177Lu-1,4,7,10-Tetraazacyclododecane-1,4,7-triacetic acid-human serum albumin-Ac-Cys-ZEGFR:1907


Leung K.

Publication Details



In vitro Rodents



Epidermal growth factor (EGF) is a growth factor composed of 53 amino acids (6.2 kDa) that is secreted by ectodermic cells, monocytes, kidneys, and duodenal glands (1). EGF stimulates growth of epidermal and epithelial cells. EGF and at least seven other growth factors and their transmembrane receptor kinases play important roles in cell proliferation, survival, adhesion, migration, and differentiation. The EGF receptor (EGFR) family consists of four transmembrane receptors: EGFR (HER1/erbB-1), HER2 (erbB-2/neu), HER3 (erbB-3), and HER4 (erbB-4) (2). HER1, HER3, and HER4 comprise three major functional domains: an extracellular ligand-binding domain, a hydrophobic transmembrane domain, and a cytoplasmic tyrosine kinase domain. No ligand has been clearly identified for HER2; however, HER2 can be activated as a result of ligand binding to other HER receptors with the formation of receptor homodimers and/or heterodimers (3). HER1 and HER2 are overexpressed on many solid tumor cells such as breast, non-small cell lung, head and neck, and colon cancers (4-6). The high levels of HER1 and HER2 expression on cancer cells are associated with a poor prognosis because high levels are related to increased proliferation (7-10).

Trastuzumab is a humanized IgG1 monoclonal antibody (mAb) against the extracellular domain of recombinant HER2 with an affinity constant (Kd) of 0.1 nM (11). Trastuzumab is approved for clinical use for anti-cancer therapies in both Europe and North America. 111In-Trastuzumab, Cy5.5-trastuzumab, and 68Ga-trastuzumab -F(ab')2 have been developed for imaging human breast cancer (12-16). However, the pharmacokinetics of the intact radiolabeled mAb, with high liver uptake and slow blood elimination, are generally not ideal for imaging. Smaller antibody fragments, such as Fab or F(ab´)2, have better imaging pharmacokinetics because they are rapidly excreted by the kidneys. A novel class of recombinant affinity ligands (Affibody molecules) for HER2 based on the Z-domain residues (58 amino acids) from one of the IgG-binding domains of staphylococcal protein A was constructed (17). Affibody molecules exhibit high binding affinity to HER2 with Kd values <100 pM. Various radiolabeled Affibody molecules have been studied in terms of their ability to image HER2 in tumors [PubMed]. The EGFR-specific Affibody ZEGFR:1907 (6.7 kDa) was identified to have good affinity (Kd = 5.4 nM) for EGFR (HER1) and labeled with 111In via isothiocyanate-benzoyl-diethylenetriaminepentaacetic acid (DTPA) to form 111In-Bz-DTPA-ZEGFR:1907 for single-photon emission computed tomography (SPECT) imaging in nude mice bearing human tumors (18). To facilitate site-specific conjugation, Ac-Cys-ZEGFR:1907 (Cys at the N terminal) was synthesized and conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7-tris-aceticacid-10-maleimidethylacetamide (maleimido-mono-amide-DOTA) to form Ac-Cys(DOTA)-ZEGFR:1907 (DOTA-ZEGFR:1907) and labeled with 64Cu (t1/2, 12.7 h) to form 64Cu-DOTA-ZEGFR:1907 for positron emission tomography imaging studies in tumor-bearing mice (19). Although high specific tumor accumulation was observed with 111In- and 64Cu-labeled ZEGFR:1907, very high kidney accumulation (>100% injected dose per gram (ID/g)) was also observed. Addition of human serum albumin (HSA) to Affibody ZHER2:342 dramatically reduced kidney accumulation and improved tumor accumulation (20). Therefore, Hoppmann et al. (21) prepared 177Lu-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-human serum albumin-ZEGFR:1907 (177Lu-DO3A-HSA-ZEGFR:1907) for use in SPECT imaging of EGFR in nude mice bearing human solid tumors showing good tumor contrast but low kidney accumulation.

