Evaluation of ligand binding and preclinical pharmacokinetics of Hermodulins: A new class of bi-specific protein antagonists that trap most growth factors for the human EGF receptor (HER) family

4076 Receptor tyrosine kinases such as members of the HER family are well-validated targets for cancer therapy. However, anti-cancer agents directed at only one HER family member often encounter endogenous or acquired resistance due to multiple HER co-activation or increased production of HER1 or HER3 ligands by tumor cells. To target multiple members of the HER family and block their function, we have developed a novel bi-specific HER1/HER3 heterodimeric ligand trap comprised of optimized HER1 and HER3 extracellular domains fused with the Fc portion of human IgG1. We investigated binding activities of a prototypic Hermodulin, RB200, using europium-labeled ligands to compare ligand binding affinities of RB200 with those of Hermodulin variants obtained via a high throughput rational mutagenesis process (optimized Hermodulins). Our data show that RB200 retains specific binding to various HER ligands, such as EGF, TGF-alpha, HB-EGF and neuregulin (NRG1-beta1) (Kd = 10 to 30 nM). Optimized Hermodulins have improved ligand binding affinities (Kd = 0.5 to 2 nM) for both HER1 and HER3 ligands with greater Bmax. We also developed a sensitive ELISA specific for Hermodulins, and studied the pharmacokinetics in rodent models in order to provide dosing strategies and interpret future studies evaluating the efficacy and tolerability of Hermodulins. RB200 was administered as a single iv or ip dose of 15-30 mg/kg in rats and mice, and plasma samples were analyzed via ELISA. RB200 exhibited high bioavailability, a low volume of distribution, and a prolonged terminal half-life consistent with expectations for Fc-fusion proteins and therapeutic monoclonal antibodies. Together these data suggest that Hermodulins act as bi-specific HER1/HER3 ligand traps and have desirable in vivo pharmacokinetic characteristics. These results support continued development of Hermodulins for cancer therapy.