1s with different N-glycan patterns by adding different types of N-glycan processing enzyme inhibitors to the hybridoma culture media, before comparing their FcγR-binding affinity using enzyme-linked immunosorbent assay (ELISA) analysis. We showed that N-glycans critically affect mIgG1 affinity to FcγRs. The removal of N-glycans nearly completely abolished mIgG1-FcγR binding. In comparison, when N-glycans are present, decreasing fucosylation levels enhanced the FcγR-binding affinity regardless of the types of N-glycans. Furthermore, high-mannose type and hybrid type N-glycans reduced FcγR-binding affinity, compared to complex type N-glycans. In conclusion, our findings clearly demonstrate that FcγR-binding affinity of mIgG1 is under the control of glycosylation. Importantly, we found that both the levels of specific glycosylation as well as the types of N-glycans affect FcγR-binding affinity. Together, these insights should greatly expand our understanding of N-glycans function in general, and assist in manipulating host immune responses by controlling antibody N-glycan patterns, which is important for designing therapeutic antibodies with improved characteristics.

Avelumab internalization by human circulating immune cells is mediated by both Fc gamma receptor and PD-L1 binding.

Avelumab is an IgG1 anti-programmed death ligand 1 (anti-PD-L1) monoclonal antibody that has been approved as a monotherapy for metastatic Merkel cell carcinoma and advanced urothelial carcinoma, and in combination with axitinib for advanced renal cell carcinoma. Avelumab is cleared faster and has a shorter half-life than other anti-PD-L1 antibodies, such as atezolizumab and durvalumab, but the mechanisms underlying these differences are unknown. IgG antibodies can be cleared through receptor-mediated endocytosis after binding of the antibody Fab region to target proteins, or via Fcγ receptor (FcγR)-mediated endocytosis. Unlike other approved anti-PD-L1 antibodies, avelumab has a native Fc region that retains FcγR binding capability. We hypothesized that the rapid clearance of avelumab might be due to the synergistic effect of both FcγR-mediated and PD-L1 target-mediated internalization. To investigate this, we performed in vitro and in vivo studies that compared engineered variants of avelumab and atezolizumab to determine mechanisms of cellular internalization. We found that both FcγR and PD-L1 binding contribute to avelumab internalization. While FcγR binding was the dominant mechanism of avelumab internalization in vitro, with CD64 acting as the most important FcγR, studies in mice and cynomolgus monkeys showed that both FcγR and PD-L1 contribute to avelumab elimination, with PD-L1 binding playing a greater role. These studies suggest that the rapid internalization of avelumab might be due to simultaneous binding of both PD-L1 and FcγR in trans. Our findings also provide a basis to alter the clearance and half-life of monoclonal antibodies in therapeutic development.

The Impact of Immunoglobulin G1 Fc Sialylation on Backbone Amide H/D Exchange.

The usefulness of higher-order structural information provided by hydrogen/deuterium exchange-mass spectrometry (H/DX-MS) for the structural impact analyses of chemical and post-translational antibody modifications has been demonstrated in various studies. However, the structure-function assessment for protein drugs in biopharmaceutical research and development is often impeded by the relatively low-abundance (below 5%) of critical quality attributes or by overlapping effects of modifications, such as glycosylation, with chemical amino acid modifications; e.g., oxidation or deamidation. We present results demonstrating the applicability of the H/DX-MS technique to monitor conformational changes of specific Fc glycosylation variants produced by in vitro glyco-engineering technology. A trend towards less H/DX in Fc Cγ2 domain segments correlating with larger glycan structures could be confirmed. Furthermore, significant deuterium uptake differences and corresponding binding properties to Fc receptors (as monitored by SPR) between α-2,3- and α-2,6-sialylated Fc glycosylation variants were verified at sensitive levels.

Crystal structure of a novel asymmetrically engineered Fc variant with improved affinity for FcγRs.

Enhancing the effector function by optimizing the interaction between Fc and Fcγ receptor (FcγR) is a promising approach to enhance the potency of anticancer monoclonal antibodies (mAbs). To date, a variety of Fc engineering approaches to modulate the interaction have been reported, such as afucosylation in the heavy chain Fc region or symmetrically introducing amino acid substitutions into the region, and there is still room to improve FcγR binding and thermal stability of the CH2 domain with these approaches. Recently, we have reported that asymmetric Fc engineering, which introduces different substitutions into each Fc region of heavy chain, can further improve the FcγR binding while maintaining the thermal stability of the CH2 domain by fine-tuning the asymmetric interface between the Fc domain and FcγR. However, the structural mechanism by which the asymmetrically engineered Fc improved FcγR binding remained unclear. In order to elucidate the mechanism, we solved the crystal structure of a novel asymmetrically engineered Fc, asym-mAb23, in complex with FcγRIIIa. Asym-mAb23 has enhanced binding affinity for both FcγRIIIa and FcγRIIa at the highest level of previously reported Fc variants. The structural analysis reveals the features of the asymmetrically engineered Fc in comparison with symmetric Fc and how each asymmetrically introduced substitution contributes to the improved interaction between asym-mAb23 and FcγRIIIa. This crystal structure could be utilized to enable us to design a more potent asymmetric Fc.

Effects of altered FcγR binding on antibody pharmacokinetics in cynomolgus monkeys.

Antibody interactions with Fcγ receptors (FcγRs), like FcγRIIIA, play a critical role in mediating antibody effector functions and thereby contribute significantly to the biologic and therapeutic activity of antibodies. Over the past decade, considerable work has been directed towards production of antibodies with altered binding affinity to FcγRs and evaluation of how the alterations modulate their therapeutic activity. This has been achieved by altering glycosylation status at N297 or by engineering modifications in the crystallizable fragment (Fc) region. While the effects of these modifications on biologic activity and efficacy have been examined, few studies have been conducted to understand their effect on antibody pharmacokinetics (PK). We present here a retrospective analysis in which we characterize the PK of three antibody variants with decreased FcγR binding affinity caused by amino acid substitutions in the Fc region (N297A, N297G, and L234A/L235A) and three antibody variants with increased FcγRIIIA binding affinity caused by afucosylation at N297, and compare their PK to corresponding wild type antibody PK in cynomolgus monkeys. For all antibodies, PK was examined at a dose that was known to be in the linear range. Since production of the N297A and N297G variants in Chinese hamster ovary cells results in aglycosylated antibodies that do not bind to FcγRs, we also examined the effect of expression of an aglycosylated antibody, without sequence change(s), in E. coli. All the variants demonstrated similar PK compared with that of the wild type antibodies, suggesting that, for the six antibodies presented here, altered FcγR binding affinity does not affect PK.