The Ratio of Immunogenicity: A quantitative metric to identify highly immunogenic protein antigens to improve tissue-engineered biomaterials

The ability to a-priori identify antigenic components responsible for driving recipient graft-specific adaptive immune destruction of tissue-engineered biomaterials is critical for their safe clinical translation and long-term function. However, despite our in-depth understanding of cellular responses toward antigenic stimuli, the question of whether individual antigens are equally stimulatory to the immune system remains largely unanswered. This lack of knowledge limits our capacity to rationally design immunologically acceptable biomaterials. Previously published decellularization/antigen removal approaches have assumed that all antigens are equally immunogenic and that reduction in extracellular matrix (ECM) scaffold total antigen content therefore correlates with the magnitude of the biomaterial-specific adaptive immune response in vivo. However, recent publications question the validity of such assumptions. Here, we describe the development and validation of a quantitative, experimentally derived metric, termed the Ratio of Immunogenicity (ROI), which can stratify antigens based on their relative immunogenicity. By combining data on the magnitude of the immune response (i.e., IgG production) and abundance (i.e. dose) of individual antigens in an in vivo ECM scaffold implantation model, a continuum of ROI values can be calculated. Critically, mitochondrial antigens are significantly enriched in the top 25% of highly immunogenic antigens (i.e., highest ROIs), while cytoplasmic, extracellular, and vesicle-associated antigens are significantly enriched in the bottom 25% of weakly immunogenic antigens (i.e., lowest ROIs). Consequently, the ROI is a powerful and versatile metric which can be utilized to identify both individual and groups of antigens which possess a relatively high capacity to stimulate graft-specific immune responses. In turn, the Ratio of Immunogenicity offers insights which can be leveraged in numerous immune-engineering contexts, including the development of immunologically acceptable, unfixed, tissue engineered biomaterials.