In AsPC-1 cells, (Fig. 1B and Supplementary Table S1) co-administration of 89Zr-MEHD7945A and unlabeled MEHD7945A at an excess of 10- and 25-fold for 1 h reduced uptake (1.97 ± 0.74%, p < 0.0001 and 1.06 ± 0.82%, p < 0.0001, respectively) compared to unblocked cells (5.55 ± 0.64%). Blocking with the anti-EGFR antibody, cetuximab, which binds to the same EGFR epitope as MEHD7945A, also significantly reduced the radiotracer uptake (1.09 ± 0.47% at 10× and 1.47 ± 0.42% at 25× blocking doses, p < 0.0001). Competitive inhibition of the tracer with the anti-HER3 antibody DL3.6b with a 10-fold excess decreased tracer binding from 5.55 ± 0.64% to 3.47 ± 0.26% (p = 0.0194), whereas treatment with 25-fold excess of DL3.6b showed no significant difference with 5.60 ± 0.51% total bound compared to control. External validation through flow cytometry was performed on separate groups of AsPC-1 cells exposed for 48 h to 25-fold higher cold doses of MEHD7945A, cetuximab and DL3.6b (Supplementary Fig. S3A and Supplementary Table S1). Control untreated cells had a median fluorescent intensity (MFI) of 340.3 ± 32.8 for EGFR-expressing cells and 79.6 ± 11.5 for HER3-expressing cells. Blocking with MEHD7945A significantly reduced EGFR expression compared to control (32.6 ± 2.8 MFI, p < 0.0001) but did not significantly reduce cells bearing HER3 expression (57.3 ± 3.7 MFI, p = 0.096). Blocking with cetuximab significantly depleted EGFR expression (0.1 ± 0.9 MFI, p = 0.0001) but not HER3 (80.4 ± 6.9 MFI). Treatment with DL3.6b did not change EGFR expression (336.0 ± 3.6) but significantly attenuated total HER3 (48.2 ± 4.9 MFI, p = 0.0128).