Despite numerous recent advances in our understanding of the molecular mechanisms underlying receptor tyrosine kinase downregulation and degradation in response to growth factor binding, relatively little is known about ligand-independent receptor tyrosine kinase degradation mechanisms. In a screen for proteins that might regulate the trafficking or localization of the ErbB3 receptor, we have identified a tripartite or RBCC (RING, B-box, coiled-coil) protein that interacts with the cytoplasmic tail of the receptor in an activation-independent manner. We have named this protein Nrdp1 for neuregulin receptor degradation protein-1. Northern blotting reveals ubiquitous distribution of Nrdp1 in human adult tissues, but message is particularly prominent in heart, brain, and skeletal muscle. Nrdp1 interacts specifically with the neuregulin receptors ErbB3 and ErbB4 and not with epidermal growth factor receptor or ErbB2. When coexpressed in COS7 cells, Nrdp1 mediates the redistribution of ErbB3 from the cell surface to intracellular compartments and induces the suppression of ErbB3 and ErbB4 receptor levels but not epidermal growth factor receptor or ErbB2 levels. A putative dominant-negative form of Nrdp1 potentiates neuregulin-stimulated Erk1͞2 activity in transfected MCF7 breast tumor cells. Our observations suggest that Nrdp1 may act to regulate steady-state cell surface neuregulin receptor levels, thereby influencing the efficiency of neuregulin signaling.
Cetuximab is a recombinant, human/mouse chimeric IgG1, monoclonal antibody (mAb) that binds to the epidermal growth factor receptor (EGFR/HER1). Cetuximab is approved for the treatment of patients with HER1-expressing metastatic colorectal cancer. Limitations in currently reported radiolabeled cetuximab for PET applications prompted the development of 86Y-CHX-A”-DTPA-cetuximab as an alternative for imaging HER1-expressing cancer. 86Y-CHX-A”-DTPA-cetuximab can also serve as a surrogate marker for 90Y therapy. Methods Bifunctional chelate, CHX-A”-DTPA was conjugated to cetuximab and radiolabeled with 86Y. In vitro immunoreactivity was assessed in HER1-expressing A431 cells. In vivo biodistribution, PET imaging and non-compartmental pharmacokinetics were performed on mice bearing HER1-expressing human colorectal (LS-174T and HT29), prostate (PC-3 and DU145), ovarian (SKOV3) and pancreatic (SHAW) tumor xenografts. Receptor blockage was demonstrated by co-injection of either 0.1 or 0.2 mg cetuximab. Results 86Y-CHX-A”-DTPA-cetuximab was routinely prepared with a specific activity of 1.5– 2 GBq/mg and in vitro immunoreactivity ranging from 65–75 %. Biodistribution and PET imaging studies demonstrated high HER1-specific tumor uptake of the radiotracer and clearance from non-specific organs. In LS-174T tumor bearing mice injected with the 86Y-CHX-A”-DTPA-cetuximab alone, 86Y-CHX-A”-DTPA-cetuximab plus 0.1 mg cetuximab or 0.2 mg cetuximab, the tumor uptake values at 3 d were 29.3 ± 4.2, 10.4 ± 0.5 and 6.4 ± 0.3 % ID/g, respectively, demonstrating dose-dependent blockage of the target. Tumors were clearly visualized 1 d after injecting 3.8–4.0 MBq 86Y-CHX-A”-DTPA-cetuximab. Quantitative PET revealed highest tumor uptake in LS-174T (29.55 ± 2.67 % ID/cc) and lowest tumor uptake in PC-3 (15.92 ± 1.55 % ID/cc) xenografts at 3 d after injection. Tumor uptake values quantified by PET were closely correlated (r2= 0.9, n=18) to values determined by biodistribution studies. Conclusion This study demonstrates the feasibility in preparation of high specific activity 86Y-CHX-A”-DTPA-cetuximab and its application for quantitative non-invasive PET imaging of HER1-expressing tumors. 86Y-CHX-A”-DTPA-cetuximab offers an attractive alternative to previously labeled cetuximab for PET and warrants further investigation for clinical translation.
The monoclonal antibody cetuximab binds to EGFR and thus provides an opportunity to create both imaging and therapies that target this receptor. The potential of cetuximab as a radioimmunoconjugate using the acyclic bifunctional chelator, CHX-A”-DTPA was investigated. The pharmacokinetic behavior in the blood was determined in mice with and without tumors. Tumor targeting and scintigraphic imaging were evaluated in mice bearing xenografts of LS-174T (colorectal), SHAW (pancreatic), SKOV3 (ovarian), DU145 (prostate) and HT-29 (colorectal). Excellent tumor targeting was observed in each of the models with peak tumor uptakes of 59.8±18.1, 22.5±4.7, 33.3±5.7, 18.2±7.8 and 41.7±10.8 %ID/g at 48-72 hr, respectively. In contrast, the highest tumor %ID/g obtained in mice bearing melanoma (A375) xenografts was 6.3±1.1 at 72 hr. The biodistribution of 111In-cetuximab was also evaluated in non-tumor bearing mice. The highest %ID/g was observed in the liver (9.3±1.3 at 24 hr) and the salivary glands 8.1±2.8 at 72 hr). Scintigraphy showed excellent tumor targeting at 24 hr. Blood pool was evident as expected but cleared over time. At 168 hr the tumor was clearly discernible with negligible background.
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