Ticks secrete a variety of salivary proteins that suppress host immune responses during blood feeding. Among these are evasins, a family of proteins that inhibit inflammatory chemokines and thereby suppress leukocyte recruitment. Their potent anti-inflammatory activity has been demonstrated in vitro and in vivo, highlighting their potential as a new class of multi-target therapeutics1.
However, wild-type evasins bind multiple chemokines, raising concerns about unwanted immune suppression in therapeutic applications. We hypothesised that the natural chemokine-binding properties of evasins could be re-engineered to create more selective inhibitors targeting disease-associated inflammatory pathways.
To explore molecular determinants of chemokine recognition and selectivity, we developed a CHO cell-surface display platform to screen a saturation mutagenesis library of the tick evasin EVA-P974 (EVA-P). The library was screened by FACS against three target chemokines (CCL2, CCL7 and CCL8) and three off-target chemokines (CCL3, CCL11 and CCL14), with cells sorted into high-, low- and non-binding populations.
Deep mutational profiling revealed EVA-P residues critical for chemokine recognition. More importantly, we identified mutations that selectively enhanced target chemokine binding while reducing off-target interactions. These results establish a framework for engineering evasin therapeutics and demonstrate how proteins evolved for parasite immune evasion can be repurposed as precision anti-inflammatory drugs.