Evaluation of Maleimide Derivative of DOTA for Site-Specific Labeling of Recombinant Affibody Molecules

Abstract

Affibody molecules are a new class of small (7 kDa) scaffold affinity proteins, which demonstrate promising properties as agents for in vivo radionuclide targeting. The Affibody scaffold is cysteine-free and therefore independent of disulfide bonds. Thus, a single thiol group can be engineered into the protein by introduction of one cysteine. Coupling of thiol-reactive bifunctional chelators can enable site-specific labeling of recombinantly produced Affibody molecules. In this study, the use of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (MMA-DOTA) for 111 In-labeling of anti-HER2 Affibody molecules His 6-Z HER2:342-Cys and Z HER2:2395-Cys has been evaluated. The introduction of a cysteine residue did not affect the affinity of the proteins, which was 29 pM for His 6-Z HER2:342-Cys and 27 pM for Z HER2:2395-Cys, comparable with 22 pM for the parental Z HER2:342. MMA-DOTA was conjugated to DTT-reduced Affibody molecules with a coupling efficiency of 93% using a 1:1 molar ratio of chelator to protein. The conjugates were labeled with 111 In to a specific radioactivity of up to 7 GBq/mmol, with preserved binding for the target HER2. In vivo, the non-His-tagged variant 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys demonstrated appreciably lower liver uptake than its His-tag-containing counterpart. In mice bearing HER2-expressing LS174T xenografts, 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys showed specific and rapid tumor localization, and rapid clearance from blood and nonspecific compartments, leading to a tumor-to-blood-ratio of 18 +/- 8 already 1 h p.i. Four hours p.i., the tumor-to-blood ratio was 138 +/- 8. Xenografts were clearly visualized already 1 h p.i.

Imaging of HER3-expressing xenografts in mice using a 99mTc(CO)3-HEHEHE-ZHER3:08699 affibody molecule

“…Taking into account that the in vivo specificity study demonstrated that radioactivity uptake in these organs was saturable, we can conclude that this uptake was receptor specific. Previously, an affibody molecule with an unrelated specificity for Taq polymerase (ZTaq), which has the same 3-helix structure and that only differs in the few amino acids in the binding region, was tested for biodistribution and tumor uptake in LS174T xenograft model [20,22]. The level of radioactivity concentration in tumors due to unspecific uptake was lower or on the level of muscles and blood.…”

“…Together with very high affinity, this permits high-contrast imaging of target expression within a few hours after injection. Multiple studies with different xenograft models have demonstrated that there is no unspecific uptake of affibody molecules in tumors, which is important to exclude false-positive findings [20,21,22]. Two clinical studies have confirmed the potential of affibody-based agents for imaging of HER2-expressing metastases in patients with disseminated breast cancer [23,24].…”

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

“…Thus, it is unlikely that 111 In and 68 Ga were released from the chelator. In addition, we have earlier directly compared an 111 In-labeled recombinant 3-helix Affibody molecule conjugated to maleimido DOTA through C-terminal cysteine with a synthetic counterpart conjugated to DOTA via an amide bond [40]. We have not found any significant difference in liver uptake between these two conjugates.…”

“…In principle, uptake in these organs might be specific. However, a numerous studies with parental Z HER2:343 and its derivatives having the same binding site have demonstrated that uptake in these organs is not saturable, and therefore nonspecific [40][41][42][43][44]. Much lower uptake was also for radiolabeled PEP09239 having the same binding site and higher affinity to HER2.…”

“…It can be due to the higher amount of impurity in the eluted fraction. Decreasing the activity to 40 MBq improved the yield considerably; analytical radiochemical yield was 92.2 ± 1.9%…”