Cells were washed twice with the same buffer, incubated for 1 h on ice with 2.5 mg/ml R-phycoerythrin-conjugated anti-human IgG (Jackson ImmunoResearch Laboratories), washed three times, and suspended in buffer containing 0.5 mg/ml propidium iodide. Analysis was performed using a FACS Calibur instrument (Becton Dickinson) and FlowJo software (Tree Star), using appropriate gates to exclude cellular debris and aggregates. Wnt chimera constructs were made by cloning full-length Wnt1 or Wnt3a upstream of full-length FZD4, FZD5, or LRP6 in pRK5 expression vector. The 24-amino acid linker (GGGSGGGT)3 was inserted between Wnt and FZD or LRP6 sequences [19]. LRP6 Antibody Generation and Testing Human LRP6 cDNA fragments encoding regions E1-E2 (amino acids A16-R657) and E3-E4 (amino acids A624-G1254) were separately cloned into a mammalian expression vector containing the HSV signal sequence and human IgG Fc region as a protein tag. LRP6 E1-E2-Fc and E3-E4-Fc proteins were expressed in CHO cells by transient transfection, purified by Protein A affinity chromatography, and used individually to screen human synthetic antibody phage libraries. After selection on immobilized LRP6 protein, phage clones were isolated and confirmed by phage ELISA for binding to the LRP6-Fc fusion protein fragment and not anther Fc fusion protein. Phage Fab clones were then reformatted for expression as human IgG1 monoclonal antibodies. Twenty-four unique clones against LRP6 E1-E2-Fc and 22 clones against E3-E4-Fc were transfected and transiently expressed in HEK293 cells, and IgG protein was purified by affinity chromatography. Subsequent large-scale antibody preparations were produced by transient transfection in CHO cells. For affinity maturation of YW211.31 antibody, three different combinations of CDR loops (H1/L3, H2/L3, and H3/L3) were targeted for randomization in separate libraries by soft-random- izing selected residues. In addition, the L1/L2/L3 CDR combination was targeted for hard randomization. In the first round of selection, phage from the randomized libraries were selected with immobilized LRP6 E3-E4-His protein, followed by five rounds of solution-phase sorting in which the concentration of E3-E4-His was gradually reduced from 300 nM to 0.5 nM, and a 100-fold excess of E3-4-Fc protein was added to deplete antibodies with faster dissociation rates. Eleven phage clones were purified, and all showed improved affinity for LRP3 E3-E4 as determined by phage competition ELISA. The sequence of these clones displayed 1 to 6 amino acid changes in CDR-H1, CDR-H3, and CDR-L3. The one-armed YW211.31 antibody variant was produced in E. coli by co-expressing the YW211.31.62 heavy and light chains with a truncated Fc domain using ‘knobs-into-holes’ engineering technology [38]. For antibody cross-linking, Fc-specific goat-anti- human IgG antibody or F(ab9)2 fragment (Sigma-Aldrich) was incubated with one-armed YW211.31 or Apomab antibody for 1 h before adding the mixture to cells. For binding affinity determination of antibodies, SPR measure- ment with a BIAcoreTM-3000 instrument was used. To measure affinity between LRP6 antibodies and LRP6 E1-E2-His protein (expressed in insect cells) or LRP6 E3-E4-His protein (expressed in CHO cells), anti-LRP6 human IgG was captured by CM5 biosensor chips coated mouse anti-human IgG to achieve approximately 100 response units. For kinetics measurements, two-fold serial dilutions of LRP6 E1-E2-His (250 nM to 3.9 nM) or E3-E4-His (31.2 nM to 0.49 nM) protein was injected in PBS with 0.05% Tween 20 at 25uC with a flow rate of 30 ml/min. Association rates (kon) and dissociation rates (koff) were calculated using a simple one-to-one Langmuir binding model (BIAcore Wnts Specify LRP6 mAb Activity Evaluation Software version 3.2). The equilibrium dissociation constant (KD) was calculated as the ratio koff/kon. LRP6 Protein Binding Assay Biolayer interferometry was performed as previously described [14]. Briefly, biotinylated, His-tagged LRP6 proteins were purified from baculovirus-infected insect cells using the AviTag system (GeneCopoeia). Binding kinetics were measured on the Octet RED System (ForteBio) using Streptavidin High Binding FA Biosensors loaded with 20 mg/ml LRP6 protein. Carrier-free purified human Wnt3a and mouse Wnt9b were obtained from R&D Systems, and purified DKK1 protein was produced as previously described [14]. Tumor and Bone Studies Tumors from MMTV-Wnt1 transgenic mice were passaged in mammary fat pads of C57BL/6 mice, mechanically and enzymatically dissociated, resuspended in Matrigel and Hank’s Balanced Salt Solution (HBSS), and injected into the mammary fat pad of athymic NCr nude mice (Taconic). Treatments were 3 initiated once tumor volumes reached 250–800 mm . For each treatment group, ten mice were administered 30 mg/kg of antibody or protein intraperitoneally (IP) every two days. Tumor volume was analyzed using caliper measurement. Ntera-2 xenograft tumor growth and in vivo studies were performed as previously described [21]. Briefly, NU/NU athymic nude mice (Charles River) were injected subcutaneously with 10 million Ntera-2 cells (in 50% Matrigel in HBSS) per mouse, divided into groups of four or five animals once mean tumor volumes reached 535–595 mm3, and injected with a single IP dose of 100 mg/kg antibody or 30 mg/kg Fzd8CRD-Fc protein. Tumor and blood serum samples were collected 16 h after treatment. Tumors were homogenized using the TissueLyser system (QIAGEN), and RNA was extracted using the RNeasy kit (QIAGEN). Calvariae were harvested and cultured as described previously [39]. Briefly, calvariae were dissected from 2-day-old mouse pups, cut into halves, and separated from dura mater, vessels, and scalp. Calvariae were cultured in tissue culture plates in BGJb medium supplemented with 0.1% bovine serum albumin and 100 U/ml each of penicillin and streptomycin for 1 day before treating with 10 mg/ml antibody or protein for 7 days. The bones were cultured in a humidified atmosphere of 5% CO2 at 37uC. Mouse calvariae were imaged with a mCT 40 (SCANCO Medical, Basserdorf, Switzerland) X-ray micro-computer tomography (micro-CT) system with the following parameters: X-ray tube energy level of 45 kV, current of 177 mA, integration time of 300 msec, and 2000 projections. Axial images were obtained at an isotropic resolution of 6 mm. A hydroxyapatite (HA) phantom was used for calibration. Micro-CT scans were analyzed with Analyze (AnalyzeDirect Inc., Lenexa, KS, USA). Maximum-intensity projections and three- dimensional surface renderings in the transverse plane were created for each sample. Parietal bone borders were manually defined within micro-CT images in order to segment the parietal region. Within this region, sample volume and mean bone mineral density (BMD) were calculated. A threshold of 0.3 gm-HA/cm3 was applied to the region in order to calculate the mean BMD of only calcified tissue within the region. The threshold was also used to calculate percentage calcified volume of the parietal region by dividing the number of calcified voxels over the total voxels for the parietal region. The following parameters were analyzed for each sample: parietal region volume, BMD of calcified voxels of the parietal region, and parietal region percentage calcified. PLoS ONE | www.plosone.org 15 September 2010 | Volume 5 | Issue 9 | e12682