Background/Objectives: Anti-drug antibody (ADA) formation can impact the safety, pharmacokinetics, and/or efficacy of biotherapeutics, including monoclonal antibodies (mAbs). Current strategies for ADA/immunogenicity risk prediction of mAbs include in silico algorithms, T cell proliferation assays, MHC-associated peptide proteomics assays (MAPPs), and dendritic cell internalization assays. However, B cell-mediated responses are not assessed in these assays. B cells are professional antigen-presenting cells (APCs) and secrete antibodies toward immunogenic mAbs. Therefore, methods to determine B cell responses would be beneficial for immunogenicity risk prediction and may provide a more comprehensive assessment of risk. Methods: We used a PBMC culture method with the addition of IL-4, IL-21, B cell activating factor (BAFF), and an anti-CD40 agonist mAb to support B cell survival and activation. Results: B cells in this assay format become activated, proliferate, and secrete IgG. A panel of 51 antibodies with varying clinical immunogenicity rates were screened in this assay with IgG secretion used as a readout for immunogenicity risk. IgG secretion differed among test articles but did not correlate with the clinical immunogenicity rating. Conclusions: This dataset highlights the challenges of developing a B cell assay for immunogenicity risk prediction and provides a framework for further refinement of a B cell-based assay for immunogenicity risk prediction of mAbs.

Food allergy has had a rapid rise in prevalence, and thus it is important to identify approaches to limit the development of food allergy early in life. Because maternal dietary supplementation with α-tocopherol (α-T), an isoform of vitamin E, during pregnancy and nursing increases neonate plasma levels of α-T and can limit neonate development of other allergies, we hypothesized that α-T can limit development of food allergy. To assess this, male mice with mutations in their skin barrier genes (FT−/− mice) were mated with wild-type females that received a diet supplemented with α-tocopherol or a control diet. Starting at postnatal day 3, these FT+/− pups were sensitized 4 to 5 times over 2.5 weeks by skin co-exposure to the food allergen peanut extract (PNE) and the environmental allergen Alternaria alternata (Alt). Control pups were exposed to saline, PNE only or Alt only. Supplementation with α-T blocked Alt+PNE sensitization (anti-PNE-specific IgE), without blocking Alt+PNE-stimulated skin IL33, Areg, OSM, CCL11, TSLP or plasma MCPT1. However, supplementation with α-T blocked mast cell activation, the increase in plasma histamine in Alt+PNE sensitized pups, histamine receptor stimulation of endothelial PKCα signaling, and ultimately oral PNE-induced anaphylaxis in Alt+PNE sensitized mice. Thus, maternal supplementation with α-tocopherol reduced development of food allergy and anaphylaxis in neonates. These results have implications for supplementation of mothers with α-tocopherol to limit development of food allergy in neonates with skin barrier mutations.

SignificanceOver 100 monoclonal antibodies have been approved by the U.S. Food and Drug Administration (FDA) for clinical use; however, a paucity of knowledge exists regarding the injection site behavior of these formulated therapeutics, particularly the effect of antibody, formulation, and tissue at the injection site. A deeper understanding of antibody behavior at the injection site, especially on blood oxygenation through imaging, will help design improved versions of the therapeutics for a wide range of diseases.AimThe aim of this research is to understand the dynamics of monoclonal antibodies at the injection site as well as how the antibody itself affects the functional characteristics of the injection site [e.g., blood oxygen saturation (sO2)].ApproachWe employed triple-wavelength equipped functional photoacoustic imaging to study the dynamics of dye-labeled and unlabeled monoclonal antibodies at the site of injection in a mouse ear. We injected a near-infrared dye-labeled (and unlabeled) human IgG4 isotype control antibody into the subcutaneous space in mouse ears to analyze the injection site dynamics and quantify molecular movement, as well as its effect on local hemodynamics.ResultsWe performed pharmacokinetic studies of the antibody in different regions of the mouse body to show that dye labeling does not alter the pharmacokinetic characteristics of the antibody and that mouse ear is a viable model for these initial studies. We explored the movement of the antibody in the interstitial space to show that the bolus area grows by ∼300 % over 24 h. We discovered that injection of the antibody transiently reduces the local sO2 levels in mice after prolonged anesthesia without affecting the total hemoglobin content and oxygen extraction fraction.ConclusionsThis finding on local oxygen saturation opens a new avenue of study on the functional effects of monoclonal antibody injections. We also show the suitability of the mouse ear model to study antibody dynamics through high-resolution imaging techniques. We quantified the movement of antibodies at the injection site caused by the interstitial fluid, which could be helpful for designing antibodies with tailored absorption speeds in the future.