Octet system data analysis software

An Octet RED96e instrument (FortéBio, Sartorius AG) was used to perform binding experiments of the antibodies to soluble murine PD-1 monoFc fusion protein. The murine PD-1 fusion protein was biotinylated using EZ-Link sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, the biotin-conjugate was added to protein in 0.1 M sodium phosphate buffer at a ratio of 1:5 molar excess. The reaction proceeded at room temperature with continuous rocking and was stopped with 10x tris-buffered saline after 30 minutes. Any remaining unbound biotin was removed by Amicon® Ultra-15 centrifugal filters (Millipore Sigma) or Zeba Spin Desalting Columns (Thermo Fisher Scientific), and the protein was buffer exchanged to PBS (Corning). The biotinylated protein was then loaded onto SA sensors at 2 μg/mL for 60 seconds. These sensors were then first dipped into PBS (Corning) for 60 s to establish a baseline and then exposed to each antibody solution (varying concentrations) for 600 seconds to measure association. The sensors were then returned to PBS (Corning) for 1800 seconds for dissociation. Curve fitting to calculate apparent K_D was performed using the Octet System Data Analysis software (FortéBio, Sartorius AG).