Joint poster with Merck: case study on protein allergy
Characterization of respiratory sensitizing properties of the protein Subtilisin, using GARD®air
Presented at the 12th World Congress on Alternatives and Animal Use in the Life Sciences 2023
Conclusion
GARDair is an experimental in vitro assay for assessment of respiratory sensitizers, based on evaluation of exposure-induced gene expression changes of genomic biomarkers in a Dendritic Cell-like cell line and machine learning-assisted classifications.
GARDair has previously been shown to be functional, having been subjected to a validating ring trial, demonstrating its capability to predict respiratory sensitization properties of low-molecular weight test chemicals.
Here, we investigated the hypothesis that protein allergens engage similar toxicity pathways as low-molecular weight chemicals and demonstrated that GARDair can be successfully used to predict the respiratory sensitizing properties of the model allergen Subtilisin.
Abstract
Sensitization is a condition induced by an immune system response to a variety of molecules, including proteins or chemicals, referred to as sensitizers. Proactive identification of sensitizers is central in hazard and risk assessment of both biologics and chemicals, for regulatory registration or to ensure occupational safety. While large investments in New Approach Methodologies for assessment of dermal sensitizers have been made, the ability to accurately predict respiratory sensitizers in vitro, including both low-molecular weight chemicals and biologics such as proteins and peptides, remains unfulfilled.
The Genomic Allergen Rapid Detection assay for hazard assessment of respiratory sensitizers (GARDair) is an experimental method, originally developed to provide binary hazard identification of chemical respiratory sensitizers. Based on the same technological framework as the OECD validated GARDskin assay (OECD TG 442E), the method evaluates the transcriptional patterns of disease-associated genes in the dendritic cell-like cell line SenzaCell™, following test item exposure. Here, we hypothesized similar toxicity pathway engagement by protein sensitizers as for low-molecular weight chemicals. Using the model protein allergen Subtilisin, a well-known inducer of occupational asthma, we here demonstrate the ability of GARDair to assess the respiratory sensitization potential of proteins.
The GARDair test results of the Subtilisin and control samples are presented in Figure 3. Subtilisin gives rise to clearly positive response signals, along with the low-molecular weight positive control (Reactive black 5), while the negative (vehicle) control does not. Evidently, the protein allergen Subtilisin triggers, at least to a limited albeit sufficient extent, similar toxicity pathways as the low-molecular weight chemical space used to define the method. While these findings are solely originating from an in vitro setting, we hypothesize that such toxicity pathways would be similarly engaged also in an in vivo setting, thereby, at least partly, explaining the allergenic effects known to be associated with Subtilisin exposure. Further, we hypothesize that such toxicity pathways would, at least partly, be associated with the GARDair GPS, which is proposed to monitor transcriptional changes induced specifically by respiratory sensitizers, related to the bridging of innate and adaptive immune functions and skewing towards Th2 type immune responses (including TSLP and IL-7R-alpha chain).
Based on the findings reported in this study, GARDair may constitute a valuable tool for assessment of respiratory sensitization properties of chemicals and proteins. Given the limitation of this study with respect to sample size, next steps include the generation of more data to support the predictive capacity, including the assessment of proteins with low (or lack of ) sensitization potential.
