89Zr-Radiolabelling of p-NCS-Bz-DFO-Anti-HER2 Affibody Immunoconjugate: Characterization and Assessment of In Vitro Potential in HER2-Positive Breast Cancer Imaging

Abstract

Background: The ⁸⁹Zr radioisotope is increasingly vital in positron emission tomography (PET), especially immuno-PET, due to its long half-life of 78.4 h, allowing extended tracking of biological processes. This makes it particularly suitable for researching medicines with slow pharmacokinetics and enhances the precision of molecular imaging, especially in oncology. Despite zirconium's potential for skeletal accumulation, effective chelation with agents like deferoxamine (DFO) enables high-resolution imaging of antigen-specific tumours, such as HER2-positive breast cancer, offering insights into tumour biology and treatment response. Methods: ⁸⁹Zr was produced at the ACSI TR-19 cyclotron via ⁸⁹Y(p,n)⁸⁹Zr reaction. Natural yttrium foils (250 μm) were irradiated with 12.9 MeV protons on target, with 100 μA·h. An HER2-targeting affibody was synthesized and conjugated with p-NCS-Bz-DFO (1:4 mass ratio) at 37 °C for 60 min (pH 9.2 ± 0.2), then purified on a PD-10 column. Radiolabelling was performed with [⁸⁹Zr]Zr-oxalate at pH ranging from 7.0 to 9.0, with concentrations from 110 to 460 MBq/mL. Results: Final activity reached 2.95 ± 0.31 GBq/batch (EOB corrected), with ≥ 99.9% radionuclide and ≥95% radiochemical purities. The anti-HER2 affibody was successfully radiolabelled with ⁸⁹Zr, resulting in a radiochemical purity of over 85% with molar activity of 26.5 ± 4.4 and 11.45 MBq/nmol at pH 7.0-7.5. In vitro tests on BT-474 and MCF-7 cell lines confirmed high uptake in HER2-positive cells, validating specificity and stability. Conclusions: The successful synthesis and labelling of the [⁸⁹Zr]Zr-p-NCS-Bz-DFO-anti-HER2 affibody are promising achievements for its further application in targeted immuno-PET imaging for HER2-positive malignancies. Further in vivo studies are needed to support its clinical translation.

Keywords: HER2-positive; affibody; breast cancer; pharmacokinetics; radiopharmaceuticals; zirconium-89.

Figure 1

Schematic representation of an affibody molecule, highlighting its small size (approx. 7.5 kDa) and alpha-helical structure, along with the key advantages of affibodies over monoclonal antibodies.

Figure 2

Purification process using the ZR cartridge [27,28].

Figure 3

Anti-HER2 affibody sequence [18].

Figure 4

Purification of mAb–chelator conjugate using PD-10 column.

Figure 5

General radiolabelling procedure of [⁸⁹Zr]Zr-NCS-Bz-DFO-affibody [30].

Figure 6

Gamma-ray spectrum of [⁸⁹Zr]Zr-oxalate solution.

Figure 7

Radio-TLC chromatograms for the [⁸⁹Zr]Zr-p-NCS-Bz-DFO-anti-HER2 affibody complex (experiment 1): (a) before purification and (b) after purification.

Figure 8

Radio-TLC chromatograms of the [⁸⁹Zr]Zr-p-NCS-Bz-DFO-anti-HER2 affibody solution from experiment 2 after (a) 1 h from the initiation of the reaction and (b) 12 h at 37 °C.

Figure 9

Influence of labelling pH on radiochemical purity over time.

Figure 10

Radio-TLC chromatograms for the [⁸⁹Zr]Zr-p-NCS-Bz-DFO-anti-HER2 affibody complex from experiment 3 for the pH labelling of 7.0–7.5 (a) after 1 h from the initiation of the reaction and (b) after 12 h at 37 °C.

Figure 11

Stability of [⁸⁹Zr]Zr-oxalate (a), [⁸⁹Zr]Zr-p-NCS-Bz-DFO (b), and (c) [⁸⁹Zr]Zr-p-NCS-Bz-DFO–anti-HER2 affibody in saline solution and human and rat serum.

Figure 12

Overlay of the uptake/retention curves of the [⁸⁹Zr]Zr-p-NCS-Bz-DFO-anti-HER2 affibody from the 3 experiments in BT-474. Arrows indicate the first point of the uptake and the retention phase, respectively.

Figure 13

Overlay of the uptake/retention curves of the [⁸⁹Zr]Zr-p-NCS-Bz-DFO-anti-HER2 affibody in BT-474 and MCF-7.