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Ac-Cys-ZEGFR:1907 was prepared with automated solid-phase peptide synthesizer (21). DOTA-N-hydroxysuccinimide eater and HSA (100:1 molar ratio) were incubated in borate buffer (pH 8.5) for 2 h at 4°C to form DO3A-HSA, which was isolated with PD-10 column. DO3A-HSA was then reacted with sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC) in molar ratio of 1:5 for 1 h at 4 °C. The resulting bioconjugate was further purified using the PD-10 column. Ac-Cys-ZEGFR:1907 was site specifically conjugated to the DOTA and Sulfo-SMCC modified HSA via the cysteine residue. The reaction was performed in a molar ratio of 1:5 of DO3A-HSA-SMCC and Ac-Cys-ZEGFR:1907 (22 nmol) for 6 h at room temperature. DO3A-HSA-ZEGFR:1907 was purified with ultracentrifugation. There were average three DO3A moieties and two Ac-Cys-ZEGFR:1907 moieties per HSA protein as determined with MALDI-TOF-MS and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Approximately 2.4 nmol of the DO3A-HSA-ZEGFR:1907 was radiolabeled with 64Cu by addition of 74 MBq (2 mCi) of 177LuCl3 in 0.1 N sodium acetate buffer (pH 5.0) and incubated for 1 h at 39°C. 177Lu-DO3A-HSA-ZEGFR:1907 was isolated with PD-10 column with >70% radiolabeling yield. 177Lu-DO3A-HSA-ZEGFR:1907 exhibited a specific activity of ~30 MBq/nmol (1.0 mCi/nmol) at the end of synthesis. 177Lu-DO3A-HSA-ZEGFR:1907 was >95% intact in mouse serum for 4 h at 37°C and >80% intact at 24-168 h.

In Vitro Studies: Testing in Cells and Tissues


Accumulation studies of 177Lu-DO3A-HSA-ZEGFR:1907 in SAS human oral squamous carcinoma cells were measured at 37°C (total cell-associated radioactivity) and 4°C (membrane-bound radioactivity) at 0.5-4 h of incubation (21). Total cell-associated radioactivity was 1.97 ± 0.08% incubation dose(ID) at 0.5 h and 8.43 ± 0.55% ID at 4 h, whereas membrane-bound radioactivity was 4-fold to 5-fold lower than the total cell-associated radioactivity. Excess unlabeled Ac-Cys-ZEGFR:1907 inhibited the accumulation at all incubation time points. There were 80% and 65% inhibition at 37°C and 4°C at 4 h, respectively. 177Lu-DO3A-HSA-ZEGFR:1907 exhibited 1.5%, 7.9%, and 38.7% internalization of ID at 1 min, 30 min and 24 h, respectively. On the other hand, 177Lu-DO3A-ZEGFR:1907 exhibited 2.3%, 9.6%, and 28.9% internalization of ID at 2 min, 30 min and 24 h, respectively.

Animal Studies



Hoppmann et al. (21) performed ex vivo biodistribution studies of 177Lu- DO3A-HSA-ZEGFR:1907 in mice (n = 4/group) bearing SAS tumors with tumor accumulation of 2.0 ± 0.8% injected dose per gram (ID/g), 3.1 ± 1.0% ID/g, 4.6 ± 0.2% ID/g, 5.2 ± 0.7% ID/g, 5.3 ± 0.4% ID/g, 3.7 ± 0.6% ID/g, and 3.1 ± 1.0% ID/g at 1, 4, 24, 48, 72, 120, and 240 h after injection, respectively. The organ with the highest radioactivity was the liver (54.9% ID/g), followed by the blood (19.1% ID/g), kidney (11.1% ID/g), spleen (~12% ID/g), lung (~7% ID/g), and muscle (<1.0% ID/g) at 4 h after injection. 177Lu-DO3A-HSA-ZEGFR:1907 exhibited a two-phase blood clearance kinetics with distribution and elimination half-life of 1.99 and 25.5 h, respectively. The tumor/muscle ratio increased from 3.3 at 1 h to 7.9 at 72-240 h, whereas the tumor/blood ratio increased from 1.7 at 48 h to 16.7 at 240 h. No blocking studies were performed.

SPECT static imaging analysis was performed in nude mice (n = 3) bearing SAS tumors at 4, 24, 48, and 72 h after intravenous injection of 7.4-11.1 MBq (0.2-0.0.3 mCi) 177Lu-DO3A-HSA-ZEGFR:1907 (~0.5 nmol) (21). The tumor was clearly visualized at 24-72 h. High accumulation levels were observed in the liver at 4-72 h. No blocking studies were performed.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

NIH Support

5R01 CA119053, P50 CA114747


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