Figure 1
Flow chart of the high throughput screening platform for non-covalently assembled immunotoxins with antibody fragments derived from synthetic antibody libraries.
The scFv binders against HER2-ECD were selected in three selection/amplification cycles from the phage-displayed GH2 scFv library (built on IGKV1-NL1*01/IGHV3-23*04 single framework) with the standard phage display selection/screening procedure (Step 1~2)27. Candidate scFvs were then screened for soluble scFv binding to HER2-ECD and for binding to Protein A and Protein L (step 3)27. Simultaneous binding to Protein A and Protein L indicates that the scFv with the IGKV1-NL1*01/IGHV3-23*04 framework is well-folded (structure shown in PDB code: 4HKZ)33. Positive clones binding to HER2-ECD, Protein A and Protein L were grown in 96-deepwell plates overnight and the culture media were filtrated with 0.45 μm filter (step 4). Cells were seeded in 96-well plates one day ahead in normal medium (10% FBS) (step 5). Soluble scFvs in medium were mixed with AL1/AL2-PE38KDEL in 1:1 (scFv:AL fragment) molar ratio for 1 hour to form the scFv-AL1-PE38KDEL or scFv-AL2-PE38KDEL immunotoxin complexes (step 6a). These complexes are stably formed because of the high affinity scFv binding site in the AL fragment (Figs 2~3) and the preselection of the positive clones with well-folded scFv structure capable of binding to Protein A and Protein L simultaneously. These immunotoxin complexes were added to cultured cells: Normal medium was replaced by 50 μL/well serum free medium before adding 50 μL immunotoxin complex to each well (step 6b). After 4 hours incubation at 37 °C, medium with immunotoxin complex was replaced by normal medium (10% FBS) and the cells were cultured for 4 more days at 37 °C (step 7). WST-1 reagent (10 μL/well) was added to the medium and incubated for 3 to 4 hours for color development (step 8). The cell viabilities were determined at OD450 nm by spectrophotometer (step 9). More experimental details are described in Methods.
