Onartuzumab

Surface expression of EGFR and c-MET was assessed using a BD Accuri™ C6 flow cytometer (BD Biosciences). Cetuximab (5 mg/mL; Merck) and onartuzumab (60 mg/mL; Genentech) served as primary antibodies for EGFR and c-MET, respectively, and mouse anti-human Fc-specific FITC-conjugated secondary antibody (clone HP-6017; Sigma-Aldrich) was used for readout of both EGFR and c-MET expression, with 10,000 events assessed per sample. The sensitivity of HCC827 and HCC827ErlRes cell lines to erlotinib and NVP-AUY-922 after 4 days of treatment was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [23 (link)].

We adhere to the nomenclature rules for radiopharmaceutical chemistry (30 (link)). Panitumumab, trastuzumab, and cetuximab were obtained from the Memorial Sloan Kettering hospital pharmacy. Onartuzumab was provided by Genentech. The antibodies were conjugated with the bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine (DFO-Bz-NCS; Macrocyclics) and then radiolabeled with ⁸⁹Zr in accordance with previously reported methods (28 (link)). The antibodies were conjugated with p-SCN-Bn-DFO in a 5:1 DFO:antibody molar ratio at 37°C for 90 min. After reaction, the conjugates were purified via a PD-10 column using Chelex (Bio-Rad) phosphate-buffered saline (0.5 g/L Chelex resin) at pH 7.4. The ⁸⁹Zr-oxalate (supplied in 1.0 M oxalic acid at Memorial Sloan Kettering Cancer Center (28 (link))) was neutralized to pH 7.0–7.5 with 1.0 M Na₂CO₃ followed by addition of the corresponding DFO–antibody conjugate in Chelex phosphate-buffered saline (pH 7.4). The mixture was incubated at 37°C for 1 h on an agitating heating block. [⁸⁹Zr]Zr-DFO-antibody and radiochemical purity was determined by instant thin-layer chromatography, and the product was used for in vivo studies.

Onartuzumab was produced and provided by Genentech (San Francisco, CA). Approximately 10 × 10⁶ cells of each PDAC line were harvested, and washed with ice-cold phosphate buffered saline (PBS) three times. Cell pellets were resuspended in FcR block (1:5 final dilution) (Miltenyi Biotec, Bergisch Gladbach, Germany) and incubated for 30 minutes on ice. Cell suspensions were then split into multiple groups, stained with fluorescently labeled Onartuzumab (3µg/mL), fluorescently-labeled non-specific IgG (3µg/mL), or incubated without antibody for 30 minutes on ice. Following incubation, cells were washed with ice cold buffer (PBS, 2% FCS, 0.1% sodium azide, 1 mM ethylenediaminetetraacetic acid (EDTA)) three times 34 (link). DAPI (Sigma-Aldrich, St. Louis, MO) was added prior to assaying samples. Single color controls were made and results were analyzed with FlowJo software (FlowJo LLC, Ashland, Oregon).