Small-Animal PET Imaging of Human Epidermal Growth Factor Receptor Positive Tumor with a ⁶⁴Cu Labeled Affibody Protein

Abstract

Epidermal growth factor receptor (EGFR) has become an attractive target for cancer molecular imaging and therapy. Affibody proteins against EGFR have been reported, and thus, we were interested in evaluating their potential for positron emission tomography (PET) imaging of EGFR positive cancer. An Affibody analogue (Ac-Cys-Z(EGFR:1907)) binding to EGFR was made through conventional solid phase peptide synthesis. The purified protein was site-specifically coupled with the 1,4,7,10-tetraazacyclododecane-1,4,7-tris-aceticacid-10-maleimidethylacetamide (maleimido-mono-amide-DOTA) to produce the bioconjugate, DOTA-Z(EGFR:1907). (64)Cu labeled probe (64)Cu-DOTA-Z(EGFR:1907) displayed a moderate specific activity (5-8 MBq/nmol, 22-35 microCi/microg). Cell uptake assays by pre-incubating without or with 300 times excess unlabeled Ac-Cys-Z(EGFR:1907) showed high EGFR-specific uptake (20% applied activity at 0.5 h) in A431 epidermoid carcinoma cancer cells. The affinity (K(D)) of (64)Cu-DOTA-Z(EGFR:1907) as tested by cell saturation analysis was 20 nM. The serum stability test showed excellent stability of the probe with >95% intact after 4 h of incubation in mouse serum. In vivo small-animal PET imaging showed fast tumor targeting, high tumor accumulation (approximately 10% ID/g at 1 h p.i.), and good tumor-to-normal tissue contrast of (64)Cu-DOTA-Z(EGFR:1907) spiked with a wide dose range of Ac-Cys-Z(EGFR:1907). Bio-distribution studies further demonstrated that the probe had high tumor, blood, liver, and kidney uptakes, while blood radioactivity concentration dropped dramatically at increased spiking doses. Co-injection of the probe with 500 microg of Ac-Cys-Z(EGFR:1907) for blocking significantly reduced the tumor uptake. Thus, (64)Cu-DOTA-Z(EGFR:1907) showed potential as a high tumor contrast EGFR PET imaging reagent. The probe spiked with 50 microg of Ac-Cys-Z(EGFR:1907) improved tumor imaging contrast which may have important clinical applications.

Preparation and Evaluation of 99mTc-Epidermal Growth Factor Receptor (EGFR)-Peptide Nucleic Acid for Visualization of EGFR Messenger RNA Expression in Malignant Tumors

“…Among the organs, the kidneys and liver saw the highest accumulation of radioactivity. This result is expected because the liver, as a major organ involved in metabolism, carries a high expression level of EGFR (36). During molecular imaging, substantial radioactivity was seen accumulated in the abdomen, consistent with the biodistribution analysis, which would render assessments of tumors relatively challenging in this region.…”

Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging

“…To develop a PET tracer based on the HAC-PD-1 scaffold, we conjugated a mutated variant, HAC-N91C, with the thiol-reactive bifunctional chelate DOTA-maleimide (22). Although the apparent hPD-L1 affinity of DOTA-HAC was weaker than its parent sequence HAC-V, DOTA-HAC nonetheless antagonized hPD-L1 1,200-fold more potently than WT PD-1 (SI Appendix, Fig.…”

Differential Receptor Tyrosine Kinase PET Imaging for Therapeutic Guidance

“…The binding affinity of radiolabeled DOTA-cetuximab-F(ab9) 2 (EGFR PET probe) or DOTA-HER3 mAb105-F(ab9) 2 (HER3 PET probe) was determined by direct radioligand binding assays, with slight modification of previously described methods (supplemental data; available at http:// jnm.snmjournals.org) (19).…”