CB213 cross-species target affinities

A FortéBio OCTET instrument was used to study the interaction between CB213 with human (R&D Systems 2319-L3) and recombinant cynomolgus monkey LAG3-Fc tagged proteins (R&D System, 8578-PD-050) and similarly for recombinant human and cynomolgus monkey Fc-tagged PD1 (R&D Systems: 1086-PD, 8578-PD-050), and for albumin from human (Sigma Aldrich A3782), cynomolgus monkey (Abcam ab184894) and mouse (Sigma Aldrich A3559). Murine his-tagged LAG3 (Acro Biosystems LA3-M52H5) and recombinant mouse-PD1-human-Fc chimaera (R&D Systems, 1021-PD-100) proteins were also assessed, as was IgG from human serum (Sigma Aldrich I2511).

Targets in sodium acetate pH 6.0 were coupled to AR2G biosensors by amine coupling using the amine reagent coupling kit (2nd generation, FortéBio). Binding was then analysed against CB213 at a range of predetermined concentrations (7-point 2-fold dilution series in 1× dPBS + 0.02% Tween + 0.1% BSA, with a top concentration of 250 nM) with an association phase of 200 seconds and a dissociation phase of 300 seconds. The kinetics and affinities of the binding interactions were modelled using a 1:1 binding model and calculated using the FortéBio Analysis Software version 8.1 or above.