Phage selection

Ph.D.™-12 Phage Display Peptide Library Kit was purchased from New England Biolabs Inc. (Beverly, MA, USA). Biopanning procedures were done according to the manufacturer’s instruction with certain modifications. Briefly, a 96-well plate was coated with 150 µL hEGFRvIII (Sino Biological #29662-H08B) (in the first two rounds at 100 μg/mL, the third round at 10 μg/mL) overnight at 4 °C. Wells were washed with PBS, blocked with blocking buffer (3% BSA in PBS), washed six times with cold PBST (PBS + 0.1% [v/v] Tween-20), then incubated with 10¹⁰ pfu phage peptide library Ph.D.™-12 for 2 h at RT. Unbound phages were removed by washing 10 times with cold PBST (PBS + 0.1% [v/v] Tween-20 in the first round and 0.5% [v/v] in the other two rounds). Bound phages were eluted with 100 μL of 0.2 M Glycine–HCl (pH 2.2), 1 mg/mL BSA and neutralized with 15 μL of 1 M Tris–HCl, pH 9.1. The elution procedure was repeated three times and the final eluate was used for amplification in Escherichia coli ER2738 culture. Recovered phages were subjected for two more rounds of biopanning with hEGFRvIII proteins (Sino Biological #29662-H08B). The eluates of each round were titrated by qPCR to enumerate phages and eluate from the third round of screening for NGS sequencing. Streptavidin was used as a biopanning control with the same condition as the target except for elution. The unbound phages were eluted with 100 μL of 0.1 M Biotin in PBS for 30 min.