The ability of mBiTEs and sBiTEs to bind to the PD-L1 and CD3 proteins on the cell surface was evaluated by flow cytometry. The ability of BiTEs to bind to the PD-L1 protein was tested in KKU055, KKU100, and KKU213A cells. Briefly, 200 ng of BiTE protein was used to stain KKU055, KKU100, and KKU213A cells, which were then analyzed for the percentage of positive cells compared to that of the commercial anti-PD-L1 monoclonal antibody (Clone 29E.2A3, BioLegend, San Diego, CA, USA). Equal concentrations of BiTEs were tested for their ability to bind to CD3 on lymphocytes, after which the percentage of positive cells was compared to that of the commercial anti-CD3 monoclonal antibody (Clone UCHT1, ImmunoTools, Friesoythe, Germany).
To study the specificity of mBiTEs and sBiTEs to PD-L1 and CD3, a blocking assay was conducted. CCA cells or lymphocytes were incubated with 200 ng of mBiTEs or sBiTEs for 1 h on ice. After washing, CCA cells were incubated with PE-conjugated anti-PD-L1 antibody, and the lymphocytes were incubated with FITC-conjugated anti-human CD3 antibody (BioLegend, San Diego, CA, USA) for 30 min on ice. The reduction in the monoclonal antibody signal was detected and compared to that of no-BiTE controls using a BD Accuri C6 Flow Cytometer and its analysis software program (BD Biosciences, San Jose, CA, USA).
To study the specificity of mBiTEs and sBiTEs to PD-L1 and CD3, a blocking assay was conducted. CCA cells or lymphocytes were incubated with 200 ng of mBiTEs or sBiTEs for 1 h on ice. After washing, CCA cells were incubated with PE-conjugated anti-PD-L1 antibody, and the lymphocytes were incubated with FITC-conjugated anti-human CD3 antibody (BioLegend, San Diego, CA, USA) for 30 min on ice. The reduction in the monoclonal antibody signal was detected and compared to that of no-BiTE controls using a BD Accuri C6 Flow Cytometer and its analysis software program (BD Biosciences, San Jose, CA, USA).