Epidermal growth factor (EGF) is one of the major factors involved in the re-epithelialization and remodeling phase of wound healing. Local and sustained presence of EGF has been shown to accelerate wound healing. However, attempts to develop EGF for clinical applications have been limited by its short circulating half-life. We hypothesized that increasing the affinity of human EGF for its receptor (EGFR) would result in enhanced biological efficacy. Yeast surface display was used to engineer EGF mutants with a 4-to 30-fold increase in receptor binding affinity (Cochran, JR, Kim, Y, Lippow, S, Rao, B, and Wittrup, KD (2006) Protein Eng. Des. Sel., 19, 245-53). Five engineered EGF mutants with a range of EGFR binding affinities were chosen for soluble expression in Saccharomyces cerevisiae to assess their effects on EGFR-mediated cellular processes related to wound healing.

EGF mutants were tested for their ability to exhibit persistent cell signaling with in vitro bioassays that were designed to mimic in vivo clearance. Fibroblast cells expressing human EGFR were pulsed with wild-type EGF or mutants for 10-15 min at 37 ºC. Media containing soluble growth factor was removed, and cells were washed and incubated at 37 ºC for the remainder of the assays. A time course monitoring EGFR downregulation indicated that EGF mutants elicited persistent cell signaling after pulsing with growth factor, in contrast to wild-type EGF. Directional cell migration was quantified using a gap-closure assay, an in vitro model for wound re-epithelialization. Engineered EGF mutants were shown to significantly enhance cell migration over wild-type EGF. Interestingly, directional cell migration levels elicited by wild-type EGF were comparable to the negative control, suggesting that wild-type EGF was removed by the wash steps of the pulse assay. Collectively, this data demonstrates that EGF mutants engineered for increased EGFR binding affinity remain bound to cell surface receptors longer than wild-type EGF, acting as a growth factor reservoir for continued stimulation of EGFR-mediated cell responses. As a prelude to pre-clinical testing of EGF mutants in a diabetic wound healing model, we demonstrated that EGF mutants elicited enhanced chemotactic cell migration over wild-type EGF on fibroblast cells expressing murine EGFR. In addition to clinical applications, we are using these engineered EGF mutants to probe structure-function relationships of EGF ligand/receptor interactions. This research was supported by a Wallace H. Coulter Foundation Translational Partnership Award.

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