Synthesis and biological evaluation of panitumumab–IRDye800 conjugate as a fluorescence imaging probe for EGFR-expressing cancers

To investigate panitumumab–IRDye800 as an intraoperative optical imaging agent for epidermal growth factor receptor (EGFR)-expressing cancers, we developed clinical-quality panitumumab–IRDye800 and evaluated its specificity and sensitivity to visualize tumors by fluorescence imaging in a variety of mouse xenograft models with different levels of EGFR-expression. Panitumumab was chemically conjugated to NIR-dye (Li-COR 800CW) at well-defined and limited substitution ratio (1:1–2) for the characterization of fluorescence signals. The yield and purity of the conjugate was 80 ± 5% and 95 ± 2%, respectively (n = 6). Quality control (QC) tests showed that the product was suitable for clinical development. Female athymic nude xenograft tumor bearing mice (n = 5 per tumor model) with very low (BT-474), moderate (MDA-MB-231), and high (MDA-MB-468) EGFR-expression levels were administered panitumumab–IRDye800 formulations (100 μg of mAb in 100 μL of 0.9% saline) via tail-vein injection. Animal imaging and biodistribution experiments were conducted on an FMT 2500 (Perkin Elmer) fluorescence scanner at 24, 48, 72, 96, and 144 hours post-injection. Immuno-fluorescence images of a panitumumab–IRDye conjugate recorded in mouse xenograft models showed a good correlation (R2 = 0.91) between EGFR-expression level and tumor uptake. Uptake of panitumumab labeled with IR-Dye or [89Zr] in different tumor xenografts with high, medium, and low EGFR expression, as measured by fluorescence or radioactive counts, is highly correlated (r2 = 0.99). This preclinical in vivo study proved that panitumumab–IRDye800 is specific and optical imaging in conjunction with this probe is sensitive enough to detect EGFR-expressing tumors.