(242d) Developing Therapeutic Proteins by Engineering Ligand-Receptor Interactions

Ligand?receptor interactions govern a myriad of cell signaling pathways that regulate homeostasis and ensure that cells respond properly to stimuli. Growth factors use these interactions to stimulate cell responses, including proliferation, migration, angiogenesis, and cell death. Proteins that inhibit these processes have potential as therapeutics for cancer, whereas proteins that stimulate these processes offer promise in regenerative medicine. Although much of the focus in this area over the past decade has been on antibodies, recently there has been increased interest in using non-antibody proteins as therapeutic agents. We will discuss our recent successes using directed evolution through yeast surface display to engineer epidermal growth factor (EGF) or hepatocyte growth factor (HGF) ligands with therapeutic potential.

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. We show that EGF mutants engineered for increased receptor binding affinity by yeast surface display elicit increased directional and chemotactic migration of fibroblast cells compared to wild-type EGF. Increased biological activity of the EGF mutants was due to their ability to bind much higher levels of cell surface receptors compared to wild-type EGF after just minutes of incubation. To move towards pre-clinical testing in murine models of wound healing, we demonstrated that EGF mutants also elicited enhanced chemotactic migration over wild-type EGF on fibroblast cells expressing murine EGFR. In full-thickness mouse wounds, one mutant in particular was shown to accelerate wound closure over wild-type EGF, demonstrating promise for clinical translation.

Met (also known as the hepatocyte growth factor receptor) is a receptor tyrosine kinase that plays a central role in controlled cell proliferation and invasion during embryonic development. Conversely, dysregulated Met activation results in a phenotype of invasion and metastasis in many human tumors. Despite its strong role in cancer progression, there are currently no FDA-approved therapeutics targeting the Met receptor. Previously, fragments of the hepatocyte growth factor were rationally engineered as Met receptor agonists and antagonists; however, these ligands bind too weakly to Met (micromolar binding affinity) to be therapeutically useful. To improve the clinical potential of these proteins, we used yeast surface display to engineer mutants with high-affinity binding to the Met receptor. Libraries of yeast-displayed mutants were screened with soluble Met receptor extracellular domain to isolate clones with high affinity binding. The soluble expression, receptor binding, and biological effects of these high-affinity Met-binding mutants will be discussed.

Funded by NIH/NCI 1R21 CA131706, the Wallace H. Coulter Foundation, and fellowship support from NIH 5T32 GM008412-15S1 (DSJ), NIH T90 DK070103 (SEB), and NSF (SSL).