Supporting Information

Transcription

1 Supporting Information Chan et al /pnas SI Text Protein Purification. PCSK9 proteins were expressed either transiently in 2936E cells (1), or stably in HepG2 cells. Conditioned culture medium was first concentrated, then diafiltered against 20 mm Tris-HCl ph 8.0, 1 M NaCl, 20 mm Imidazole until a diafiltration factor of 10 was achieved. Purification of recombinant polyhistidine-tagged PCSK9 proteins was carried out by sequential Ni-IMAC capture and size exclusion chromatography on HisTrap FF Crude and Superdex 200 columns, respectively (GE Healthcare). mab1 was purified by capture on MabSelect SuRE (GE Healthcare), and nonspecific binders were removed by a 10 column volume wash with 1 Dulbecco s Phosphate Buffered Saline (Mediatech). Bound IgGs were eluted with 0.5% acetic acid ph 3.5 and immediately neutralized with 1 M Tris-HCl ph 7.9. The Mab Select SuRE eluate was bound to SP HP Sepharose in the presence of 10 mm sodium acetate ph 5.2, and eluted using a linear gradient of 0 to 500 mm NaCl in 20 column volume. Purified antibody was formulated in 10 mm sodium acetate ph 5.2, 9% sucrose, and homogeneity was assessed by dynamic light scattering and HPLC size exclusion analysis. The IgG2 isotype of mab1 has a molecular weight of 143, and an isoelectric point (pi) of PCSK9 ELISA. Levels of human PCSK9 in adeno-associated virus (AAV)-treated mouse sera was determined by sandwich ELISA. Clear 96-well plates were coated overnight (4 C) with mab2, a monoclonal anti-pcsk9 antibody (Amgen reagent). After thorough washing, plates were incubated for 2hwithAAVmouse serum samples diluted in general assay diluent. A rabbit polyclonal biotinylated anti-human PCSK9 antibody (D8774, Amgen Inc.) was added at 1 g/ml (in 1% BSA/PBS), followed by neutravidin-hrp at 200 ng/ml (in 1% BSA/PBS). Bound PCSK9 was detected by incubation with TMB substrate and absorbance measurement at 650 nm. A standard curve generated with recombinant human PCSK9 was used to determine the corresponding concentration of PCSK9 in the serum samples. The intra-assay coefficient of variation (CV) ranged from % and the inter-assay CV ranged from 1 6%. The levels of secreted PCSK9 in HepG2 conditioned medium and the levels of mouse serum PCSK9 were determined in a similar manner. For mouse serum PCSK9, mab1 was used as the capture antibody and the standard curve was generated using recombinant mouse PCSK9. The intra-assay CV ranged from % and the inter-assay CV ranged from 1 8%. The concentration of free PCSK9 in cynomolgus monkey sera was determined by sandwich ELISA using mab1 as capture antibody and biotinylated mab3, a monoclonal anti-pcsk9 antibody, as detection reagent. The acceptance of assay data required fulfillment of the same criteria described below for the antibody assay. Serum Antibody Concentrations. Serum concentrations of mab1 were determined by sandwich ELISA, using polyclonal goat anti-human IgG and goat anti-human IgG-HRP (Jackson ImmunoResearch) for mouse studies, and anti-human Fc mab and HRP labeled anti-human Fc mab (Amgen Inc.) for the cynomolgus monkey study. Acceptance of assay data required that a minimum of 6 standard levels met the following criteria: (i) 20% bias (recovery) between the mean assay concentration and the nominal concentration, and (ii) 20% CV between replicate assay concentrations. Additionally, a minimum of 4 out of 6 replicate Quality Control (QC) samples and at least 1 QC at each level (low, mid, and high range) must have met the criteria stated in (i) and (ii). Data were reduced using Watson version data reduction package with a logistic (autoestimate) regression of separately prepared standard curves. Noncompartmental pharmacokinetic analyses were performed on mean concentration-time data using WinNonlin Enterprise software (Version 5.1.1). AUC 0 - values were calculated using the log-linear trapezoidal method, and reported to 3 significant figures. 1. Durocher Y, Perret S, Kamen A (2002) High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293- EBNA1 cells. Nucleic Acids Res 30(2):E9. 1of8

