Combinatorial Screening of Anti-RAS VH/VL

Combinations of VH and VL variants (anti-RAS VH, VHdm and VHmut, anti-RAS VL#204, non-specific VL#I21 and anti-LMO2 VLs #819, #826 and #827^{15 (link)}) were cloned into the triplex vector^{16 (link)} via NotI/SfiI and BamHI/EcoRV restriction sites. HEK293T cells were transiently co-transfected in triplicate with the 400 ng triplex plasmids encoding the various combinations of both VH-VP16 and VL-Gal4DBD, alongside 200 ng pG5-Luc reporter and 200 ng pPGK-KRAS₁₆₆(G12V)-2A-Puro RAS antigen plasmid. Cells were analyzed for luciferase activity 48 hours post-transfection using the Dual-Glo Luciferase assay (Promega), with luminescence measurements read by a M2e Spectramax plate reader (Molecular Devices). The ratio of Renilla to Firefly luciferase was calculated. Fold luciferase activity was calculated relative to the empty vector control.