In vivo targeting of HER2-positive tumor using 2-helix affibody molecules

Molecular imaging of human epidermal growth factor receptor type 2 (HER2) expression has drawn significant attention because of the unique role of the HER2 gene in diagnosis, therapy and prognosis of human breast cancer. In our previous research, a novel cyclic 2-helix small protein, MUT-DS, was discovered as an anti-HER2 Affibody analog with high affinity through rational protein design and engineering. MUT-DS was then evaluated for positron emission tomography (PET) of HER2-positive tumor by labeling with two radionuclides, Ga-68 and F-18, with relatively short half-life (t (1/2) < 2 h). In order to fully study the in vivo behavior of 2-helix small protein and demonstrate that it could be a robust platform for labeling with a variety of radionuclides for different applications, in this study, MUT-DS was further radiolabeled with Cu-64 or In-111 and evaluated for in vivo targeting of HER2-positive tumor in mice. Design 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated MUT-DS (DOTA-MUT-DS) was chemically synthesized using solid phase peptide synthesizer and I-2 oxidation. DOTA-MUT-DS was then radiolabeled with Cu-64 or In-111 to prepare the HER2 imaging probe (Cu-64/In-111-DOTA-MUT-DS). Both biodistribution and microPET imaging of the probe were evaluated in nude mice bearing subcutaneous HER2-positive SKOV3 tumors. DOTA-MUT-DS could be successfully synthesized and radiolabeled with Cu-64 or In-111. Biodistribution study showed that tumor uptake value of Cu-64 or In-111-labeled DOTA-MUT-DS was 4.66 +/- A 0.38 or 2.17 +/- A 0.15%ID/g, respectively, in nude mice bearing SKOV3 xenografts (n = 3) at 1 h post-injection (p.i.). Tumor-to-blood and tumor-to-muscle ratios for Cu-64-DOTA-MUT-DS were attained to be 3.05 and 3.48 at 1 h p.i., respectively, while for In-111-DOTA-MUT-DS, they were 2.04 and 3.19, respectively. Co-injection of the cold Affibody molecule Z(HER2:342) with Cu-64-DOTA-MUT-DS specifically reduced the SKOV3 tumor uptake of the probe by 48%. In-111-DOTA-MUT-DS displayed lower liver uptake at all the time points investigated and higher tumor to blood ratios at 4 and 20 h p.i., when compared with Cu-64-DOTA-MUT-DS. This study demonstrates that the 2-helix protein based probes, Cu-64/In-111 DOTA-MUT-DS, are promising molecular probes for imaging HER2-positive tumor. Two-helix small protein scaffold holds great promise as a novel and robust platform for imaging and therapy applications.