Serum 3BNC117 Quantification Protocol

Serum concentrations of 3BNC117 were measured at multiple time points postinfusion using a validated luciferase-based neutralization assay in TZM.bl cells as previously described (Sarzotti-Kelsoe et al., 2014 (link)). Briefly, serum samples were tested using a primary 1:20 dilution with fivefold titration series against HIV-1 Env pseudovirus Q769.d22, which is highly sensitive to neutralization by 3BNC117. Env pseudoviruses were produced using an ART-resistant backbone vector that reduces background inhibitory activity of antiretroviral drugs if present in the serum sample (SG3ΔEnv/K101P.Q148H.Y181C, M.S. Seaman, unpublished data). 3BNC117 clinical drug product was also tested in every assay setup using a primary concentration of 10 µg/ml with fivefold titration series. The serum concentration of 3BNC117 for each sample was calculated as follows: serum ID₅₀ titer (dilution) × 3BNC117 IC₅₀ titer (μg/ml) = serum concentration of 3BNC117 (μg/ml). Murine leukemia virus was used as a negative control. All assays were performed in a laboratory meeting GCLP standards. The half-life of 3BNC117 was calculated using Phoenix WinNonLin Build 8 (Certara).