Small Amounts of Sub-Visible Aggregates Enhance the Immunogenic Potential of Monoclonal Antibody Therapeutics

Purpose Determine the effect of minute quantities of sub-visible aggregates on the in vitro immunogenicity of clinically relevant protein therapeutics. Methods Monoclonal chimeric (rituximab) and humanized (trastuzumab) antibodies were subjected to fine-tuned stress conditions to achieve low levels (<3% of total protein) of subvisible aggregates. The effect of stimulating human dendritic cells (DC) and CD4(+) Tcells with the aggregates was measured in vitro using cytokine secretion, proliferation and confocal microscopy. Results Due to its intrinsic high clinical immunogenicity, aggregation of rituximab had minimal effects on DC activation and T cell responses compared to monomeric rituximab. However, in the case of trastuzumab (low clinical immunogenicity) small quantities of aggregates led to potent CD4(+) Tcell proliferation as a result of strong cytokine and co-stimulatory signals derived from DC. Consistent with this, confocal studies showed that stir-stressed rituximab was rapidly internalised and associated with late endosomes of DC. Conclusions These data link minute amounts of aggregates with activation of the innate immune response, involving DC, resulting in T cell activation. Thus, when protein therapeutics with little or no clinical immunogenicity, such as trastuzumab, contain minute amounts of sub-visible aggregates, they are associated with significantly increased potential risk of clinical immunogenicity.