^99mTc-maEEE-Z_HER2:342, an Affibody Molecule-Based Tracer for the Detection of HER2 Expression in Malignant Tumors

Abstract

Detection of HER2-overexpression in tumors and metastases is important for the selection of patients who will benefit from trastuzumab treatment. Earlier investigations showed successful imaging of HER2-positive tumors in patients using indium- or gallium-labeled Affibody molecules. The goal of this study was to evaluate the use of (99m)Tc-labeled Affibody molecules for the detection of HER2 expression. The Affibody molecule Z(HER2:342) with the chelator sequences mercaptoacetyl-Gly-Glu-Gly (maGEG) and mercaptoacetyl-Glu-Glu-Glu (maEEE) was synthesized by peptide synthesis and labeled with technetium-99m. Binding specificity, cellular retention, and in vitro stability were investigated. The biodistribution of (99m)Tc-maGEG-Z(HER2:342) and (99m)Tc-maEEE-Z(HER2:342) was compared with (99m)Tc-maGGG-Z(HER2:342) in normal mice, and the tumor targeting properties of (99m)Tc-maEEE-Z(HER2:342) were determined in SKOV-3 xenografted nude mice. The results showed that the Affibody molecules were efficiently labeled with technetium-99m. The labeled conjugates were highly stable in vitro with preserved HER2-binding capacity. The use of glutamic acid in the chelator sequences for (99m)Tc-labeling of Z(HER2:342) reduced the hepatobiliary excretion 3-fold with a single Gly-to-Glu substitution and 10-fold with three Gly-to-Glu substitutions. (99m)Tc-maEEE-Z(HER2:342) showed a receptor-specific tumor uptake of 7.9 +/- 1.0 %IA/g and a tumor-to-blood ratio of 38 at 4 h pi. Gamma-camera imaging with (99m)Tc-maEEE-Z(HER2:342) could detect HER2-expressing tumors in xenografts already at 1 h pi. It was concluded that peptide synthesis for the coupling of chelator sequences to Affibody molecules for (99m)Tc labeling is an efficient way to modify the in vivo kinetics. Increased hydrophilicity, combined with improved stability of the mercaptoacetyl-triglutamyl chelator, resulted in favorable biodistribution, making (99m)Tc-maEEE-Z(HER2:342) a promising tracer for clinical imaging of HER2 overexpression in tumors.

Incorporation of a Triglutamyl Spacer Improves the Biodistribution of Synthetic Affibody Molecules Radiofluorinated at the N-Terminus via Oxime Formation with 18F-4-Fluorobenzaldehyde

“…Apparently, incorporation of the triglutamyl spacer reduced the on-rate of the binding, which caused increase of the dissociation constant from 49 pM for FBO-PEP4313 to 180 pM for FBO-E 3 -PEP4313. This was not entirely unexpected, as N-terminal modification has earlier caused some modulation of affinity of Affibody molecules 34,38 . However, our recent study 39 Reduction of the renal uptake was an interesting effect of the spacer.…”

“…Tc at the N-terminus by increasing the hydrophilicity of the peptide-based chelators via incorporation of glutamyl residues 34,35 .…”

Evaluation of backbone-cyclized HER2-binding 2-helix Affibody molecule for In Vivo molecular imaging

“…In addition, different chemistry was used for conjugation of the DOTA chelator. Earlier experience with Affibody molecules has suggested that even smaller changes in composition (substitution of one to three amino acids) could cause appreciable differences in biodistribution, such as the level of hepatic uptake [25,26] or percentage of hepatobiliary excretion [27][28][29]. Moreover, coupling of different nuclides using the same chelator could cause differences in organ uptake and blood clearance rate of Affibody molecules [21,29,30].…”

“…in DCM was added (40 min) to the peptide-resin for specific acylation, followed by deprotonation and cyclization of the linker with 0.5 M DIEA in DCM [28].…”

HER2/neu: an increasingly important therapeutic target. Part 1: basic biology & therapeutic armamentarium

“…These conjugates include affitoxin [101], pseudomonas exotoxin [102,103], ricin A chain [104], various probes (radioactive and nonradioactive) [104][105][106][107][108][109][110][111][112], photosensitizers [113][114][115], RNAse [116,117], cytokines [118] or chemotherapeutic agents [119][120][121][122]. A Phase II study of a novel future science group HER2/neu: an increasingly important therapeutic target.…”