Construction of tebotelimab
Tebotelimab is a cynomolgus monkey, cross-reactive, Fc-bearing (IgG4) DART molecule comprising bispecificity for two checkpoint molecules, PD-1 (CD279) and LAG-3 (CD223). Tebotelimab was assembled upon completion of humanization. Larger chain, comprising LAG-3 VL-linker-PD-1 VH-E coil-IgG4Hinge-Fc(S228P), was assembled by overlapping PCR to combine the V-region segments and restriction site cloning to add the E coil-Hinge-Fc segment. Smaller chain, comprising PD-1 VL-linker-LAG-3 VH-K coil, was assembled by overlapping PCR with restriction site cloning. Tebotelimab was captured from conditioned culture medium using MabSelect Protein A affinity chromatography (Cytiva). After column equilibration in phosphate buffer saline (PBS), the conditioned medium was loaded at a capacity of 10–30 mg of tebotelimab per milliliter of protein A resin at a flow rate of 100 cm h