Labeling a TCO-functionalized single domain antibody fragment with 18F via inverse electron demand Diels Alder cycloaddition using a fluoronicotinyl moiety-bearing tetrazine derivative

Abstract

Single domain antibody fragments (sdAbs) exhibit a rapid tumor uptake and fast blood clearance amenable for labeling with ¹⁸F (t_½ = 110 min) but suffer from high kidney accumulation. Previously, we developed a method for ¹⁸F-labeling of sdAbs via trans-cyclooctene (TCO)-tetrazine (Tz) inverse electron demand Diel's Alder cycloaddition reaction (IEDDAR) that incorporated a renal brush border enzyme (RBBE)-cleavable linker. Although >15 fold reduction in kidney activity levels was achieved, tumor uptake was compromised. Here we investigate whether replacing the [¹⁸F]AlF-NOTA moiety with [¹⁸F]fluoronicotinyl would rectify this problem. Anti-HER2 sdAb 5F7 was first derivatized with a TCO-containing agent that included the RBBE-cleavable linker GlyLys (GK) and a PEG chain, and then subjected to IEDDAR with 6-[¹⁸F]fluoronicotinyl-PEG₄-methyltetrazine to provide [¹⁸F]FN-PEG₄-Tz-TCO-GK-PEG₄-5F7 ([¹⁸F]FN-GK-5F7). For comparisons, a control lacking GK linker and 5F7 labeled using residualizing N-succinimidyl 3-guanidinomethyl-5-[¹²⁵I]iodobenzoate (iso-[¹²⁵I]SGMIB) also were synthesized. Radiochemical purity, affinity (K_D) and immunoreactive fraction of [¹⁸F]FN-GK-5F7 were 99%, 5.4 ± 0.7 nM and 72.5 ± 4.3%, respectively. Tumor uptake of [¹⁸F]FN-GK-5F7 in athymic mice bearing subcutaneous SKOV3 xenografts (3.7 ± 1.2% ID/g and 3.4 ± 1.0% ID/g at 1 h and 3 h, respectively) was 2- to 3-fold lower than for co-injected iso-[¹²⁵I]SGMIB-5F7 (6.9 ± 1.9 %ID/g and 8.7 ± 3.0 %ID/g). However, due to its 6-fold lower kidney activity levels, tumor-to-kidney ratios for [¹⁸F]FN-GK-5F7 were 3-4 times higher than those for co-injected iso-[¹²⁵I]SGMIB-5F7 as well as those observed for the ¹⁸F conjugate lacking the RBBE-cleavable linker. Micro-PET/CT imaging of [¹⁸F]FN-GK-5F7 in mice with SKOV-3 subcutaneous xenografts clearly delineated tumor as early as 1 h with minimal activity in the kidneys; however, there was considerable activity in gallbladder and intestines. Although the tumor uptake of [¹⁸F]FN-GK-5F7 was unexpectedly disappointing, incorporating an alternative RBBE-cleavable linker into this labeling strategy may ameliorate this problem.

Keywords: Fluorine-18; HER2; Inverse electron-demand Diels-Alder reaction; PET imaging; Renal brush border enzyme-cleavable linker; Single domain antibody fragment (VHH, Nanobody).

Figure 1.

In vitro quality control data for [¹⁸F]FN-PEG₄-Tz-TCO-GK-PEG₄-5F7 ([¹⁸F]8). (A) SDS-PAGE/phosphor imaging shows only one band corresponding to the molecular weight of 5F7 sdAb (right lane); molecular weight markers are shown for comparison (left lane). (B) Data from the saturation binding assay performed using HER2-positive SKOV-3 cells to determine affinity (C) Data from the immunoreactivity assay to determine binding to HER2 ECD at infinite antigen excess.

Figure 2.

Paired-label uptake of iso-[¹²⁵I]SGMIB-5F7 ([¹²⁵I]10) and either (a) [¹⁸F]FN-PEG₄-Tz-TCO-PEG₄-5F7 ([¹⁸F]9) or (b) [¹⁸F]FN-PEG₄-Tz-TCO-GK-PEG₄-5F7 ([¹⁸F]8) in SKOV-3 cells. Cells were incubated with [¹²⁵I]10 (yellow) and either [¹⁸F]9 (blue) or [¹⁸F]8 (red) in the presence or absence of excess trastuzumab at 37 °C and processed at 1, 2, and 4 h as described in the text. Data (mean ± SD) are percentage of initially added activity that remained specifically (total minus nonspecific) bound to cells.

Figure 3.

Tumor-to-tissue ratios obtained 1 and 3 h after injection of [¹⁸F]FN-PEG₄-Tz-TCO-PEG₄-5F7 ([¹⁸F]9, blue) and [¹⁸F]FNPEG₄-Tz-TCO-GK-PEG₄-5F7 ([¹⁸F]8, red) in athymic mice bearing subcutaneous SKOV-3 human ovarian cancer xenografts from the two paired-label biodistribution studies.

Figure 4.

Maximum intensity projection images obtained by microPET/CT imaging of a representative mouse bearing SKOV-3 human ovarian cancer xenograft 1, 2, and 3 h after administration of [¹⁸F]FN-PEG₄-Tz-TCO-GK-PEG₄-5F7 ([¹⁸F]8). Positions of tumor (T), kidney (K), Intestine (Int) and gall bladder (gb) are indicated.

Scheme 1.

Scheme for the synthesis of FN-Tz and [¹⁸F]FN-Tz^a

Scheme 2.

Pre-derivatization of 5F7 with 5 (or TCO-PEG₄-NHS Ester) and subsequent ¹⁸F-labeling via IEDDAR with [¹⁸F]4^a.