Wnts Specify LRP6 mAb Activity Figure 6. LRP6 antibodies block binding of Wnt isoforms to different sites. (A, B) Biolayer interferometry with biotinylated LRP6 E1-E4 protein immobilized on Streptavidin biosensors indicates that YW211.31.57 antibody inhibits binding of Wnt3a, but not Wnt9b, protein to LRP6 (A), and YW210.09 antibody inhibits only Wnt9b binding (B). Antibody binding to LRP6 protein was allowed to reach equilibrium, and the subsequent wavelength shift in the interference pattern is shown for association and dissociation phases of Wnt protein binding. (C, D) Smaller, non-overlapping fragments of LRP6 separate the binding of different antibodies and Wnt isoforms. Wnt3a binds to the E3-E4 region, and this interaction is blocked by either the intact or one-armed (OA) YW211.31 antibody (C). YW210.09 antibody binds the LRP6 E1-E2 protein fragment and competes with Wnt9b binding (D). (E) YW211.31.57 and YW210.09 antibodies can bind together to immobilized LRP6 E1-E4 protein when added sequentially in either order, confirming separate epitopes. (F) When either of these antibodies, or the one-armed YW211.31 antibody, is bound to LRP6 E1-E4 protein, DKK1 protein binding to LRP6 is antagonized. doi:10.1371/journal.pone.0012682.g006 inoma xenografts driven by autocrine Wnt signaling of an unknown Wnt isoform [21]. For MMTV-Wnt1 tumors, rapid and sustained tumor regression was observed with YW210.09 antibody treatment, similar to Fzd8CRD-Fc protein, whereas no effect on tumor growth was evident with YW211.31.57 antibody (Figure 7A). These results are consistent with the antibody effects described above on Wnt target gene expression for MMTV-Wnt1 tumor cells treated in tissue culture. For Ntera-2 xenograft tumors, RNA extracted from tumors treated with antibodies YW211.31.57, one-armed YW211.31, or the combination of YW211.31.57 and YW210.09 reveals reduced expression of Wnt target gene SP5 to 41–57% the level of PBS- treated tumors, whereas Fzd8CRD-Fc protein treatment reduced SP5 expression to 8.0% (Figure 7B). Axin2 expression was reduced to only 56% by Fzd8CRD-Fc, and no significant changes in Axin2 expression were detected with any of the antibody treatments (data Figure 7. Wnt-driven signaling and growth in tumors, and mineral density in bone, are modulated by specific LRP6 antibodies. (A) MMTV-Wnt1 allograft tumors show regression of growth when mice are treated with YW210.09 antibody, similar to that observed with Fzd8CRD-Fc protein. YW211.31.57 antibody did not alter tumor growth under these conditions compared to control buffer (PBS) or anti-gD antibody treatment. Mice were administered 30 mg/kg of antibody or protein every two days (arrowheads). (B) Ntera-2 xenograft tumors display reduced expression of SP5 mRNA by qPCR analysis in mice treated with intact or one-armed (OA) YW211.31 antibody, but not with YW210.09 antibody. SP5 mRNA levels were normalized to GAPDH mRNA levels within the same tumor, and additionally normalized to PBS-treated tumors. All treatments except YW210.09 display p-value ,0.005 by ANOVA compared to PBS control. (C) YW210.09, but not YW211.31.62, antibody treatment of mouse calvaria explants in culture significantly increases bone mineral density (BMD) of calcified parietal bone, similar to treatment with RANK-Fc protein. All treatments were 10 mg/ml antibody or protein for 7 d. Data points represent eight calvaria halves from four mice for each treatment group; mean and standard error of the mean are shown as horizontal and vertical lines, respectively. Only YW210.09 and RANK-Fc treatments differ significantly from untreated samples with p-values less than 0.01 and 0.05, respectively, by Dunnett’s test (,0.05 for both by t-test). doi:10.1371/journal.pone.0012682.g007 PLoS ONE | www.plosone.org 11 September 2010 | Volume 5 | Issue 9 | e12682