Pharmacokinetics of vopratelimab and nivolumab

Blood samples were collected for vopratelimab concentration determinations during cycle 1 prior to dosing, at 0.5, 1, 1.5 (vopratelimab monotherapy), or 2.5 (vopratelimab in combination with nivolumab), 4 and 6 hours postdose, and 2, 8, and 15 days postdose. For subsequent cycles, samples were collected predose and 1 hour postdose. All times were relative to the start of the vopratelimab infusion times. Blood samples for nivolumab concentration determinations were collected at the same timepoints as for vopratelimab.

Vopratelimab concentrations were quantified in serum using a validated electrochemiluminescent assay. Briefly, vopratelimab was captured on meso scale discovery (MSD) plates coated with a mouse anti-human vopratelimab mAb. Detection was via the same mAb conjugated to ruthenium. The assay was validated to a lower limit of quantitation (LLOQ) of 750 pg/mL.

Nivolumab concentrations were measured in serum using a validated ELISA. Briefly, nivolumab was captured on 96-well plates coated with either PD-1-human IgG1 fusion protein or his-tagged recombinant PD-1 protein and detected using a mouse anti-human IgG4 mAb conjugated to horseradish peroxidase (HRP). The assay was validated to an LLOQ of 100 ng/mL.

Samples for vopratelimab anti-drug antibodies (ADA) and neutralizing ADA were collected predose on day 1 of each cycle. ADA to vopratelimab were detected via an electrochemiluminescent assay using a three-tier procedure that consisted of screening without added vopratelimab, screening with added vopratelimab to confirm specificity, and serial dilution of positive samples to determine the titer. Briefly, clinical samples or controls were diluted 1:50 into acetic acid to disrupt existing ADA/drug complexes, then incubated with a neutralizing solution containing biotinylated vopratelimab (bt-vopratelimab) and ruthenium-labeled vopratelimab (ru-vopratelimab). For specificity screening and titering, unlabeled vopratelimab was also included in the assay solution. Bridging complexes of bt-vopratelimab-ADA-ru-vopratelimab were captured on streptavidin-coated plates and ru-vopratelimab was quantitated by electrochemiluminescence. Samples testing positive in both screening steps were considered positive and corresponding titers were determined by serial dilution and reported. Otherwise, samples were reported as negative.

ADA-positive samples were further assayed for neutralizing activity. Briefly, samples were acid treated, then incubated with bt-vopratelimab in a neutralizing buffer. The resulting complexes were captured on a streptavidin-coated plate, washed, and then acid treated to dissociate ADA. The acid-treated samples were incubated with ru-vopratelimab in neutralizing buffer, transferred to an MSD-streptavidin plate coated with biotin-conjugated ICOS to capture uncomplexed ru-vopratelimab. Captured ru-vopratelimab was quantitated by electrochemiluminescence. A maximal signal was observed in absence of neutralizing ADA, and the amount of neutralizing activity was estimated on the basis of the decline in signal.

Pharmacokinetic parameters were calculated for vopratelimab and nivolumab for cycle 1 by noncompartmental analysis using Phoenix WinNonlin (v. 8.2). C_max, C_trough, and the AUC from the start of dosing to the last measured timepoint (AUC_last) were calculated for all patients. For patients treated with nivolumab, the AUC from dosing through day 14 was also calculated for comparison with historical data for nivolumab administered every 2 weeks. When the data were adequate to estimate the terminal elimination half-life, half-life values were also reported for vopratelimab. Actual sampling times were used for the analysis. C_max, C_trough, and AUC values were summarized by geometric mean and geometric coefficient of variation. Half-life was summarized by arithmetic mean and SD.