Small amounts of sub-visible aggregates enhance the immunogenic potential of monoclonal antibody therapeutics

Pharm Res. 2015 Apr;32(4):1383-94. doi: 10.1007/s11095-014-1541-x.


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.

MeSH terms

  • CD4-Positive T-Lymphocytes / drug effects*
  • CD4-Positive T-Lymphocytes / immunology
  • Cells, Cultured
  • Cytokines / immunology
  • Dendritic Cells / drug effects*
  • Dendritic Cells / immunology
  • Drug Stability
  • Humans
  • Immunity, Innate / drug effects
  • Lymphocyte Activation / drug effects*
  • Protein Aggregates / immunology*
  • Rituximab / immunology*
  • Trastuzumab / immunology*


  • Cytokines
  • Protein Aggregates
  • Rituximab
  • Trastuzumab