Size-exclusion chromatogram with multi angle light scattering detection of 833c, fresolimumab, 833c&Freso and 833cX&Freso.

Average hydrodynamic radius (nm): 833c = 5.2, fresolimumab = 5.0, 833c&Freso = 6.1, and 833Xc&Freso = 6.1. (TIF)</p

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Lung targeting of 833c&Freso but not fresolimumab.

After bleomycin treatment, rats were injected iv with radio-iodinated antibodies 125I-833c&Freso, 125I-833cX&Freso, and 125I-fresolimumab (see Methods). (A, C, E) SPECT/CT images of rats taken at 1* and 24 h. Maximum intensity projection (MIP), coronal, sagittal, and transverse cross sections are shown. (B, D, F) Planar F067-scintigraphy imaging at the indicated timepoints. A-F: N = 2–3 rats per group (G) Biodistribution analysis of radioiodinated antibodies. Radioactivity was counted in the indicated organs excised from bleomycin-exposed rats. Uptake was quantified as the percentage of injected dose per gram tissue (means ± SD). N = 3–10 rats per group. *Imaging started at 30 min and takes approximately 30 to 90 min.</p

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Therapeutic effect of 833c&Freso on lung pathology and edema from bleomycin.

Rats were treated with 10 μg/kg 833c&Freso iv on day 2 after bleomycin challenge. Lungs excised on day 3 were sectioned and stained with H&E. Light microscopy images (×20) showing lung histopathology (A-D) vs. normal (E). Tissue damage was clearly evident but varied within each group, ranging from low (A, C) to high (B, D). (F) Lung damage in each 20x microscopy field was scored as described in Methods. (G) Wet to dry lung weight ratio, n = 10 total rats per group. Scatter graphs indicate means ± SD. Percentage (%) inhibition of lung injury scores (F) and edema (G) is indicated. **P< 0.01, and *** P< 0.001 were obtained vs bleo control by one-way ANOVA followed by Dunnett’s test.</p

Dose optimization of 833c&Freso.

Rats were injected with 833c&Freso at the indicated doses (μg/kg) on day 1 and day 2 (D1&2) after bleomycin (bleo) exposure. BALf was collected on day 3. (A) Total and differential leukocyte profile in BALf. (B) Relative cell numbers normalized to 10 μg/kg. Data pooled from 1–4 independent experiments with n = 5–22 total rats per group. Scatter graphs of each data point with indicated means ± SD. *P (TIF)</p

Genetic engineering and characterization of 833c&Freso.

(A) Plasmid map for 833c&Freso heavy chain expression. Fresolimumab single-chain variable fragment (scFv) was cloned into the 3’ end of the 833c CH3 domain. (B) 833c&Freso schematic: scFv is attached to the CH3 domain at the 3’ end. Red: human domains, blue: murine domains. CR: constant region, VH and VL: variable regions. (C) Size exclusion chromatography and (D) Reducing SDS-PAGE of purified 833c&Freso. (E) ELISA binding to rat aminopeptidase P2 of 833c&Freso (triangles), 833cX&Freso (circles) and 833c (squares) (see Methods). (F) ELISA binding to TGF-β1 of 833c&Freso (triangle, black), 833cX&Freso (triangle, gray), fresolimumab (squares) and huIgG4, a isotype-matched control IgG for fresolimumab (open circles) (see Methods).</p

833c&Freso reduces TGF-β-mediated disease biomarkers and signaling in the lung.

Biochemical assays were performed on lung samples taken from untreated rats and rats treated as indicated with 833c&Freso on day 2 after bleomycin challenge. BALf or lung tissue was harvested on day 3. (A) Concentration of the indicated biomarkers detected in BALf. Data pooled from 2–3 independent experiments with n = 10–15 total rats per group. (B) Western blot of lung tissue homogenates using antibodies to the indicated proteins. GAPDH: loading control. Data pooled from 2–3 independent experiments with n = 6–11 total rats per group. (C) Relative expression of indicated proteins derived from western blot image analysis. Smad 2/3 protein phosphorylation: ratio of P- to T Smad 2/3. Graphs show means ± SD. Percentage (%) inhibition of biomarkers in BALf (A) and proteins expressed in the lung (C) are shown. *P< 0.03, **P< 0.01, and *** P< 0.001 were obtained vs bleo control by one-way ANOVA followed by Dunnett’s test.</p

833c&Freso does not affect PDGF/AKT/mTOR axis signaling in the lung.

Rats were treated with 10 μg/kg 833c&Freso iv on day 2 post-bleomycin. Lung tissue was harvested on day 3. (A) Lung tissue homogenates were subjected to western blot analysis using antibodies to indicated proteins. (B) Relative expression of PDGFRβ, P-AKT to AKT and P-p70S6K to p70S6K derived from western blot image analysis. Scatter graphs of each data point with indicated means ± SD. N = 3–5 rats per group. *** P (TIF)</p

Therapeutic effects of 833&Freso on bleomycin-induced lung infiltration of inflammatory cells detected in BALf.

833c&Freso at the indicated doses (μg/kg) was injected iv into rats on the indicated days after bleomycin challenge (A-D) day 2 (D2), (C-D) day 1 (D1), (C-D) day 1 and 2 (D1&2)). BALf was collected on day 3. Total leukocyte and neutrophil count per ml of BALf (A,C) and percentage inhibition of the bleomycin effect on total leucocyte and neutrophil infiltration (B). Data pooled from 2 independent experiments totaling n = 10 rats per group. (D) Relative inhibition is normalized to single treatment on D2, with n = 5 rats per group. Scatter graphs showing each data point with indicated means ± SD. *P< 0.03, **P< 0.01, and *** P< 0.001 vs. bleo only, by using one-way ANOVA followed by Dunnett’s test, except as indicated otherwise (A and B) when comparing dose differences with Student’s unpaired t test.</p