Engineering Murine Cross-Reactivity Into an Affibody to Human Death Receptor 5

Interspecies cross-reactive protein therapeutics that target conserved epitopes across species are critical for translational research. The present study showcases the engineering of an affibody molecule, originally discovered for binding to human death receptor 5 (hDR5) with 94 nM affinity, to simultaneously acquire cross-reactivity to murine DR5 and enhance its binding affinity to human DR5. DR5 plays a pivotal role in metabolic dysfunction-associated steatohepatitis (MASH) by mediating hepatocyte apoptosis and inflammation. Utilizing a rationally designed library guided by enrichment information and a helix-walking mutagenesis strategy, combined with alternating binding selections between human and murine DR5, we evolved affibody variants exhibiting significantly improved binding to both receptors. Deep sequencing revealed amino acid preferences in the paratope, and the dominant variant, ABY_DR5-A, demonstrated over 1000-fold and 16-fold affinity improvements to murine and human DR5, respectively, with equilibrium dissociation constants of 15 and 5.8 nM. ABY_DR5-A exhibited nanomolar IC₅₀ values for antagonism of TRAIL-induced DR5 signaling, measured via caspase 8 activation, in both murine and human cells, albeit with incomplete inhibition. This engineered affibody provides a promising candidate for therapeutic development targeting DR5-mediated liver disease. Further functional characterization and pharmacokinetic optimization are required to advance these findings toward preclinical evaluation in murine MASH models and, ultimately, clinical applications.