Influence of composition of cysteine-containing peptide-based chelators on biodistribution of 99mTc-labeled anti-EGFR affibody molecules

Maryam Oroujeni,Bogdan Mitran,Xenia Steinhardt,Javad Garousi,John Löfblom,Ken G Andersson,Anzhelika Vorobyeva,Anna Orlova,Mohamed Altai,Vladimir Tolmachev

doi:10.1007/s00726-018-2571-1

Maryam Oroujeni, Bogdan Mitran + Show 8 more

Open Access

https://doi.org/10.1007/s00726-018-2571-1

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Journal: Amino acids	Publication Date: May 4, 2018
Citations: 18	License type: open-access

Affiliation: Uppsala University, KTH Royal Institute of Technology

Abstract

Epidermal growth factor receptor (EGFR) is overexpressed in a number of cancers and is the molecular target for several anti-cancer therapeutics. Radionuclide molecular imaging of EGFR expression should enable personalization of anti-cancer treatment. Affibody molecule is a promising type of high-affinity imaging probes based on a non-immunoglobulin scaffold. A series of derivatives of the anti-EGFR affibody molecule ZEGFR:2377, having peptide-based cysteine-containing chelators for conjugation of 99mTc, was designed and evaluated. It was found that glutamate-containing chelators Gly-Gly-Glu-Cys (GGEC), Gly-Glu-Glu-Cys (GEEC) and Glu-Glu-Glu-Cys (EEEC) provide the best labeling stability. The glutamate containing conjugates bound to EGFR-expressing cells specifically and with high affinity. Specific targeting of EGFR-expressing xenografts in mice was demonstrated. The number of glutamate residues in the chelator had strong influence on biodistribution of radiolabeled affibody molecules. Increase of glutamate content was associated with lower uptake in normal tissues. The 99mTc-labeled variant containing the EEEC chelator provided the highest tumor-to-organ ratios. In conclusion, optimizing the composition of peptide-based chelators enhances contrast of imaging of EGFR-expression using affibody molecules.

Highlights

Aberrant expression of growth-controlling receptors is a part of cellular phenotypes for several kinds of cancer
Protein purity was determined to ≥ 97% for all of the produced proteins (Supplemental Fig. 2) and the affinity to recombinant human Epidermal growth factor receptor (EGFR) was measured to low nanomolar affinity (Table 1)
Results of microSPECT/CT imaging (Figs. 7, 8) demonstrated that visualization of EGFR expression in tumors using affibody molecules labeled with 99mTc using peptide-based glutamate-containing chelators is feasible

Summary

Introduction

Aberrant expression of growth-controlling receptors is a part of cellular phenotypes for several kinds of cancer. Studies using HER2-targeting affibody molecules demonstrated that it is possible to modulate the residualizing properties of the 99mTc label, its retention in excretory organs and the predominant excretion pathway by varying the composition of the peptide-based chelator (Wållberg et al 2011; Altai et al 2012) This permitted to obtain excellent contrast of imaging. A panel of engineered anti-EGFR affibody molecules was created, containing the same amino acid sequence at the N-terminus (AEN–), as well as APKC, GGGC, GGEC, GEEC and EEEC peptide-based chelators (Fig. 1b) at the C-terminus. Labeling of these affibody molecules with 99mTc was optimized, and their stability, in vitro and in vivo targeting properties were investigated

Materials and methods

Results

Discussion

Compliance with ethical standards