Assessment of pharmacokinetics in cynomolgus monkey

Nine male cynomolgus monkeys were administered a single intravenous bolus dose of 0.07, 0.97, or 10.39 mg/kg TNB-585 (Alta Science Preclinical Seattle, Everett, Washington, USA). Blood samples collected from a peripheral vein were processed to serum on day −12 (predose), and on days 1 (1 hour and 6 hours postdose), 2, 3, 5, 7, 15 and 21. Concentrations of TNB-585 in serum were measured by ELISA. In brief, a rabbit anti-idiotype antibody against the anti-PSMA arm was coated at 1 µg/mL onto 96-well plates and incubated overnight at 4°C. The plates were then washed three times with wash buffer (TBST +0.1% Tween-20) and blocked using 1% milk for 1 hour at room temperature. Following incubation, the plates were washed, and 100 µL of diluted serum was added to each well and incubated for 2 hours at room temperature. After three washes, a rat anti-idiotype antibody against the anti-CD3 arm was added at 1 µg/mL for 30 min. Binding was detected by incubation with anti-rat IgG2a-HRP followed by addition of ELISA Ultra TMB substrate. After sufficient color developed, 100 µL of stop solution was added to each well. Absorbance was read at 450 nm on a SpectraMax i3x plate reader. TNB-585 concentrations in serum were determined by interpolation from the standard curve. Non-compartmental PK parameters were estimated using Phoenix WinNonlin V.8.1 software (Certara USA, Inc, Princeton, New Jersey, USA).