title: Small amounts of sub-visible aggregates enhance the immunogenic potential of monoclonal antibody therapeutics.
abstract: OBJECTIVE
  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.
authors.affiliation: Antitope Ltd, Babraham Research Campus, Babraham, Cambridge, CB22 3AT, UK.
authors.forename: Maryam
authors.initials: M
authors.lastname: Ahmadi
authors.forename: Christine J
authors.initials: CJ
authors.lastname: Bryson
authors.forename: Edward A
authors.initials: EA
authors.lastname: Cloake
authors.forename: Katie
authors.initials: K
authors.lastname: Welch
authors.forename: Vasco
authors.initials: V
authors.lastname: Filipe
authors.forename: Stefan
authors.initials: S
authors.lastname: Romeijn
authors.forename: Andrea
authors.initials: A
authors.lastname: Hawe
authors.forename: Wim
authors.initials: W
authors.lastname: Jiskoot
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Baker
authors.forename: Mark H
authors.initials: MH
authors.lastname: Fogg
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects* / Q000276:immunology; D002478:Cells, Cultured; D016207:Cytokines / Q000276:immunology; D003713:Dendritic Cells / Q000187:drug effects* / Q000276:immunology; D004355:Drug Stability; D006801:Humans; D007113:Immunity, Innate / Q000187:drug effects; D008213:Lymphocyte Activation / Q000187:drug effects*; D066329:Protein Aggregates / Q000276:immunology*; D000069283:Rituximab / Q000276:immunology*; D000068878:Trastuzumab / Q000276:immunology*
chemical_list: D016207:Cytokines; D066329:Protein Aggregates; D000069283:Rituximab; D000068878:Trastuzumab