When cleaved linker is considered as a conjugated species, SG3544 ADC exhibited no retro-Michael deconjugation in mouse serum, whereas SG3249 ADC exhibited 38% retro-Michael deconjugation, indicating that a more stable thiol linkage resulted from the N-phenyl maleimide drug-linker

When cleaved linker is considered as a conjugated species, SG3544 ADC exhibited no retro-Michael deconjugation in mouse serum, whereas SG3249 ADC exhibited 38% retro-Michael deconjugation, indicating that a more stable thiol linkage resulted from the N-phenyl maleimide drug-linker. L, diluted from 5000X stock with water, Invitrogen, Carlsbad, CA, USA) in a 96-well RT-PCR plate. Samples were then loaded into a CFX96 Real-Time System equipped with a C1000 Thermal Cycler instrument (BioRad, Hercules, CA, USA) and the temperature was equilibrated to 20 C for 10 min. Fluorescence intensity was measured at 20 C and the fluorescence intensity is reported as the average measurement value of five samples standard deviation. 2.7. ADC Serum Stability ADCs were incubated in mouse and rat sera SAR405 R enantiomer to challenge the stability of the thiosuccinimide linkage. ADC samples were added to normal mouse or rat serum (Jackson Immunoresearch, West Grove, PA, USA) to achieve a final concentration of 0.2 mg/mL (1.33 M antibody), with the total volume of ADC solution added to serum being less than 10%. The ADC-serum mixture was sterile filtered and incubated at 37 C for seven days in SAR405 R enantiomer a sealed container without stirring. Total human antibody (PBD-conjugated and unconjugated) was recovered from serum by immunoprecipitation using Fc-specific anti-human IgG-agarose resin (Sigma-Aldrich, St. Louis, MO, USA). Resin was rinsed twice with PBS, once with IgG elution buffer, and then twice more with PBS. ADC-containing serum samples were then combined with anti-human IgG resin (100 L of ADC-serum mixture, 50 L resin slurry) and gently mixed for 15 min at room temperature. Resin was recovered by centrifugation and then washed twice with PBS. The resin pellet was resuspended in 100 L IgG elution buffer (Thermo Scientific, Waltham, MA, USA) and further incubated for 5 min SAR405 R enantiomer at room temperature. Resin was removed by centrifugation and then 20 L of 10X glycol buffer 1 (New England Biolabs, Ipswich, MA, USA) was added to the supernatant. Recovered human antibody solution was sterile filtered and deglycosylated by adding 1.5 uL Remove-iT? EndoS (200K units/mL, New England Biolabs, Ipswich, MA, USA), and followed by further incubation at 37 C for 1 h. Deglycosylated ADCs were reduced with TCEP and analyzed by LC/MS. Percent conjugated antibody was determined from peak heights of mass spectra similar to a previously described method [29]. 2.8. Cytotoxicity Analysis Site-specific A07-108 T289C ADCs were evaluated for in vitro potency against receptor positive MDA-MB-361 breast cancer cells. Cells were plated in 80 L of Leibovitzs L-15 culture medium containing 20% FBS into 96-well flat-bottomed plates at 5000 cells/well. Cells were allowed to adhere overnight. A 5X concentration stock solution of each ADC was prepared by diluting the test articles in culture medium. Twenty microliters of each test article were added to cells in duplicate in a stepwise 1:4 serial dilution series. Treated cells were cultured at 37 C/0% CO2 for six days, and cell viability was assessed with the Cell Titer-Glo (CTG) Luminescent Viability Assay from Promega. 100 L of reconstituted CTG reagent was added to each well and the plate was mildly shaken for 10 min at room temperature. The luminescence of each sample at 560 nm was read using a Perkin Elmer EnVision luminometer (Waltham, MA, USA). Percent cell viability was calculated by the following formula: (average luminescence of treated samples/average luminescence of untreated control samples) 100. EC50 values were determined using logistic non-linear regression analysis with GraphPad Prism v7.02 software (La Jolla, CA, USA). 2.9. Tumor Growth Inhibition All of the animal procedures were performed in accordance with appropriate regulatory standards under protocols approved by the MedImmune Institutional Animal Care and Use Committee. In vivo efficacy studies were performed using five- to six-week-old female athymic nude mice (Harlan Sprague Dawley Inc., Indianapolis, IN, USA). Sixty day 0.36 mg slow release estradiol pellets were implanted subcutaneously into the dorsal flank of mice the day before tumor cell inoculation. Ten million MDA-MB-361 cells in 50% Matrigel were injected subcutaneously into the 2nd mammary fat pad of mice to generate tumors. When tumors reached approximately 200 mm3 mice were randomized based upon tumor volume and assigned into groups (= 10, each group). IgG DICER1 control- or A07-108-PBD ADCs were administered at 0.3 or 1 mg/kg intravenously. Tumors were measured twice weekly with calipers and tumor volumes were calculated using the formula: V = ? L W2 (1) where L = length; W = width. Tumor growth graphs were.