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 sub-visible aggregates. The effect of stimulating human dendritic cells (DC) and CD4+ T cells 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+ T cell 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.
CITATION STYLE
Ahmadi, M., Bryson, C. J., Cloake, E. A., Welch, K., Filipe, V., Romeijn, S., … Fogg, M. H. (2015). Small amounts of sub-visible aggregates enhance the immunogenic potential of monoclonal antibody therapeutics. Pharmaceutical Research, 32(4), 1383–1394. https://doi.org/10.1007/s11095-014-1541-x
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