F 2653

Omalizumab, ligelizumab, and quilizumab were prepared as IgG1-format antibodies, as described above. Edox cells were harvested and incubated with antibodies at the concentration corresponding to saturation concentration for staining (10 nM for 15cl12, 3.33 nM for omalizumab, 1.5 nM for ligelizumab, and 100 nM for quilizumab), in RPMI with 10% FCS and 2 mM L-glutamine, sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin, at 200,000 cells/well, on ice, and at 37 °C for 4 h. Samples were then placed on ice for 5 min, resuspended in 10 µg/mL anti-FLAG antibody (RRID: AB_439712, F-3040, Sigma-Aldrich) in 2% BSA–PBS, and incubated on ice for 30 min. The binding of anti-FLAG was detected with goat anti-mouse-FITC conjugate (AB_259490, F-2653, Sigma-Aldrich), diluted 1:200 in 2% BSA–PBS, after 30 min incubation on ice. Cells were resuspended in 200 µL ice-cold PBS and analyzed with a Guava^® easyCyte™ Flow Cytometer (Luminex). A decrease in fluorescent signal upon treatment at 37 °C was calculated as (1-(MFI at 37 °C/MFI on ice)) × 100.

Omalizumab, ligelizumab, and quilizumab were prepared as IgG1-format antibodies, as described above. Edox cells were harvested and incubated with antibodies at the concentration corresponding to saturation concentration for staining (10 nM for 15cl12, 3.33 nM for omalizumab, 1.5 nM for ligelizumab, and 100 nM for quilizumab), in RPMI with 10% FCS and 2 mM L-glutamine, sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin, at 200,000 cells/well, on ice, and at 37 °C for 4 h. Samples were then placed on ice for 5 min, resuspended in 10 µg/mL anti-FLAG antibody (RRID: AB_439712, F-3040, Sigma-Aldrich) in 2% BSA–PBS, and incubated on ice for 30 min. The binding of anti-FLAG was detected with goat anti-mouse-FITC conjugate (AB_259490, F-2653, Sigma-Aldrich), diluted 1:200 in 2% BSA–PBS, after 30 min incubation on ice. Cells were resuspended in 200 µL ice-cold PBS and analyzed with a Guava^® easyCyte™ Flow Cytometer (Luminex). A decrease in fluorescent signal upon treatment at 37 °C was calculated as (1-(MFI at 37 °C/MFI on ice)) × 100.

Hamster tumours and spleens collected next day after the last treatment, were mechanically disrupted into single cell suspensions, filtered through 70μm filters and then used for downstream analysis. Samples were stained with antibodies for CD8⁺ (PE, 12-0080-82; eBioscience, San Diego, CA, USA), CD4⁺ (PE-Cyanine 7, 25-0041-82; eBioscience, San Diego, CA, USA), and MHC II⁺ cells (FITC, 11-5980-82; eBioscience, San Diego, CA, USA). NK⁺ cells were labelled with the polyclonal antibody anti-Asialo-GM1 (Alexa Fluor-488, 53-6507-80; eBioscience, San Diego, CA, USA), and macrophages and dendritic cells (Mac-2) cells with anti-Galectin 3 (PE, 12-5301-82; eBioscience, San Diego, CA, USA). For analysis of PD-L1 on Syrian hamster adherent PBMCs, freshly isolated PBMCs were cultured in flat bottom 6 well plates overnight to allow adherent cells to attach to the bottom. The next day adherent cells were collected and stained with parental hybridoma culture supernatants followed by secondary antibody staining (secondary antibody (Anti-Mouse IgG (Fab specific) F(ab′)2 fragment–FITC antibody produced in goat, F2653; Sigma-Aldrich). Cell fluorescence for all experiments was detected using BD Accuri C6 (BD Biosciences) collecting at least 50,000 events per sample.