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.

Automated high-throughput purification of antibody fragments to facilitate evaluation in functional and kinetic based assays.

Screening antibodies from phage displayed in vitro libraries and from affinity maturation of lead antibodies requires testing of antibody fragments (scFvs and Fabs) for function and binding affinities. Crude scFv or Fab periplasmic preparations from Escherichia coli are often not pure and/or concentrated enough for use in functional and affinity assays. We have developed an automated high-throughput approach for small and large-scale expression and purification of His-tagged scFvs and Fabs using the Qiagen BioRobot 3000 LS with optimized application software. This automated procedure enabled us to rapidly evaluate antibody fragments in functional and surface plasmon resonance (SPR) assays. We have used these procedures to make thousands of purified scFv/Fabs for several antibody maturation campaigns and significantly decreased the time needed to select the best candidates. The assay results from these purified samples were used to prioritize candidates before converting them to IgG. This protocol can process up to 300 small-scale and up to 72 large-scale scFvs or Fabs per week per full-time employee (FTE).