Labeling Single Domain Antibody Fragments with Fluorine-18 Using 2,3,5,6-Tetrafluorophenyl 6-[18F]Fluoronicotinate Resulting in High Tumor-to-Kidney Ratios

Abstract

ImmunoPET agents are being investigated to assess the status of epidermal growth factor receptor 2 (HER2) in breast cancer patients with the goal of selecting those likely to benefit from HER2-targeted therapies and monitoring their progress after these treatments. We have been exploring the use of single domain antibody fragments (sdAbs) labeled with ¹⁸F using residualizing prosthetic agents for this purpose. In this study, we have labeled two sdAbs that bind to different domains on the HER2 receptor, 2Rs15d and 5F7, using 2,3,5,6-tetrafluorophenyl 6-[¹⁸F]fluoronicotinate ([¹⁸F]TFPFN) and evaluated their HER2 targeting properties in vitro and in vivo. The overall decay-corrected radiochemical yield for the synthesis of [¹⁸F]TFPFN-2Rs15d and [¹⁸F]TFPFN-5F7 was 5.7 ± 3.6 and 4.0 ± 2.0%, respectively. The radiochemical purity of labeled sdAbs was >95%, immunoreactive fractions were about 60%, and affinity was in the low nanomolar range. Intracellularly trapped activity from [¹⁸F]TFPFN-2Rs15d and [¹⁸F]TFPFN-5F7 in HER2-expressing SKOV-3 ovarian and BT474M1 breast carcinoma cells were similar to the sdAbs labeled using the previously validated radioiodination residualizing prosthetic agents N-succinimidyl 4-guanidinomethyl-3-[¹²⁵I]iodobenzoate ([¹²⁵I]SGMIB) and N-succinimidyl 3-guanidinomethyl-5-[¹²⁵I]iodobenzoate ( iso-[¹²⁵I]SGMIB). Intracellular activity was about 2-fold higher for radiolabeled 5F7 compared with 2Rs15d for both ¹⁸F and ¹²⁵I. While tumor uptake of both [¹⁸F]TFPFN-2Rs15d and [¹⁸F]TFPFN-5F7 was comparable to those for the coadministered ¹²⁵I-labeled sdAb, renal uptake of the ¹⁸F-labeled sdAbs was substantially lower. In microPET images, the tumor was clearly delineated in SKOV-3 and BT474 xenograft-bearing athymic mice with low levels of background activity in normal tissues, except the bladder. These results indicate that the [¹⁸F]TFPFN prosthetic group could be a valuable reagent for developing sdAb-based immunoPET imaging agents.

Keywords: 2,3,5,6-etrafluorophenyl 6-fluoronicotinate; HER2; N-succinimidyl 4-fluorobenzoate; fluorine-18; immunoPET; single domain antibody fragment.