To determine whether 89Zr-MEHD7945A binds to either EGFR or HER3 alone or both, in vivo blocking studies were conducted. Blocking EGFR with cetuximab in AsPC-1 tumors (Fig. 4A and Supplementary Table S3) lowered the uptake of 89Zr-MEHD7945A by almost 3-fold (19.30 ± 5.60%ID/g vs. 7.72 ± 6.70%ID/g, p = 0.0457). In contrast, blocking the HER3 epitope with DL3.6b did not alter tumor accumulation of 89Zr-MEHD7945A (18.79 ± 8.75%ID/g).Figure 4In vivo competitive inhibition. In AsPC-1 xenografts, blocking with cetuximab (EGFR block) showed an almost 2-fold decrease in 89Zr-MEHD7945A uptake, whereas blocking HER3 with DL3.6b did not change probe uptake. (A) In BxPC-3 xenografts, blocking with cetuximab (EGFR block) showed a slight decrease in 89Zr-MEHD7945A, whereas blocking with DL3.6b (HER3 block) showed a statistically significant, increase in probe accumulation. (B) IHC staining in BxPC-3 tumors blocked with 25× cetuximab (25x EGFR, left), 25x DL3.6b (25x HER3, middle) or left unblocked (right) were assessed by IHC for EGFR (top) and HER3 (bottom) expression, and showed an increase in EGFR and HER3 in both blocked cohorts. (C) Tumor sections depicted for IHC are shown in 100×. Densitometry analysis of western blots on tumor lysates (n = 2) from AsPC-1 (left) and BxPC-3 (right) that were untreated, exposed to EGFR-block with cetuximab and a HER3-block with DL3.6b. (D) Densitometry is shown as a ratio of target protein/loading control.