Purified EDEM3 or EDEM1 alone produces determinant oligosaccharide structures from M8B in mammalian glycoprotein ERAD.

Sequential mannose trimming of N-glycan, from M9 to M8B and then to oligosaccharides exposing the α1,6-linked mannosyl residue (M7A, M6, and M5), facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). We previously showed that EDEM2 stably disulfide-bonded to the thioredoxin domain-containing protein TXNDC11 is responsible for the first step (George et al., 2020). Here, we show that EDEM3 and EDEM1 are responsible for the second step. Incubation of pyridylamine-labeled M8B with purified EDEM3 alone produced M7 (M7A and M7C), M6, and M5. EDEM1 showed a similar tendency, although much lower amounts of M6 and M5 were produced. Thus, EDEM3 is a major α1,2-mannosidase for the second step from M8B. Both EDEM3 and EDEM1 trimmed M8B from a glycoprotein efficiently. Our confirmation of the Golgi localization of MAN1B indicates that no other α1,2-mannosidase is required for gpERAD. Accordingly, we have established the entire route of oligosaccharide processing and the enzymes responsible

Characterization of anti-SARS-CoV-2 monoclonal antibodies focusing on antigen binding, neutralization, and FcγR activation via formation of immune complex

ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV−2) causes coronavirus disease 2019 (COVID−19). Antibodies induced by SARS-CoV−2 infection or vaccination play pivotal roles in the body’s defense against the virus; many monoclonal antibodies (mAbs) against SARS-CoV−2 have been cloned, and some neutralizing mAbs have been used as therapeutic drugs. In this study, we prepared an antibody panel consisting of 31 clones of anti-SARS-CoV−2 mAbs and analyzed and compared their biological activities. The mAbs used in this study were classified into different binding classes based on their binding epitopes and showed binding to the SARS-CoV−2 spike protein in different binding kinetics. A multiplex assay using the spike proteins of Alpha, Beta, Gamma, Delta, and Omicron variants clearly showed the different effects of variant mutations on the binding and neutralization activities of different binding classes of mAbs. In addition, we evaluated Fcγ receptor (FcγR) activation by immune complexes consisting of anti-SARS-CoV−2 mAb and SARS-CoV−2 pseudo-typed virus, and revealed differences in the FcγR activation properties among the binding classes of anti-SARS-CoV−2 mAbs. It has been reported that FcγR-mediated immune-cell activation by immune complexes is involved in the promotion of immunopathology of COVID−19; therefore, differences in the FcγR-activation properties of anti-SARS-CoV−2 mAbs are among the most important characteristics when considering the clinical impacts of anti-SARS-CoV−2 mAbs

Development of a cell-based assay measuring the activation of FcγRIIa for the characterization of therapeutic monoclonal antibodies.

Antibody-dependent cellular cytotoxicity (ADCC) is one of the important mechanisms of action of the targeting of tumor cells by therapeutic monoclonal antibodies (mAbs). Among the human Fcγ receptors (FcγRs), FcγRIIIa is well known as the only receptor expressed in natural killer (NK) cells, and it plays a pivotal role in ADCC by IgG1-subclass mAbs. In addition, the contributions of FcγRIIa to mAb-mediated cytotoxicity have been reported. FcγRIIa is expressed in myeloid effector cells including neutrophils and macrophages, and it is involved in the activation of these effector cells. However, the measurement of the cytotoxicity via FcγRIIa-expressing effector cells is complicated and inconvenient for the characterization of therapeutic mAbs. Here we report the development of a cell-based assay using a human FcγRIIa-expressing reporter cell line. The FcγRIIa reporter cell assay was able to estimate the activation of FcγRIIa by antigen-bound mAbs by a very simple method in vitro. The usefulness of this assay for evaluating the activity of mAbs with different abilities to activate FcγRIIa was confirmed by the examples including the comparison of the activity of the anti-CD20 mAb rituximab and its Fc-engineered variants, and two anti-EGFR mAbs with different IgG subclasses, cetuximab (IgG1) and panitumumab (IgG2). We also applied this assay to the characterization of a force-oxidized mAb, and we observed that oxidation significantly decreased the FcγRIIa activation by EGFR-bound cetuximab. These results suggest that our FcγRIIa reporter assay is a promising tool for the characterization of therapeutic mAbs, including Fc-engineered mAbs, IgG2-subclass mAbs, and their product-related variants

Establishment of the FcγR-expressing reporter cell lines.

<p>(A) The cell surface expressions of FcγRs in Jurkat/FcγR cells were analyzed by flow cytometric analysis. (B) The crosslinking of FcγRs by anti-FcγR monoclonal antibodies induced the tyrosine-phosphorylated proteins in Jurkat/FcγR cells. (C) Bridging between calcein-labeled Daudi and calcein-violet-labeled Jurkat/FcγR cells via rituximab was analyzed by flow cytometry. (D) The crosslinking of FcγRs by anti-FcγR monoclonal antibodies induced the luciferase activities in Jurkat/FcγR/NFAT-Luc cells. The assays were performed in triplicate, and the data are the mean ± SEM.</p

Activation of Jurkat/FcγR/NFAT-Luc cells by EGFR-bound cetuximab and panitumumab.

<p>A431 and Jurkat/FcγR/NFAT-Luc cells were co-cultured in the presence of serially diluted cetuximab (IgG1 subclass) or panitumumab (IgG2 subclass). The luciferase activity (i.e., the fold increase compared to the control sample without mAbs) is represented on the graphs. The assays were performed in triplicate, and the data are the mean ± SEM. Panitumumab activated Jurkat/FcγRIIa/NFAT-Luc cells more strongly than cetuximab (p<0.0001, Graphpad Prism Software).</p