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

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.Electronic supplementary materialThe online version of this article (10.1007/s00726-018-2571-1) contains supplementary material, which is available to authorized users.

Preclinical Evaluation of [68Ga]Ga-DFO-ZEGFR:2377: A Promising Affibody-Based Probe for Noninvasive PET Imaging of EGFR Expression in Tumors

“…This is a good match for Ga 3+ , which is typically hexacoordinated. In addition, the use of Ga 3+ instead of Zr 4+ decreases the positive charge on C-terminus of ZEGFR:2377, which is favorable for biodistribution [ 32 , 44 ].…”

Imaging using radiolabelled targeted proteins: radioimmunodetection and beyond

“…4c and d) (Garousi et al 2017b). Labelling of the same affibody molecule with 68 Ga using DFO provided (Oroujeni et al 2018a(Oroujeni et al , 2018b also higher uptake in tumour than in liver (Table 1 and Fig. 4e).…”

“…This enables site-specific incorporation of a variety of chelators and prosthetic groups for radiolabelling in any desirable position (Orlova et al 2010;Perols et al 2012;Rosik et al 2014); -Introduction of a unique cysteine into the cysteine-free affibody scaffold permits site-specific labelling of recombinantly-produced affibody molecules using maleimido-mediated thiol-directed chemistry. This might be used for coupling of different chelators for radiometals (Tolmachev et al 2008;Tolmachev et al 2012;Altai et al 2013) or prosthetic groups for radiohalogens (Namavari et al 2008;Tolmachev et al 2009b;Kramer-Marek et al 2008;Su et al 2014); -Placement of cysteine at C-terminus provides N 3 S chelators (formed by thiol group of cysteine and amide nitrogens from adjacent amino acids) for site-specific labelling with 99m Tc, 188 Re or 186 Re (Altai et al 2012;Altai et al 2014;Oroujeni et al 2018aOroujeni et al , 2018b).…”

“…It has been demonstrated that it is possible to find an amount of injected protein, which could saturate EGFR in liver without saturating receptors in tumours (Tolmachev et al 2010a). Further studies demonstrated that the interaction of radiolabelled affibody molecules with EGFR on hepatocytes is not the only mechanism of (Oroujeni et al 2018a(Oroujeni et al , 2018b hepatic uptake. The physicochemical properties of a chelator-radionuclide combination has also a strong influence on hepatic uptake.…”

Affibody Molecules as Targeting Vectors for PET Imaging

“…Due to the slow internalization, at least some radiolabeled affibody molecules remain bound to receptors on hepatocyte membranes for a while and dissociate when the blood concentration decreases due to clearance. Therefore, the liver acts as a depot, and the blood clearance of anti-EGFR affibody molecules is slower than clearance of affibody molecules to other targets [23,[48][49][50]. An optimal imaging time might be 24 h after injection in this case.…”