Internalization of 89Zr-MEHD7945A in all cell lines was conducted at 37 °C (Fig. 1A, left). BxPC-3 displayed the highest uptake from 3.29 ± 0.28% at 1 h to almost a two-fold increase at 24 h with 5.91 ± 0.05% of the tracer internalized. In AsPC-1, the tracer was steadily internalized over time (2.58 ± 0.23% at 1 h, 3.23 ± 0.26% at 4 h and 4.70 ± 0.52% at 24 h) while the negative control cell line, Mia PACA2 demonstrated lower uptake across all time points (1.15 ± 0.06% at 1 h, 1.76 ± 0.17% at 4 h and 3.04 ± 0.21% at 24 h respectively). Significantly lower accumulation was observed in all cell lines at 4 °C, supporting an endocytotic mechanism of internalization (Fig. 1A, right).Figure 1In vitro characterization. 89Zr-MEHD7945A demonstrated higher internalization rates in BxPC-3 and AsPC-1 at 37 °C (left) compared to the EGFR/HER3-negative Mia PACA2 pancreatic cancer cell line. 89Zr-MEHD7945A showed a decrease in internalization at 4 °C (right) in all cell lines. (A) 89Zr-MEHD7945A demonstrated successful blocking with cold MEHD7945A and cetuximab at 10× and 25× doses. Blocking with DL3.6b at 10× lowered the uptake of the probe; binding was sustained at 25× dose of the anti-HER3 mAb. In both AsPC-1 and BxPC-3. (B) Non-linear regression analysis determined two sets of KD and Bmax for AsPC-1 with an. (C) The KD and Bmax values for BxPC-3 (D) are within the same range as the established values in AsPC-1 with an IC50 ~ 0.37 nM. (*Denote p < 0.01, ǂdenote p < 0.05, compared to no block).