Antibody construction
mAbs against PD-1 and LAG-3 were generated using standard hybridoma technology from mice immunized with human PD-1 extracellular domain molecule and human LAG-3 extracellular domain molecule. Hybridomas were generated and screened to select final mAbs. The mAbs were humanized using the framework homology-based humanization method. The humanized mAbs were used for DART molecule generation. The mAbs and DART molecule conversion was engineered by standard molecular cloning, sequencing and mutagenesis methods. mAbs against PD-1 and LAG-3 were evaluated and selected for DART molecule conversion based on binding, biophysical and functional blocking against their respective receptor/ligand axes and functional activity in reactivation of prior superantigen-stimulated T cells or in antigen-specific recall assays.