Radiolabeled Affibody−Albumin Bioconjugates for HER2-Positive Cancer Targeting

Abstract

Affibody molecules have received significant attention in the fields of molecular imaging and drug development. However, Affibody scaffolds display an extremely high renal uptake, especially when modified with chelators and then labeled with radiometals. This unfavorable property may impact their use as radiotherapeutic agents in general and as imaging probes for the detection of tumors adjacent to kidneys in particular. Herein, we present a simple and generalizable strategy for reducing the renal uptake of Affibody molecules while maintaining their tumor uptake. Human serum albumin (HSA) was consecutively modified by 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS ester) and the bifunctional crosslinker sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC). The HER2 Affibody analog, Ac-Cys-ZHER2:342, was covalently conjugated with HSA, and the resulting bioconjugate DOTA-HSA-ZHER2:342 was further radiolabeled with 64Cu and 111In and evaluated in vitro and in vivo. Radiolabeled DOTA-HSA-ZHER2:342 conjugates displayed a significant and specific cell uptake into SKOV3 cell cultures. Positron emission tomography (PET) investigations using 64Cu-DOTA-HSA-ZHER2:342 were performed in SKOV3 tumor-bearing nude mice. High tumor uptake values (> 14% ID/g at 24 h and 48 h) and high liver accumulations but low kidney accumulations were observed. Biodistribution studies and single-photon emission computed tomography (SPECT) investigations using 111In-DOTA-HSA-ZHER2:342 validated these results. At 24 h post injection, the bio distribution data revealed high tumor (16.26% ID/g) and liver uptake (14.11% ID/g) but relatively low kidney uptake (6.06% ID/g). Blocking studies with co-injected, non-labeled Ac-Cys-ZHER2:342 confirmed the in vivo specificity of HER2. Radiolabeled DOTA-HSA-ZHER2:342 Affibody conjugates are promising SPECT and PET-type probes for the imaging of HER2 positive cancer. More importantly, DOTA-HSA-ZHER2:342 is suitable for labeling with therapeutic radionuclides (e.g. 90Y or 177Lu) for treatment studies. The approach of using HSA to optimize the pharmacokinetics and biodistribution profile of Affibodies, may be extended to the design of many other targeting molecules.

Site-Specific Radiometal Labeling and Improved Biodistribution Using ABY-027, A Novel HER2-Targeting Affibody Molecule–Albumin-Binding Domain Fusion Protein

“…Recently, Hoppman et al (33) evaluated the alternative approach of direct chemical coupling of Z HER2:342 to HSA. We have made a comparison of the biodistribution of these different constructs, using the data from 48 h after injection because this was the only common time point (Table 1).…”

Positron Emission Tomography Imaging with 18F-Labeled ZHER2:2891 Affibody for Detection of HER2 Expression and Pharmacodynamic Response to HER2-Modulating Therapies

“…In this context previous Affibodies labeled with 68 Ga or 111 In showed approximately 10-fold higher renal localization than that seen in tumor (34,35,38), precluding imaging of tumors in the region around the kidney, as well as having an impact on dosimetry. Bioconjugation of the Affibody molecule with albumin, histidine containing tags or 18 F radiolabels have been proposed as alternative approaches to avoid tubular reabsorption and permitting rapid glomerular filtration (26,(39)(40)(41). Using a radiohalogen strategy with 18 F, we demonstrated rapid renal clearance of […”

177Lu–DO3A–HSA–ZEGFR:1907: characterization as a potential radiopharmaceutical for radionuclide therapy of EGFR-expressing head and neck carcinomas

“…Bioconjugation of HSA with DOTA and Ac-Cys-Z EGFR:1907 The conjugation of DOTA-NHS ester and Ac-Cys-Z EGFR:1907 was performed in the same fashion as described previously for preparation of HSA-Z HER2 bioconjugates [24]. Briefly, the HSA was conjugated to DOTA-NHS ester in 200 lL borate buffer (50 mM, pH 8.5) in a molar ratio of 1:100.…”

“…In vitro cell uptake assays of both 64 Cu-DO3A-HSA-Z EGFR:1907 and 177 Lu-DO3A-HSA-Z EGFR:1907 were performed as previously described [24]. Briefly, SAS cells (2 9 10 5 per well) were seeded in 12-well tissue culture plates and allowed to attach overnight.…”

“…We have recently demonstrated that conjugation of human serum albumin (HSA) with an anti-human epidermal growth factor receptor 2 (HER2) Affibody (Z HER2 ) can produce HSA-Z HER2 bioconjugates which exhibit improved pharmacokinetics in terms of high tumor uptake, but dramatically reduced kidney uptake [24]. These promising results have encouraged us to further explore whether radiometal-labeled HSA-Z EFGR bioconjugates ( Fig.…”