Wnts Specify LRP6 mAb Activity YW211.31.57 antibody on autocrine Wnt signaling in Hs578T (A) or EKVX (C) cells. (E–F) Crosslinking of the one-armed YW211.31 antibody partially restores the potentiating activity observed with the whole antibody in HT-1080 cells for both autocrine Wnt signaling (F) and signaling induced by Wnt2 transfection (E). Similar results are observed with crosslinking by either the whole IgG or F(ab9)2 fragments of anti-human-Fc antibodies (NA indicates no crosslinking antibody added). All antibodies were used at 1 mg/ml. (G) HEK293 cells stably integrated with Wnt luciferase reporter were transfected for Wnt1 expression and treated with either 0.4 mg/ml DKK1 protein and increasing concentrations of YW211.31 intact or one-armed antibody, or with 10 mg/ml antibody and increasing concentrations of DKK1. Single-protein control treatments use 10 mg/ml antibody, 0.4 mg/ml DKK1, or 10 mg/ml Fzd8CRD-Fc. Wnt luciferase units were normalized to total protein concentration of the lysate, and additionally normalized to signal in cells not transfected for Wnt1 expression. doi:10.1371/journal.pone.0012682.g004 E1-E4 protein without competition and when added in either order (Figure 6E). Competition for binding between YW211.31.57 antibody and only Wnt3a, and between YW210.09 and only Wnt9b, at different sites on LRP6 protein correlate with the inhibitory activity of each antibody against signaling by a specific Wnt isoform. Bourhis et al. (2010) showed by biolayer interferometry that purified DKK1 protein could bind both E3-E4 and E1-E2 fragments of LRP6, and that this interaction could inhibit binding of Wnt3a and Wnt9b to these respective protein regions [14]. We find that YW211.31.57 and YW210.09 antibodies can each inhibit DKK1 binding to LRP6 E1-E4 protein (Figure 6F). As expected, YW211.31.57 antibody also inhibits DKK1 binding to LRP6 E3- E4 protein, and YW210.09 antibody blocks binding of DKK1 to the E1-E2 fragment (data not shown). The one-armed YW211.31 antibody fully retains this inhibitory activity, even though it cannot potentiate Wnt signaling nor significantly antagonize exogenous DKK1 activity on Wnt signaling in cells. This result suggests that the ability to block DKK1 binding to LRP6, at least in vitro, does not contribute significantly to antibody-mediated potentiation of Wnt signaling, or to antagonism of DKK1 activity. Rather, dimerization of coreceptor that is also able to bind Wnt primarily mediates both these activities. LRP6 Antibodies are Active on Wnt-driven Tumors and Bone Formation To begin to explore the anti-tumor therapeutic efficacy of the LRP6 antibodies, we treated two models of Wnt ligand-driven tumors, MMTV-Wnt1 transgenic mammary tumor allografts dependent on Wnt1 expression and Ntera-2 human teratocar- Figure 5. LRP6 antibody combinations can inhibit signaling induced by multiple Wnt isoforms. (A) The combination of YW211.31.57 and YW210.09 antibodies inhibits signaling in HEK293 cells stably integrated with Wnt luciferase reporter that have been transfected with expression constructs for either Wnt3a, Wnt1, or both Wnt3a and Wnt1. All antibodies and proteins were used at 10 mg/ml each. Treatment with either YW211.31.57 or YW210.09 antibody does not significantly change the level of signaling induced by the combination of both Wnt isoforms, possibly because signaling by one Wnt is antagonized while the other Wnt is potentiated. (B–C) As with individual antibody treatments, the combination of YW211.31.57 and YW210.09 antibodies potentiates autocrine Wnt signaling in Hs578T (B) or EKVX (C) cells. doi:10.1371/journal.pone.0012682.g005 PLoS ONE | www.plosone.org 9 September 2010 | Volume 5 | Issue 9 | e12682