Fig. 3. CA-170 specifically binds to PD-L1 overexpressing cells, but fails to prevent PD-1:PD-L1 complex formation.

a Full-length human PD-L1 was overexpressed in CHO-K1 cells by lentiviral transduction. Single clones of PD-L1 overexpressing cells were isolated by limiting dilution method. The expression of PD-L1 on the parental and PD-L1 overexpressed single clones was determined by staining with anti-human PD-L1–PeCy7 antibody followed by flow cytometry. b The T₂ values obtained following the recording of ¹³C-¹H HSQC spectra with delay and without the delay from parental and PD-L1 overexpressing CHO-K1 cells treated with indicated concentrations of CA-170 are shown from three independent trials. c Schematic representation of the data observed from the NMR study from PD-L1 overexpressed cells and parental cells. d Anti-PD1 antibody or CA-170 were tested at the indicated concentrations in a TR-FRET-based PD-1:PD-L1 complex formation assay. The results are represented as mean of duplicates. e A schematic depicting the proposed mechanism-of-action for CA-170 in blocking PD-L1 action. In conjunction with the blocking of PD-L1 signaling as observed in functional assays and binding to PD-L1 in the cellular context without preventing the assembly of PD-1:PD-L1 complex formation support the formation of a defective ternary complex as the mechanism of action of CA-170.