Abstract
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.
Highlights
Each of the three scFv phage display libraries was subjected to three rounds of solution-based panning against biotinylated rhesus and mouse DKK1 proteins
A total of 176 and 88 hits from the second and third round of panning, respectively, was isolated from each of the three libraries. These individual clones from the second and third round pannings were assayed for ELISAbased binding to rhesus and mouse DKK1 to allow for selection of antibody sequences conferring pan-species applicability
Among the hits selected from all libraries, 56.6% dual ELISA positive rhesus/mouse cross reactive hits were observed from the round 2 pannings, whereas 88% dual positive hits were observed from the round 3 pannings with the largest
Summary
DKK1 Proteins—Rhesus DKK1 cDNA was cloned by RTPCR from rhesus macaque (Macaca mulatta) bone tissue and cloned into the pcDNA3.1-Myc-His expression vector (Invitrogen). Selection of scFv and IgG Antibodies in in Vitro Cell-based DKK1 Binding Assays—Rhesus DKK1 protein was labeled by Lys-directed conjugation of Eu3ϩ chelate following the manufacturer’s instruction (PerkinElmer Life Sciences) and purified with Sephadex G-25 PD-10 columns (GE Healthcare). Eu3ϩ-labeled DKK1 protein diluted in DELFIA assay buffer without detergent (PerkinElmer Life Sciences) was added at a final assay concentration of 100 pM, and the binding reaction was continued at 37 °C for 15 min. A range of 1–100 ng of the respective recombinant proteins was added to nitrocellulose membranes (Millipore), and anti-DKK1 IgGs or mouse anti-His-tag antibody (Invitrogen, 1 g/ml) was allowed to bind overnight. Media were replenished after 2 days, and treatment begun by the addition of IgG (2.7– 80 nM) followed by rhDKK1 protein (50 nM) and by control CM or Wnt3A-CM to a final concentration of 10 ng/ml. Where appropriate. a one-way ANOVA followed by Dunnett’s post-hoc test to detect group differences was calculated using Graphpad Prism 5.0 (GraphPad Software, La Jolla, CA)
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