Figure 4. Dynamic changes in tumor PD-L1 expression and its engagement by AtzMab detected using [⁶⁴Cu]WL12.

(A) Flow cytometry histogram showing increased PD-L1 cell surface expression in A549-iPDL1 cells treated with doxycycline for 6 hours and 72 hours. (B) WL12 inhibits (5 nM) binding of Cy5-conjugated AtzMab, AveMab, and DurMab (2 nM) to A549-iPDL1 cells treated with doxycycline for 72 hours. (C) [⁶⁴Cu]WL12 binding to A549-iPDL1 cells (72-hour doxycycline) is significantly reduced in the presence of 60 nM AtzMab, compared with controls. (D and E) [⁶⁴Cu]WL12 uptake in A549-iPDL1 xenografts is significantly lower in mice receiving intravenous AtzMab 24 hours prior to radiotracer injection, compared with saline controls and similar to parent A549 xenografts. Volume-rendered whole-body PET-CT images (D), and ex vivo quantification (E) at 2 hours after [⁶⁴Cu]WL12 injection (n = 10/group). (F) IHC staining for PD-L1 of the corresponding tumors. Scale bars: 100 μm. Box-and-whisker graphs showing minimum to maximum and all data points, with the horizontal line representing the median. ****P < 0.0001; NS, not significant, by 1-way ANOVA and Sidak’s multiple comparisons test in C and E.