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 cross-linker sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC). The HER2 Affibody analogue, Ac-Cys-Z(HER2:342), was covalently conjugated with HSA, and the resulting bioconjugate DOTA-HSA-Z(HER2:342) was further radiolabeled with ⁶⁴Cu and ¹¹¹In and evaluated in vitro and in vivo. Radiolabeled DOTA-HSA-Z(HER2:342) conjugates displayed a significant and specific cell uptake into SKOV3 cell cultures. Positron emission tomography (PET) investigations using ⁶⁴Cu-DOTA-HSA-Z(HER2:342) were performed in SKOV3 tumor-bearing nude mice. High tumor uptake values (>14% ID/g at 24 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-Z(HER2:342) validated these results. At 24 h post injection, the biodistribution data revealed high tumor (16.26% ID/g) and liver (14.11% ID/g) uptake but relatively low kidney uptake (6.06% ID/g). Blocking studies with coinjected, nonlabeled Ac-Cys-Z(HER2:342) confirmed the in vivo specificity of HER2. Radiolabeled DOTA-HSA-Z(HER2:342) Affibody conjugates are promising SPECT and PET-type probes for the imaging of HER2 positive cancer. More importantly, DOTA-HSA-Z(HER2:342) 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.

FIGURE 1

Schematic structure of a radiolabeled Affibody-HSA conjugate. Red regions indicate the lysine residues of HSA suitable for the conjugation with DOTA (hexagon) and HER2-Affibody molecules (violet structures). The yellow symbols indicate the radiometals 111In and 64Cu, respectively. 108×101mm (150 × 150 DPI)

FIGURE 2

Synthetic schemes for the chemical conjugation of DOTA, ZHER2:342 and HSA. (1) HSA, the red regions indicate the lysine residues; (2) HSA conjugated with DOTA-NHS to produce DOTA-HSA; (3) DOTA-HSA conjugated with the bifunctional crosslinker Sulfo-SMCC to generate DOTA-HSA-SMCC; (4) Chemically conjugated ZHER2:342 (violet structure) with DOTA-HSA-SMCC to obtain the final product DOTA-HSA-ZHER2:342. 258×154mm (150 × 150 DPI)

FIGURE 3

Analysis of Affibody-HSA bioconjugates. (A) MALDI-TOF-MS of DOTA-HSA-ZHER2:342. The number of Affibody molecules per HSA is calculated to be 1 to 5. (a), DOTA-HSA-SMCC 68.56 kDa; (b), DOTA-HSA-ZHER2:342(1) (MW=75.03); (c), DOTA-HSA-ZHER2:342(2) (MW=82.09); (d), DOTA-HSA-ZHER2:342(3) (MW= 89.19); (e), DOTA-HSA-ZHER2:342(4) (95.8 kDa) and (f), DOTA-HSA-ZHER2:342(5) (MW= 102.19) (B) SDS-PAGE analysis of several fractions obtained from the chemical conjugation procedure of HSA with ZHER2:342. 1, HSA; 2, Ac-Cys-ZHER2:342; 3, reaction mixture of DOTA-HSA and ZHER2:342 (DOTA-HSA-ZHER2:342); 4, flow-through fraction after centrifugation using microcentrifuge tubes; 5, purified DOTA-HSA-ZHER2:342. 241×120mm (150 × 150 DPI)

FIGURE 4

Uptake of 64Cu-DOTA-HSA-ZHER2:342 (A) and 111In-DOTA-HSA-ZHER2:342 (B) in SKOV3 cells over time at 37 °C in presence or absence of non-radioactive Ac-Cys-ZHER2:342. All results are expressed as percentage of applied radioactivity and are mean of six measurements ± SD. 133×180mm (150 × 150 DPI)

FIGURE 5

Micro-PET imaging in nude mice bearing SKOV3 tumors. Representative decay corrected coronal (top) and transaxial (bottom) PET images at 1 h, 4 h, 24 h and 48 h after tail vein injection of 64Cu-DOTA-HSA-ZHER2:342 (A) and of 64Cu-DOTA-HSA as control (B). Arrows indicate the location of tumors. (C) Tumor time-activity curves derived from multiple-time-point small-animal PET images after tail vein injection of 64Cu-DOTA-HSA-ZHER2:342 and of 64Cu-DOTA-HSA. (D) Time–activity curves of muscle, blood, liver and kidney derived from multiple-time-point small-animal PET images after tail vein injection of 64Cu-DOTA-HSA-ZHER2:342. Data presented in (C) and (D) are shown as mean ± SD% ID/g (n = 3). 290×184mm (150 × 150 DPI)

FIGURE 6

Small Animal SPECT/CT of a mouse bearing SKOV3 tumor xenograft at 24 h and 96 h after administration of 111In-DOTA-HSA-ZHER2:342. T, tumor; L, liver. Red color indicates highest radioactivity concentration. 99×106mm (150 × 150 DPI)

FIGURE 7

Estimated radiation absorbed doses in major organs of a human adult male after intravenous injection of 111In-DOTA-HSA-ZHER2:342 based on the biodistribution data obtained in SKOV3-tumor bearing mice. Abbreviations: LLI, lower large intestine; ULI: upper large intestine. 163×91mm (150 × 150 DPI)