Fluorine-18 Labeling of the HER2-Targeting Single-Domain Antibody 2Rs15d Using a Residualizing Label and Preclinical Evaluation

Abstract

Purpose Our previous studies with F-18-labeled anti-HER2 single-domain antibodies (sdAbs) utilized 5F7, which binds to the same epitope on HER2 as trastuzumab, complicating its use for positron emission tomography (PET) imaging of patients undergoing trastuzumab therapy. On the other hand, sdAb 2Rs15d binds to a different epitope on HER2 and thus might be a preferable vector for imaging in these patients. The aim of this study was to evaluate the tumor targeting of F-18 -labeled 2Rs15d in HER2-expressing breast carcinoma cells and xenografts. Procedures sdAb 2Rs15d was labeled with the residualizing labels N-succinimidyl 3-((4-(4-[18F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([18F]RL-I) and N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB), and the purity and HER2-specific binding affinity and immunoreactivity were assessed after labeling. The biodistribution of I-125- and F-18-labeled 2Rs15d was determined in SCID mice bearing subcutaneous BT474M1 xenografts. MicroPET/x-ray computed tomograph (CT) imaging of [18F]RL-I-2Rs15d was performed in this model and compared to that of nonspecific sdAb [18F]RL-I-R3B23. MicroPET/CT imaging was also done in an intracranial HER2-positive breast cancer brain metastasis model after administration of 2Rs15d-, 5F7-, and R3B23-[18F]RL-I conjugates. Results [18F]RL-I was conjugated to 2Rs15d in 40.8 ± 9.1 % yield and with a radiochemical purity of 97–100 %. Its immunoreactive fraction (IRF) and affinity for HER2-specific binding were 79.2 ± 5.4 % and 7.1 ± 0.4 nM, respectively. [125I]SGMIB was conjugated to 2Rs15d in 58.4 ± 8.2 % yield and with a radiochemical purity of 95–99 %; its IRF and affinity for HER2-specific binding were 79.0 ± 12.9 % and 4.5 ± 0.8 nM, respectively. Internalized radioactivity in BT474M1 cells in vitro for [18F]RL-I-2Rs15d was 43.7 ± 3.6, 36.5 ± 2.6, and 21.7 ± 1.2 % of initially bound radioactivity at 1, 2, and 4 h, respectively, and was similar to that seen for [125I]SGMIB-2Rs15d. Uptake of [18F]RL-I-2Rs15d in subcutaneous xenografts was 16–20 %ID/g over 1–3 h. Subcutaneous tumor could be clearly delineated by microPET/CT imaging with [18F]RL-I-2Rs15d but not with [18F]RL-I-R3B23. Intracranial breast cancer brain metastases could be visualized after intravenous administration of both [18F]RL-I-2Rs15d and [18F]RL-I-5F7. Conclusions Although radiolabeled 2Rs15d conjugates exhibited lower tumor cell retention both in vitro and in vivo than that observed previously for 5F7, given that it binds to a different epitope on HER2 from those targeted by the clinically utilized HER2-targeted therapeutic antibodies trastuzumab and pertuzumab, F-18-labeled 2Rs15d has potential for assessing HER2 status by PET imaging after trastuzumab and/or pertuzumab therapy.

Clinical Translation of [68Ga]Ga-NOTA-anti-MMR-sdAb for PET/CT Imaging of Protumorigenic Macrophages

“…), which is about 20-fold lower than that seen with [ 99m Tc]Tc(CO) 3 -anti-MMR-sdAb and 10-fold lower than with the here presented [ 68 Ga]Ga-NOTA-anti-MMR-sdAb [11]. Similar lower kidney retention for 18 Flabeled sdAb has also been reported for anti-HER2, although [13][14][15]. Depending on the chemical structure created, different catabolites will be produced in the kidney cells, with some 18 F-catabolites clearing faster from kidneys.…”

“…sdAbs, such as anti-MMR-sdAbs, are antigen-binding fragments derived from heavy-chain only antibodies of the Camelidae. Compared to conventional antibodies or antibody fragments, sdAbs are small (12)(13)(14)(15) and they have the ability to bind antigens on hidden or unusual epitopes [5]. sdAbs have proven their role in molecular imaging and targeted radionuclide therapy in a preclinical setting [6][7][8][9], and a 68 Ga-labeled compound for positron emission tomography (PET)/X-ray computed tomography (CT) imaging of HER2 was found safe and it is use straightforward in a phase I clinical trial, with low radiation burden for the patient [10].…”

Development of Radiotracers for Breast Cancer—The Tumor Microenvironment as an Emerging Target

“…[ 18 F]F-RL-I-2Rs15d was shown to detect HER2 + brain lesions proving the ability of this tracer to cross the blood–brain barrier. However, [ 18 F]F-RL-I-2Rs15d exhibited lower tumor cell retention both in vitro and in vivo than 5F7 therefore further optimization is required [ 23 ].…”

Early Phase I Study of a 99mTc-Labeled Anti–Programmed Death Ligand-1 (PD-L1) Single-Domain Antibody in SPECT/CT Assessment of PD-L1 Expression in Non–Small Cell Lung Cancer

“…Single-domain antibodies (sdAbs) are the smallest naturally derived antigen-binding fragments from camelid antibodies and demonstrate great potential for molecular imaging in both preclinical and clinical models (18)(19)(20)(21). Because of their low molecular weight (;15 kDa), sdAbs rapidly clear from the circulation via the kidneys while retaining high target-binding potential.…”