Biomarker context-of-use: how organizational design can impact the implementation of the appropriate biomarker assay strategy.

Since 2011, the European Bioanalysis Forum has been discussing the topic of context-of-use for biomarker assays, in support of a cross-industry implementation of its principles. The discussions have led to the acknowledgement of the challenges that we face as an industry in implementing these principles. In addition to scientific recommendations, the European Bioanalysis Forum has addressed these challenges by providing recommendations on organizational design, and what works in both sponsor and contract research organizations, to support and enable context-of-use across biomarker strategies. Here, we highlight the key considerations for organizational design to help ensure that biomarker assays are characterized and validated according to the right context-of-use, to ensure that the right decisions based on the biomarker data can be made during drug development.

Massive immune response against IVIg interferes with response against other antigens in mice: A new mode of action?

Administration of high dose intravenous immunoglobulin (IVIg) is widely used in the clinic to treat autoimmune and severe inflammatory diseases. However, its mechanisms of action remain poorly understood. We assessed the impact of IVIg on immune cell populations using an in vivo ovalbumin (Ova)-immunization mouse model. High dose IVIg significantly reduced the Ova-specific antibody response. Intriguingly, the results obtained indicate an immediate and massive immune reaction against IVIg, as shown by the activation and expansion of B cells and CD4+ T cells in the spleen and draining lymph nodes and the production of IVIg-specific antibodies. We propose that IVIg competes at the T-cell level with the response against Ova to explain the immunomodulatory properties of IVIg. Two monoclonal antibodies did not succeeded in reproducing the effects of IVIg. This suggests that in addition to the mouse response against human constant domains, the enormous sequence diversity of IVIg may significantly contribute to this massive immune response against IVIg. While correlation of these findings to IVIg-treated patients remains to be explored, our data demonstrate for the first time that IVIg re-directs the immune response towards IVIg and away from a specific antigen response.

Tregitopes and impaired antigen presentation: Drivers of the immunomodulatory effects of IVIg?

INTRODUCTION: Although intravenous immunoglobulin (IVIg) is commonly used in the clinic to treat various autoimmune and severe inflammatory diseases, the mode of action is not fully elucidated. This work investigates two proposed mechanisms: (1) the potential role of regulatory T-cell epitopes (Tregitopes) from the constant domain of IgG in the immunosuppressive function of IVIg; and (2) a potential impact of IVIg on the ability of antigen presenting cells (APCs) to present peptides. METHODS AND RESULTS: Investigation of the HLA class II peptide repertoire from IVIg-loaded dendritic cells (DCs) via MHC-associated peptide proteomics (MAPPs) revealed that numerous IgG-derived peptides were strongly presented along the antibody sequence. Surprisingly, Tregitopes 167 and 289 did not show efficient natural presentation although they both bound to HLA class II when directly loaded as "naked" peptides on human DCs. In addition, both Tregitopes could not reproduce the inhibitory effect of IVIg in a human in vitro T-cell proliferation assay as well as in vivo in mice. MAPPs data demonstrate that presentation of peptides from several antigens remained unchanged even when competed with high doses of IVIg, in both human and mouse. CONCLUSION: These data suggest that the effects mediated by IVIg are not caused by Tregitopes nor by impaired antigen presentation.

Aggregation of human recombinant monoclonal antibodies influences the capacity of dendritic cells to stimulate adaptive T-cell responses in vitro.

Subvisible proteinaceous particles which are present in all therapeutic protein formulations are in the focus of intense discussions between health authorities, academics and biopharmaceutical companies in the context of concerns that such particles could promote unwanted immunogenicity via anti-drug antibody formation. In order to provide further understanding of the subject, this study closely examines the specific biological effects proteinaceous particles may exert on dendritic cells (DCs) as the most efficient antigen-presenting cell population crucial for the initiation of the adaptive immune response. Two different model IgG antibodies were subjected to three different types of exaggerated physical stress to generate subvisible particles in far greater concentrations than the ones typical for the currently marketed biotherapeutical antibodies. The aggregated samples were used in in vitro biological assays in order to interrogate the early DC-driven events that initiate CD4 T-cell dependent humoral adaptive immune responses--peptide presentation capacity and co-stimulatory activity of DCs. Most importantly, antigen presentation was addressed with a unique approach called MHC-associated Peptide Proteomics (MAPPs), which allows for identifying the sequences of HLA-DR associated peptides directly from human dendritic cells. The experiments demonstrated that highly aggregated solutions of two model mAbs generated under controlled conditions can induce activation of human monocyte-derived DCs as indicated by upregulation of typical maturation markers including co-stimulatory molecules necessary for CD4 T-cell activation. Additional data suggest that highly aggregated proteins could induce in vitro T-cell responses. Intriguingly, strong aggregation-mediated changes in the pattern and quantity of antigen-derived HLA-DR associated peptides presented on DCs were observed, indicating a change in protein processing and presentation. Increasing the amounts of subvisible proteinaceous particles correlated very well with the pronounced increase in the peptide number and clusters presented in the context of class II HLA-DR molecules, suggesting a major involvement of a mass-action mechanism of altering the presentation.