Nanobodies (Nb; aka VHH fragments) are an attractive platform for the development of ²¹¹At targeted radiotherapeutics because their small size (~15 kDa) should offer i.v. pharmacokinetics well matched to the 7.2-h half-life of ²¹¹At. The goal of this study was to label anti-HER2 Nb, 5F7 with ²¹¹At using the residualizing agent N-succinimidyl 3-[²¹¹At]astato-4-guanidinomethylbenzoate (SAGMB).

SAGMB was synthesized by astatodestannylation as reported (1) and coupled to 5F7. Labeled Nb was evaluated for radiochemical purity (RCP); affinity and internalization/cell processing was determined using HER2+ BT474M1 breast cancer cells. Immunoreactive fraction (IRF) was evaluated using magnetic beads coated with HER2 extracellular domain. Paired-label biodistribution directly compared the in vivo behavior of SAGMB-5F7 to that of its ¹³¹I analogue SGMIB-5F7 (2) in athymic mice with BT474M1 xenografts.

Efficiency of labeling Nb 5F7 with SAGMB was 38.4 ± 15.6 % (n=3) while TCA precipitation, SDS-PAGE, and ITLC indicated RCP >93%. The K_d for SAGMB-5F7 was 4.5 ± 0.4 nM and its IRF was 93.5 ± 1.1 %, demonstrating excellent preservation of HER2 binding after ²¹¹At labeling. The percentage of initially bound activity that was internalized was 81.9 ± 1.6, 79 ± 0.1, 78.9 ± 1, 78.7 ± 0.2 and 77 ± 0.9% for SGMAB-5F7 compared with 82.5 ± 1.3, 80.5 ± 0.6, 81.5 ± 1.1, 82.6 ± 0.2, and 82.7 ± 0.7 % for SGMIB-5F7, at 1, 2, 4, 6 and 24 h, respectively. The tumor uptake of SGMAB-5F7 was 15.7 ± 1.4, 15.7 ± 1.7, 15.5 ± 3.1 and 9.4 ± 1.2 % ID/g at 1, 2, 4, 6 and 21 h, respectively, compared with 15.1 ± 1.3, 16.3 ± 1.9, 15.4 ± 3.1 and 11.8 ± 1.5 % ID/g for SGMIB-5F7. Except in kidneys, rapid clearance of ²¹¹At and ¹³¹I from normal tissues was observed, with tumor:blood ratios for SGMAB-5F7 increasing from 12:1 at 1 h to 58:1 at 21 h.

These results are highly encouraging and suggest that SAGMB-5F7 warrants further investigation as an α-particle emitting radiotherapeutic for treating patients with HER2+ malignancies.

This work was supported by Grants CA42324 and CA154291 from NIH.