Increasing the Net Negative Charge by Replacement of DOTA Chelator with DOTAGA Improves the Biodistribution of Radiolabeled Second-Generation Synthetic Affibody Molecules

Abstract

A promising strategy to enable patient stratification for targeted therapies is to monitor the target expression in a tumor by radionuclide molecular imaging. Affibody molecules (7 kDa) are nonimmunoglobulin scaffold proteins with a 25-fold smaller size than intact antibodies. They have shown an apparent potential as molecular imaging probes both in preclinical and clinical studies. Earlier, we found that hepatic uptake can be reduced by the incorporation of negatively charged purification tags at the N-terminus of Affibody molecules. We hypothesized that liver uptake might similarly be reduced by positioning the chelator at the N-terminus, where the chelator-radionuclide complex will provide negative charges. To test this hypothesis, a second generation synthetic anti-HER2 ZHER2:2891 Affibody molecule was synthesized and labeled with (111)In and (68)Ga using DOTAGA and DOTA chelators. The chelators were manually coupled to the N-terminus of ZHER2:2891 forming an amide bond. Labeling DOTAGA-ZHER2:2891 and DOTA-ZHER2:2891 with (68)Ga and (111)In resulted in stable radioconjugates. The tumor-targeting and biodistribution properties of the (111)In- and (68)Ga-labeled conjugates were compared in SKOV-3 tumor-bearing nude mice at 2 h postinjection. The HER2-specific binding of the radioconjugates was verified both in vitro and in vivo. Using the DOTAGA chelator gave significantly lower radioactivity in liver and blood for both radionuclides. The (111)In-labeled conjugates showed more rapid blood clearance than the (68)Ga-labeled conjugates. The most pronounced influence of the chelators was found when they were labeled with (68)Ga. The DOTAGA chelator gave significantly higher tumor-to-blood (61 ± 6 vs 23 ± 5, p < 0.05) and tumor-to-liver (10.4 ± 0.6 vs 4.5 ± 0.5, p < 0.05) ratios than the DOTA chelator. This study demonstrated that chelators may be used to alter the uptake of Affibody molecules, and most likely other scaffold-based imaging probes, for improvement of imaging contrast.

Al18F-Labeling Of Heat-Sensitive Biomolecules for Positron Emission Tomography Imaging

“…Affibody molecules are non-immunoglobulin-based scaffold proteins characterized by high target specificity and binding affinity (Kd in the low-nanomolar to picomolar range) 52 , 53 . These small proteins have been generated against different cancer-associated molecular targets and have been labeled with different radionuclides ( 68 Ga, 18 F, 64 Cu, 99m Tc, and 111 In) suitable for immuno-PET and SPECT 54 - 59 . As for nanobodies, the half-life of fluorine-18 perfectly matches the fast pharmacokinetics of affibody molecules 60 .…”

Benefit of Later-Time-Point PET Imaging of HER3 Expression Using Optimized Radiocobalt-Labeled Affibody Molecules

“…We and others have previously investigated the potential of radiolabeled affibody molecules as HER3-targeted imaging agents for PET and SPECT [15,16,[20][21][22]27]. It has been shown that an increase in the negative charge of the radionuclide-chelator complex can improve tumor-to-liver contrast for affibody molecules [26,36]. In addition, retention of activity in tumors is better than in normal tissues for several types of imaging probes.…”

“…It was hypothesized that an increased negative charge can reduce unspecific hepatic uptake and, therefore, enhance the tumorto-liver contrast [27]. This hypothesis was further supported by comparing the effect of differently charged radiometal-chelator complexes on the biodistribution of ZHER3 labeled with 68 Ga and 111 In and HER2-targeting affibody molecules [26,31,36].…”

PET of HER2 Expression with a Novel 18FAl Labeled Affibody

“…SKOV-3 tumor is always employed for evaluating the biologic properties of radiolabeled HER2 affibody due to abundant HER2 expression 33,34,35. Thus the HER2 targeting characteristic of 18 FAl-NOTA-MAL-MZHER 2:342 was initially determined using SKOV-3 models.…”