Abstract
Therapeutic antibodies have the potential to induce immunogenicity leading to the development of anti-drug antibodies (ADA) that consequently may result in reduced serum drug concentrations, a loss of efficacy or potential hypersensitivity reactions. Among other factors, aggregated antibodies have been suggested to promote immunogenicity, thus enhancing ADA production. Dendritic cells (DC) are the most efficient antigen-presenting cell population and are crucial for the initiation of T cell responses and the subsequent generation of an adaptive immune response. This work focuses on the development of predictive in vitro assays that can monitor DC maturation, in order to determine whether drug products have direct DC stimulatory capabilities. To this end, four independent laboratories aligned a common protocol to differentiate human monocyte-derived DC (moDC) that were treated with either native or aggregated preparations of infliximab, natalizumab, adalimumab, or rituximab. These drug products were subjected to different forms of physical stress, heat and shear, resulting in aggregation and the formation of subvisible particles. Each partner developed and optimized assays to monitor diverse end-points of moDC maturation: measuring the upregulation of DC activation markers via flow cytometry, analyzing cytokine, and chemokine production via mRNA and protein quantification and identifying cell signaling pathways via quantification of protein phosphorylation. These study results indicated that infliximab, with the highest propensity to form aggregates when heat-stressed, induced a marked activation of moDC as measured by an increase in CD83 and CD86 surface expression, IL-1β, IL-6, IL-8, IL-12, TNFα, CCL3, and CCL4 transcript upregulation and release of respective proteins, and phosphorylation of the intracellular signaling proteins Syk, ERK1/2, and Akt. In contrast, natalizumab, which does not aggregate under these stress conditions, induced no DC activation in any assay system, whereas adalimumab or rituximab aggregates induced only slight parameter variation. Importantly, the data generated in the different assay systems by each partner site correlated and supported the use of these assays to monitor drug-intrinsic propensities to drive maturation of DC. This moDC assay is also a valuable tool as an in vitro model to assess the intracellular mechanisms that drive DC activation by aggregated therapeutic proteins.
Highlights
The clinical use of therapeutic antibodies has enabled significant improvements in the treatment of an increasing number of severe diseases
The presence of aggregates in biotherapeutics has been correlated with anti-drug antibodies (ADA) development in patients and many efforts are currently ongoing in an attempt to dissect the cellular mechanisms involved in immunogenicity
The aim of this study was to optimize in vitro methods to evaluate the potential of therapeutic antibodies and aggregated preparations on therapeutic antibodies to induce dendritic cells (DC) maturation, as these professional antigenpresenting cells have a pivotal role in triggering adaptive immune responses that would in fine lead to ADA production [27]
Summary
The clinical use of therapeutic antibodies has enabled significant improvements in the treatment of an increasing number of severe diseases. All biopharmaceuticals have immunogenic potential in patients, leading to the development of anti-drug antibodies (ADA) that may have neutralizing effects on the drug, resulting in reduced effective concentrations of the therapeutic biopharmaceutical in serum, and a potentially reduced clinical response [1, 2]. Immunogenicity of therapeutic antibodies has been studied in the context of inflammatory diseases. ADA development in patients has been reported with variable frequencies, depending on clinical studies that include different patient populations, as well as on the employed detection method. The humanization status of the administrated antibody allows for a potential reduction in immunogenicity, it is rarely abolished. ADA frequencies against chimeric antibodies such as infliximab or rituximab may vary from 10 to 50% [6,7,8], whereas ADA frequencies in patients treated with the fully human antibody adalimumab may range from 20 to 25% [6, 9, 10] and patients treated with the humanized antibody natalizumab may develop ADA with a frequency of ∼6–10% [11, 12]
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