Pe conjugated anti human fc antibody

DNA sequences of designs were purchased from Twist Bioscience containing homology overhangs for cloning. DNA was transformed with linearized pCTcon2 (Addgene, 41843) or a modified pNTA vector with V5 tag into EBY-100 yeast using the Frozen-EZ Yeast Transformation II Kit (Zymo Research). Transformed yeast were passaged once in minimal glucose medium (SDCAA) before induction of surface display in minimal galactose medium (SGCAA) overnight at 30 °C. Transformed cells were washed in cold PBS with 0.05–0.1% BSA and incubated with the binding target for 2 h at 4 °C. Cells were washed once and incubated for an additional 30 min with the appropriate antibodies (Supplementary Table 5). Cells were washed and analysed using the Gallios flow cytometer (Beckman Coulter). For quantitative binding measurements, binding was quantified by measuring the fluorescence of a PE-conjugated anti-human Fc antibody (Invitrogen) detecting the Fc-fused protein target. Yeast cells were gated for the displaying population only (V5, MYC or HA positive) (Extended Data Fig. 3a).

Antibodies for APC-CD3, PE-CD4, PE/Cy7-CD8, Alexa Fluor647-CD56, PE-CD107a, PE-CCR7, PE/Cy7-CD62L, PE/Cy7-perforin, PE-granzyme B, PE-PD-L1 were purchased from BioLegend. Goat anti-B7-H3 antibody (MAB1027) was purchased from R&D System. Humanized anti-B7-H3 IgG (8H9) was expressed and purified in the laboratory (for details, see Additional file 1). Mouse anti-CD16 antibody (3G8) was purchased from BD Biosciences. PE-conjugated anti-human Fc antibody and HRP anti-human IgG antibody were purchased from Invitrogen. HRP-conjugated rabbit anti-goat IgG antibodies were purchased from Jackson ImmunoResearch. Rabbit antihuman PD-L1 antibody (13,684), HRP-conjugated anti-β-actin and anti-GAPDH antibodies were purchased from Cell Signaling.