2 A OD log [mab1] pm B RFU RFU log [mab1] nm PCSK9: mab1: Fig. S1. In vitro efficacy of mab1. (A) mab1 inhibits binding of PCSK9 to the LDLR (amino acid ) in a dose-dependent manner. The data shown yielded an IC 50 value of 2.27 nm. (B) mab1 blocked the PCSK9-mediated reduction of BODIPY-LDL uptake by HepG2 cells (25 g/ml hupcsk9 50 g/ml mab1 in right panel), yielding an EC 50 value of nm for this particular experiment (left panel). RFU is relative fluorescence units. 2of8

3 Fig. S2. Stereoview of the PCSK9:Fab1 interface with 2F o -F c electron density contoured at 1.5. The PCSK9 catalytic domain is colored tan and the prodomain is colored blue. Fab1 is colored purple. 3of8

4 Serum Concentration of mab1 (ng/ml) Time following injection (hours) Fig. S3. Circulating mab1 concentration in C57BL/6 mice following a single i.v. injection (10 mg/kg) in mice. Serum levels of mab1, measured by sandwich ELISA. Results are expressed as the mean SD, n 7. 4of8

5 Serum Total Cholesterol (mg/dl) ** ** *** ** *** Anti-KLH antibody, 10 mg/kg mab1, 3 mg/kg mab1, 6 mg/kg mab1, 10 mg/kg Time following injection (days) Fig. S4. mab1 in C57BL/6 mice. Increasing doses of mab1 prolonged the duration of the TC lowering effect. Mice were administered a single injection of 3 mg/kg, 6 mg/kg, or 10 mg/kg mab1, or 10 mg/kg anti-klh antibody. Results are expressed as the mean SEM for each time-point and treatment group. (**) indicates P 0.01 and (***) indicates P for mab1 versus anti-klh control antibody at the same time point, n 6 per group. 5of8

6 A Serum Total Cholesterol (mg/dl) Anti-KLH antibody, 10 mg/kg mab1, 10 mg/kg Anti-KLH antibody, 30 mg/kg mab1, 30 mg/kg *** * *** Time following injection (hours) B Serum HDL Cholesterol (mg/dl) ** Anti-KLH antibody, 10 mg/kg mab1, 10 mg/kg Anti-KLH antibody, 30 mg/kg mab1, 30 mg/kg *** * Time following injection (hours) Fig. S5. Changes in serum TC and HDL-C following i.v. administration of mab1 to mice expressing hupcsk9 by AAV. (A) mab1 dose-dependently lowered serum TC and (B) HDL-C (n 7 per treatment group). Results are expressed as the mean SEM. (***) indicates P 0.001, (**) indicates P 0.01 and (*) indicates P 0.05 versus anti-klh control antibody at the same time point and dose. 6of8

7 A 200 Anti-KLH antibody, 3 mg/kg mab1, 3 mg/kg Serum Triglycerides (% of predose) Time following injection (days) B 000 Serum concentration of mab1 (ng/ml) 00 0 t 1/2 = hours Time following injection (days) Fig. S6. mab1 in cynomolgus monkeys. (A) No significant changes were observed in serum triglycerides following mab1 injection. (B) Serum mab1 concentration was monitored throughout the study, and terminal half-life (t1/2) was determined to be 61 9h. 7of8

8 Table S1. Data collection and refinement statistics Crystal 1: FR-E Crystal 2: ALS Data collection Wavelength (Å) Space group C2 C2 Cell dimensions a, b, c (Å) , , , , 69.88,, ( ) 90, , 90 90, , 90 Resolution (Å) ( ) ( ) Completeness 99.4 (99.9) 99.9 () Redundancy 2.7 (2.7) 3.5 (3.6) Rmerge 15.2 (40.8) 7.9 (58.9) I / I 6.3 (2.1) 11.0 (2.5) Refinement Resolution (Å) Reflections Total 107,765 Working set 102,389 Test set 5,376 R work /R free 0.191/0.209 No. of atoms Protein 7630 Ion 1 Water 568 B-factors Protein Ion Water R.m.s deviations Bond lengths (Å) Bond angles ( ) One crystal was used to collect data for each data set. Values in parentheses are highest-resolution shell. Crystal 1: FR-E was collected on a Rigaku RE-E x-ray source. Crystal 2: ALS was collected at the Berkeley Advanced Light Source beamline Values in parenthesis are highest resolution shell. Rms deviations x i x 2 /N. 8of8