Subvisible proteinaceous particles which are present in all therapeutic protein formulations are in the focus of intense discussions between health authorities, academics and biopharmaceutical companies in the context of concerns that such particles could promote unwanted immunogenicity via anti-drug antibody formation. In order to provide further understanding of the subject, this study closely examines the specific biological effects proteinaceous particles may exert on dendritic cells (DCs) as the most efficient antigen-presenting cell population crucial for the initiation of the adaptive immune response. Two different model IgG antibodies were subjected to three different types of exaggerated physical stress to generate subvisible particles in far greater concentrations than the ones typical for the currently marketed biotherapeutical antibodies. The aggregated samples were used in in vitro biological assays in order to interrogate the early DC-driven events that initiate CD4 T-cell dependent humoral adaptive immune responses – peptide presentation capacity and co-stimulatory activity of DCs. Most importantly, antigen presentation was addressed with a unique approach called MHC-associated Peptide Proteomics (MAPPs), which allows for identifying the sequences of HLA-DR associated peptides directly from human dendritic cells.The experiments demonstrated that highly aggregated solutions of two model mAbs generated under controlled conditions can induce activation of human monocyte-derived DCs as indicated by upregulation of typical maturation markers including co-stimulatory molecules necessary for CD4 T-cell activation. Additional data suggest that highly aggregated proteins could induce in vitro T-cell responses. Intriguingly, strong aggregation-mediated changes in the pattern and quantity of antigen-derived HLA-DR associated peptides presented on DCs were observed, indicating a change in protein processing and presentation. Increasing the amounts of subvisible proteinaceous particles correlated very well with the pronounced increase in the peptide number and clusters presented in the context of class II HLA-DR molecules, suggesting a major involvement of a mass-action mechanism of altering the presentation.

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In this study we show that glycosylation is relevant for immune recognition of therapeutic antibodies, and that defined glycan structures can modulate immunogenicity. Concerns regarding immunogenicity arise from the high heterogeneity in glycosylation that is difficult to control and can deviate from human glycosylation if produced in non-human cell lines. While non-human glycosylation is thought to cause hypersensitivity reactions and immunogenicity, less is known about effects of Fc-associated glycan structures on immune cell responses. We postulated that glycosylation influences antigen recognition and subsequently humoral responses to therapeutic antibodies by modulating 1) recognition and uptake by dendritic cells (DCs), and 2) antigen routing, processing and presentation. Here, we compared different glycosylation variants of the antibody rituximab (RTX) in in vitro assays using human DCs and T cells as well as in in vivo studies.We found that human DCs bind and internalize unmodified RTX stronger compared to its aglycosylated form suggesting that glycosylation mediates uptake after recognition by glycan-specific receptors. Furthermore, we show that DC-uptake of RTX increases or decreases if glycosylation is selectively modified to recognize activating (by mannosylation) or inhibitory lectin receptors (by sialylation). Moreover, glycosylation seems to influence antigen presentation by DCs because specific glycovariants tend to induce either stronger or weaker T cell activation. Finally, we demonstrate that antibody glycosylation impacts anti-drug antibody (ADA) responses to RTX in vivo. Hence, defined glycan structures can modulate immune recognition and alter ADA responses. Glyco-engineering may help to

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