Production of a human neutralizing monoclonal antibody and its crystal structure in complex with ectodomain 3 of the interleukin-13 receptor α1.

Gene deletion studies in mice have revealed critical roles for IL (interleukin)-4 and -13 in asthma development, with the latter controlling lung airways resistance and mucus secretion. We have now developed human neutralizing monoclonal antibodies against human IL-13Rα1 (IL-13 receptor α1) subunit that prevent activation of the receptor complex by both IL-4 and IL-13. We describe the crystal structures of the Fab fragment of antibody 10G5H6 alone and in complex with D3 (ectodomain 3) of IL-13Rα1. Although the structure showed significant domain swapping within a D3 dimer, we showed that Arg(230), Phe(233), Tyr(250), Gln(252) and Leu(293) in each D3 monomer and Ser(32), Asn(102) and Trp(103) in 10G5H6 Fab are the key interacting residues at the interface of the 10G5H6 Fab-D3 complex. One of the most striking contacts is the insertion of the ligand-contacting residue Leu(293) of D3 into a deep pocket on the surface of 10G5H6 Fab, and this appears to be a central determinant of the high binding affinity and neutralizing activity of the antibody.

Generation and selection of novel fully human monoclonal antibodies that neutralize Dickkopf-1 (DKK1) inhibitory function in vitro and increase bone mass in vivo.

Wnt/LRP5 signaling is a central regulatory component of bone formative and resorptive activities, and the pathway inhibitor DKK1 is a suppressor of bone formation and bone mass accrual in mice. In addition, augmented DKK1 levels are associated with high bone turnover in diverse low bone mass states in rodent models and disease etiologies in human. However, examination of the precise role of DKK1 in the normal skeleton and in higher species requires the development of refined DKK1-specific pharmacological tools. Here, we report the strategy resulting in isolation of a panel of fully human anti-DKK1 antibodies applicable to studies interrogating the roles of mouse, rhesus, and human DKK1. Selected anti-DKK1 antibodies bind primate and human DKK-1 with picomolar affinities yet do not appreciably bind to DKK2 or DKK4. Epitopes mapped within the DKK1 C-terminal domain necessary for interaction with LRP5/6 and consequently effectively neutralized DKK1 function in vitro. When introduced into naïve normal growing female mice, IgGs significantly improved trabecular bone volume and structure and increased both trabecular and cortical bone mineral densities in a dose-related fashion. Furthermore, fully human DKK1-IgG displayed favorable pharmacokinetic parameters in non-human primates. In summary, we demonstrate here a rate-limiting function of physiologic DKK1 levels in the regulation of bone mass in intact female mice, amendable to specific pharmacologic neutralization by newly identified DKK1-IgGs. Importantly the fully human IgGs display a profile of attributes that recommends their testing in higher species and their use in evaluating DKK1 function in relevant disease models.

Evidence that inhibition of insulin receptor signaling activity by PC-1/ENPP1 is dependent on its enzyme activity.

Plasma cell membrane glycoprotein-1 or ectonucleotide pyrophosphatase/phosphodiesterase (PC-1/ENPP1) has been shown to inhibit insulin signaling, and its genetic polymorphism or increased expression is associated with type 2 diabetes in humans. Therefore, PC-1 inhibition represents a potential strategy in treating diabetes. Since patients with phosphodiesterase/pyrophosphatase deficient PC-1 manifest abnormal calcification, enhancing insulin signaling by inhibiting PC-1 for the treatment of diabetes will be feasible only if PC-1 phosphodiesterase/pyrophosphatase activity needs not be significantly diminished. However, whether inhibition of insulin receptor signaling by PC-1 is dependent upon its phosphodiesterase/pyrophosphatase activity remains controversial. In this study, the extracellular domain of the human PC-1 in its native form or with a T256A or T256S mutation was overexpressed and purified. Enzymatic assays showed that both mutants have less than 10% of the activity of the wild-type protein. In HEK293 cells stably expressing recombinant insulin receptor or insulin-like growth factor 1 (IGF1) receptor, transient expression of wild-type full length PC-1 (PC-1.FL.WT) but not the T256A or T256S mutants inhibits insulin signaling without affecting IGF1 signaling. Western blot and FACS analysis showed that the wild-type and mutant full length PC-1 proteins are expressed at similar levels in the cells, and were localized to the similar levels on the cell surface. Overexpression of PC-1.FL.WT did not affect insulin receptor mRNA level, total protein and cell surface levels. Together, these results suggest that the inhibition of insulin signaling by PC-1 is somewhat specific and is dependent upon the enzymatic activity of the phosphodiesterase/pyrophosphatase.