A 2-helix small protein labeled with 68Ga for PET imaging of HER2 expression

Abstract

Affibody molecules are a class of scaffold proteins being developed into a generalizable approach to targeting tumors. Many 3-helix-based Affibody proteins have shown excellent in vivo properties for tumor imaging and therapy. By truncating one alpha-helix that is not responsible for receptor recognition in the Affibody and maturating the protein affinity through synthetic strategies, we have successfully identified in our previous research several small 2-helix proteins with excellent binding affinities to human epidermal growth factor receptor type 2 (HER2). With preferential properties such as faster blood clearance and tumor accumulation, lower immunogenic potential, and facile and economically viable synthetic schemes, we hypothesized that these 2-helix protein binders could become excellent molecular imaging probes for monitoring HER2 expression and modulation.

Methods: In this study, a 2-helix small protein, MUT-DS, was chemically modified with a metal chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). DOTA-MUT-DS was then site-specifically radiolabeled with an important PET radionuclide, (68)Ga. The resulting radiolabeled anti-HER2 2-helix molecule was further evaluated as a potential molecular probe for small-animal PET HER2 imaging in a SKOV3 tumor mouse model.

Results: The 2-helix DOTA-MUT-DS showed high HER2-binding affinity (dissociation constant, 4.76 nM). The radiolabeled probe displayed high stability in mouse serum and specificity toward HER2 in cell cultures. Biodistribution and small-animal PET studies further showed that (68)Ga-DOTA-MUT-DS had rapid and high SKOV3 tumor accumulation and quick clearance from normal organs. The specificity of (68)Ga-DOTA-MUT-DS for SKOV3 tumors was confirmed by monitoring modulation of HER2 protein on treatment of tumor mice with heat shock protein 90 inhibitor 17-N,N-dimethyl ethylene diamine-geldanamycin in vivo.

Conclusion: This proof-of-concept research clearly demonstrated that synthetic 2-helix (68)Ga-DOTA-MUT-DS is a promising PET probe for imaging HER2 expression in vivo. The Affibody-derived small 2-helix protein scaffold has great potential for developing targeting agents for a variety of tumor-associated biomarkers.

Figure 1

Three-helix Affibody and 2-helix protein scaffold–based PET probes for HER2 imaging. Black dots and red regions indicate amino acid residues responsible for receptor binding.

Figure 2

HPLC radiochromatograms of purified ⁶⁸Ga-DOTA-MUT-DS (A) and radiolabeled probe after 40 min of incubation with mouse serum (B).

Figure 3

Biosensor binding studies of DOTA-MUT-DS.

Figure 4

Cell uptake of ⁶⁸Ga-DOTA-MUT-DS in SKOV3 cells over time at 37°C with or without nonradioactive Affibody molecule Z_HER2:477. All results are expressed as mean of triplicate measurement ± SD.

Figure 5

Representative decaycorrected coronal (top) and transaxial (bottom) PET images of nude mice bearing SKOV3 tumor on right shoulder at 0.5, 1, and 2 h after tail vein injection of ⁶⁸Ga-DOTA-MUT-DS: nontreated group (A) and treated group (B) (images were acquired after 24 h of treatment with 17-DMAG; arrows indicate location of tumors). (C) Quantification analysis of tumor uptake before and after 17-DMAG treatment. Data are expressed as mean of 3 mice ± SD.

Figure 6

Representative Western blot detection of HER2 expression in tumor tissue samples. SKOV3 tumor homogenates were prepared and 50 μg of protein were detected with rabbit antihuman HER2 monoclonal antibody. L1 = lane 1, nontreated tumor; L2 = lane 2, tumor treated with 17-DMAG.