In Vivo Evaluation of Cysteine-Based Chelators for Attachment of ^99mTc to Tumor-Targeting Affibody Molecules

Abstract

Affibody molecules present a new class of affinity proteins, which utilizes a scaffold based on a 58-amino acid domain derived from protein A. The small (7 kDa) Affibody molecule can be selected to bind to cell-surface targets with high affinity. An Affibody molecule (Z HER2:342 ) with a dissociation constant (K d ) of 22 pM for binding to the HER2 receptor has been reported earlier. Preclinical and pilot clinical studies have demonstrated the utility of radiolabeled Z HER2:342 in imaging of HER2-expressing tumors. The small size and cysteine-free structure of Affibody molecules enable complete peptide synthesis and direct incorporation of radionuclide chelators. The goal of this study was to evaluate if incorporation of the natural peptide sequences cysteine-diglycine (CGG) and cysteine-triglycine (CGGG) sequences would enable labeling of Affibody molecules with 99m Tc. In a model monomeric form, the chelating sequences were incorporated by peptide synthesis. The HER2-binding affinity was 280 and 250 pM for CGG-Z HER2:342 and CGGG-Z HER2:342, respectively. Conjugates were directly labeled with 99m Tc with 90% efficiency and preserved the capacity to bind specifically to HER2-expressing cells. The biodistribution in normal mice showed a rapid clearance from the blood and the majority of organs (except kidneys). In the mice bearing SKOV-3 xenografts, tumor uptake of 99m Tc-CGG-Z HER2:342 was HER2-specific and a tumorto-blood ratio of 9.2 was obtained at 6 h postinjection. Gamma-camera imaging with 99m Tc-CGG-Z HER2:342 clearly visualized tumors at 6 h postinjection. The results show that the use of a cysteine-based chelator enables 99m Tclabeling of Affibody molecules for imaging.

Molecular Design and Optimization of 99mTc-Labeled Recombinant Affibody Molecules Improves Their Biodistribution and Imaging Properties

“…The use of amino acids with hydrophilic (polar or charged) side chains in the N-terminal chelators provided reduced liver uptake and low levels of hepatobiliary excretion (14,(16)(17)(18)22), whereas the composition of the C-terminus was shown to be less influential in that aspect (23). However, in agreement with previously published data on chelator stability and biodistribution of 99m Tc-labeled proteins (24)(25)(26), the use of an N 3 S cysteine-based chelator at the C-terminus provided appreciably less release of 99m Tc-pertechnetate in the circulation than did the SN 3 chelator at the N-terminus (15,19,21). Furthermore, Ala 1 ,Glu 2 at the N-terminus has been shown to be associated with low hepatic uptake and low hepatobiliary excretion (19,(21)(22)(23).…”

“…99m Tc-Z HER2:V2 provided one of the lowest levels of renal radioactivity among all previously evaluated 99m Tclabeled Affibody molecules (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). It is likely that the low retention of 99m Tc-Z HER2:V2 -associated radioactivity in kidneys was due to the absence of stabilizing effects from adjacent electron-donating side chains.…”

“…Because hexahistidine tags cause an elevated hepatic uptake of Affibody molecules (12,19,22,23) and their use, thus, should be avoided, a novel antiidiotypic purification strategy has been developed and applied to the constructs used in this study (28). Further, N-terminal-placed peptide-based chelators with nonpolar or noncharged side chains were associated with elevated hepatobiliary excretion (13)(14)(15)(16). This problem seems to have been solved by the placement of peptidebased chelators at the C-terminus and by improving the design of the N-terminus (AEN-) of the Z HER2:VX constructs, adopted from earlier studies (19,22,31).…”

“…Recently, several approaches to label the HER2-binding Affibody molecule Z HER2:342 and its derivatives with 99m Tc have been evaluated (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Variants and positioning of histidine tags, as well as mercaptoacetyl-or cysteine-containing peptide-based chelators, were evaluated for the coordination of 99m Tc.…”

Direct Site-Specific Radiolabeling of an Affibody Protein with 4-[18F]Fluorobenzaldehyde via Oxime Chemistry

“…Affibody molecules [3][4], a novel class of proteins with molecular weight range of 6.5 to 14 kDa, have been engineered via phage display technology and have the potential to bind a variety of targets. Affibodies, specifically targeted against human epidermal growth factor receptor 2 (HER2, also known as HER2neu) labeled primarily with single-photon isotopes, including 99m Tc [5], 125 I [6], and 111 In [7], have been reported, all of which show the affibodies to have in vivo pharmacokinetics compatible with short-lived isotopes. The molecular size in combination with the high in vivo stability of the affibodies show both blood clearance and target uptake in the minutes time frame with imaging possible in an hour or less.…”