Chemokines are important mediators of leukocyte recruitment in inflammation; however, chemokines also contribute to inflammatory diseases when they are not properly regulated. Due to its complexity, there is a scarcity of successful treatments targeting the chemokine system1. Evasins are a family of tick salivary proteins that bind and inhibit chemokines, effectively inhibiting inflammation in vitro and in vivo2. However, wild-type evasins are typically too broadly selective. Therefore, evasins must be engineered to have the desired specificity for disease-associated chemokines and to minimise off-target immune suppression.
We plan to use mammalian cell surface display to screen libraries of combinatorially mutated evasins, with the purpose of defining sequence binding requirements to different chemokines and developing targeted evasins through in vitro evolution. To this end, we will describe methods we have established for a) amplifying a library of evasin variants without losing its complexity, b) efficiently transfecting mammalian cells, and c) detection of HA-tagged evasins on the cell surface.
Importantly, we successfully detected HA-tagged evasin displayed on the surface of Flp-In CHO cells with up to ~98% efficiency and demonstrated comparable levels of chemokine binding by the displayed evasins. In the course of this research, we found that this high level of HA tag detection was only reached by incubating the cells with sodium chlorate, providing evidence of tyrosine sulfation of the HA tag impairing antibody binding ability. This phenomenon has wide-reaching potential implications for the interpretation of research that relies on HA tag detection of secreted proteins. These findings represent important steps in developing and optimising the evasin combinatorial engineering pipeline to produce engineered disease-specific evasins.