Mechanisms of therapeutic antibodies immunogenicity: infliximab oligomeric aggregates increase T-cell response and uptake by dendritic cells involving mannose-sensitive receptor CD206.

Introduction: Aggregation of therapeutic antibodies (Ab)s is now recognized as a major product-related factor associated with increased potential of immunogenicity. Regulatory agencies have established limits for the presence of (sub)visible particles in commercialized products. These particles are considered a potential risk factor for the development of unwanted immune responses. However, little is known concerning the role of oligomeric Abs aggregates of nanometric size in Abs' immunogenicity. The objective of this work was to better understand the role and the mechanisms of T-cell response to nanometric Ab aggregates using infliximab (IFX) as a model. Method: We produced nanosized IFX aggregates by exposing native IFX to ultraviolet light. These aggregates were characterized using size exclusion chromatography (SEC), dynamic light scattering (DLS) and field flow fractionation coupled with a multi-angle light scattering detector (FFF-MALS). We then investigated the impact of IFX aggregation on its uptake by dendritic cells and the subsequent activation of IFX-specific T-cell responses. Results: Using a human in vitro autologous dendritic cell (DC)/T-cells coculture assay, we showed that aggregation increased the frequency of responsive T cells to IFX. Moreover, we observed exclusive T-cell responses to IFX aggregates and different TCR clonotypes responding to each form of IFX. Using fluorescent native or aggregated IFX, we found a significantly higher internalization rate of aggregated IFX by DCs in a mannose-dependent manner. Confocal microscopy experiments showed that aggregates were rapidly internalized and associated with markers of early endosomes and lysosomes as well as antigen presentation molecules, compared to the native form of IFX. Discussion: Our results suggest that internalization of IFX oligomeric aggregates via the mannose receptor pathway significantly contributes to enhanced IFX uptake by DCs and increased routing into the degradative pathway for processing into peptides. Increased presentation of immunogenic peptides may contribute to the augmentation of T-cell recruitment with aggregates compared to the native form. .