The elicitation of anti-drug antibodies (ADA) against biotherapeutics can have detrimental effects on drug safety, efficacy, and pharmacokinetics. The immunogenicity of biotherapeutics is, therefore, an important issue. There is evidence that protein aggregation can result in enhanced immunogenicity; however, the precise immunological and biochemical mechanisms responsible are poorly defined. In the context of biotherapeutic drug development and safety assessment, understanding the mechanisms underlying aggregate immunogenicity is of considerable interest. This review provides an overview of the phenomenon of protein aggregation, the production of unwanted aggregates during bioprocessing, and how the immune response to aggregated protein differs from that provoked by non-aggregated protein. Of particular interest is the nature of the interaction of aggregates with the immune system and how subsequent ADA responses are induced. Pathways considered here include ‘classical’ activation of the immune system involving antigen presenting cells and, alternatively, the breakdown of B-cell tolerance. Additionally, methods available to screen for aggregation and immunogenicity will be described. With an increased understanding of aggregation-enhanced immune responses, it may be possible to develop improved manufacturing and screening processes to avoid, or at least reduce, the problems associated with ADA.

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The induction phase of contact sensitization is associated with the movement of epidermal Langerhans cells (LC ) from the skin and their migration, via afferent lymphatics, to draining lymph nodes where they accumulate as immunostimulatory dendritic cells (DC ). It has been demonstrated previously that tumour necrosis factor-a ( TNF-a) provides an important signal for LC migration and that in the absence of this cytokine, movement of LC from the epidermis to regional lymph nodes is inhibited. Recent evidence indicates that interleukin-1b (IL-1b), a cytokine produced in murine epidermis exclusively by LC, may also play a role in LC migration. The purpose of the investigations described here was to clarify, using relevant neutralizing anticytokine antibodies, the contributions made by TNF-a and IL-1b to the migration of LC from the epidermis. It was found that like anti-TNF-a, anti-IL-1b administered systemically to mice (by intraperitoneal injection), prior to skin sensitization with the contact allergen oxazolone, resulted in a marked inhibition of DC accumulation in draining lymph nodes. It was shown also that anti-IL-1b inhibited TNF-a-induced LC migration and DC accumulation and that, in similar fashion, the stimulation of LC migration and DC accumulation induced by IL-1b was compromised by prior treatment with anti-TNF-a. Based upon these data it is proposed that the stimulation of LC migration in response to skin sensitization requires the receipt by LC of two independent signals, one provided by TNF-a and the other by IL-1b. Morphological analyses of LC in epidermal sheets prepared from animals exposed to these cytokines with or without prior systemic treatment with anti-cytokine antibody suggested that the changes induced in LC by TNF-a and IL-1b may include the altered expression of adhesion molecules and acquisition of the ability to interact with and pass through the basement membrane.

The goal of eliminating animal testing in the predictive identification of chemicals with the intrinsic ability to cause skin sensitization is an important target, the attainment of which has recently been brought into even sharper relief by the EU Cosmetics Directive and the requirements of the REACH legislation. Development of alternative methods requires that the chemicals used to evaluate and validate novel approaches comprise not only confirmed skin sensitizers and non-sensitizers but also substances that span the full chemical mechanistic spectrum associated with skin sensitization. To this end, a recently published database of more than 200 chemicals tested in the mouse local lymph node assay (LLNA) has been examined in relation to various chemical reaction mechanistic domains known to be associated with sensitization. It is demonstrated here that the dataset does cover the main reaction mechanistic domains. In addition, it is shown that assignment to a reaction mechanistic domain is a critical first step in a strategic approach to understanding, ultimately on a quantitative basis, how chemical properties influence the potency of skin sensitizing chemicals. This understanding is necessary if reliable non-animal approaches, including (quantitative) structure-activity relationships (Q)SARs, read-across, and experimental chemistry based models, are to be developed.