Development and characterization of dendritic cell internalization and activation assays contributing to the immunogenicity risk evaluation of biotherapeutics.

Introduction: Immunogenicity refers to the ability of a substance, such as a therapeutic drug, to elicit an immune response. While beneficial in vaccine development, undesirable immunogenicity can compromise the safety and efficacy of therapeutic proteins by inducing anti-drug antibodies (ADAs). These ADAs can reduce drug bioavailability and alter pharmacokinetics, necessitating comprehensive immunogenicity risk assessments starting at early stages of drug development. Given the complexity of immunogenicity, an integrated approach is essential, as no single assay can universally recapitulate the immune response leading to the formation of anti-drug antibodies. Methods: To better understand the Dendritic Cell (DC) contribution to immunogenicity, we developed two flow cytometry-based assays: the DC internalization assay and the DC activation assay. Monocyte-derived dendritic cells (moDCs) were generated from peripheral blood mononuclear cells (PBMCs) and differentiated over a five-day period. The internalization assay measured the accumulation rate of therapeutic antibodies within moDCs, while the activation assay assessed the expression of DC activation markers such as CD40, CD80, CD86, CD83, and DC-SIGN (CD209). To characterize these two assays further, we used a set of marketed therapeutic antibodies. Results: The study highlights that moDCs differentiated for 5 days from freshly isolated monocytes were more prone to respond to external stimuli. The internalization assay has been shown to be highly sensitive to the molecule tested, allowing the use of only 4 donors to detect small but significant differences. We also demonstrated that therapeutic antibodies were efficiently taken up by moDCs, with a strong correlation with their peptide presentation on MHC-II. On the other hand, by monitoring DC activation through a limited set of activation markers including CD40, CD83, and DC-SIGN, the DC activation assay has the potential to compare a series of compounds. These two assays provide a more comprehensive understanding of DC function in the context of immunogenicity, highlighting the importance of both internalization and activation processes in ADA development. Discussion: The DC internalization and activation assays described here address key gaps in existing immunogenicity assessment methods by providing specific and reliable measures of DC function. The assays enhance our ability to pre-clinically evaluate the immunogenic potential of biotherapeutics, thereby improving their safety and efficacy. Future work should focus on further validating these assays and integrating them into a holistic immunogenicity risk assessment framework.

Monitoring extracellular ion and metabolite dynamics with recombinant nanobody-fused biosensors.

Ion and analyte changes in the tumor microenvironment (TME) alter the metabolic activity of cancer cells, promote tumor cell growth, and impair anti-tumor immunity. Consequently, accurate determination and visualization of extracellular changes of analytes in real time is desired. In this study, we genetically combined FRET-based biosensors with nanobodies (Nbs) to specifically visualize and monitor extracellular changes in K+, pH, and glucose on cell surfaces. We demonstrated that these Nb-fused biosensors quantitatively visualized K+ alterations on cancer and non-cancer cell lines and primary neurons. By implementing a HER2-specific Nb, we generated functional K+ and pH sensors, which specifically stained HER2-positive breast cancer cells. Based on the successful development of several Nb-fused biosensor combinations, we anticipate that this approach can be readily extended to other biosensors and will open new opportunities for the study of extracellular analytes in advanced experimental settings.