Discovery and optimization of a pH-responsive ultra-long-acting VHH-based growth hormone mimetic

Weekly formulations of long-acting growth hormone (LAGH) effectively treat growth hormone (GH) deficiency in both children and adults while offering improved convenience compared to daily GH. To further enhance patient convenience and compliance, longer-acting GH preparations have become a key research focus. However, due to the inherent short half-life of protein-based drugs, no ultra-long-acting growth hormone or analog products (with dosing intervals longer than once weekly) are currently available worldwide. In this study, we developed a potentially ultra-long-acting growth hormone receptor (GHR) agonist pH.VHH03 with GH-mimetic activity by engineering hinge region and variable region of a VHH antibody. Through optimization of the hinge region, the in vitro cell proliferation activity of this molecule was significantly enhanced. As a result, it demonstrated LAGH-like effects in promoting body weight gain and stimulating IGF-1 secretion in rats. Furthermore, pH-dependent binding was introduced into the variable region. The optimized molecule exhibited a 227-fold difference in dissociation rates between neutral and acidic conditions. This modification prolonged its in vivo efficacy in rats from 6 days to over 15 days, far exceeding the 3-day duration observed with PEGylated GH. Subsequent in vivo experiments in rats confirmed that the final optimized molecule dose-dependently promoted body weight gain, insulin-like growth factor 1 (IGF-1) secretion, tibial growth, and significantly increased growth plate thickness. The in vivo profile fully replicated the physiological activities of GH. Moreover, at medium to high doses, it induced a notably flatter and sustained IGF-1 response compared to PEGylated GH, suggesting a potentially longer duration of pharmacological activities in humans.