Astatine-211 labeled anti-HER2 5F7 single domain antibody fragment conjugates: radiolabeling and preliminary evaluation.

Introduction: Derived from heavy chain only camelid antibodies, ~15-kDa single-domain antibody fragments (sdAbs) are an attractive platform for developing molecularly specific imaging probes and targeted radiotherapeutics. The rapid tumor accumulation and normal tissue clearance of sdAbs might be ideal for use with At, a 7.2-h half-life α-emitter, if appropriate labeling chemistry can be devised to trap At in cancer cells after sdAb binding. This study evaluated two reagents, [At]SAGMB and iso-[At]SAGMB, for this purpose.

Methods: [At]SAGMB and iso-[At]SAGMB, and their radioiodinated analogues [I]SGMIB and iso-[I]SGMIB, were synthesized by halodestannylation and reacted with the anti-HER2 sdAb 5F7. Radiochemical purity, immunoreactivity and binding affinity were determined. Paired-label internalization assays on HER2-expressing BT474M1 breast carcinoma cells directly compared [I]SGMIB-5F7/[At]SAGMB-5F7 and iso-[I]SGMIB-5F7/iso-[At]SAGMB-5F7 tandems. The biodistribution of the two tandems was evaluated in SCID mice with subcutaneous BT474M1 xenografts.

Results: Radiochemical yields for Boc-iso-[At]SAGMB and Boc-[At]SAGMB synthesis, and efficiencies for coupling of iso-[At]SAGMB and [At]SAGMB to 5F7 were similar, with radiochemical purities of [At]SAGMB-5F7 and iso-[At]SAGMB-5F7 >98%. iso-[At]SAGMB-5F7 and [At]SAGMB-5F7 had immunoreactive fractions >80% and HER2 binding affinities of less than 5 nM. Internalization assays demonstrated high intracellular trapping of radioactivity, with little difference observed between corresponding At- and I-labeled 5F7 conjugates. Higher BT474M1 intracellular retention was observed from 1-6 h for the iso-conjugates (iso-[At]SAGMB-5F7, 74.3 ± 2.8%, vs. [At]SAGMB-5F7, 63.7 ± 0.4% at 2 h) with the opposite behavior observed at 24 h. Peak tumor uptake for iso-[At]SAGMB-5F7 was 23.4 ± 2.2% ID/g at 4 h, slightly lower than its radioiodinated counterpart, but significantly higher than observed with [At]SAGMB-5F7. Except in kidneys and lungs, tumor-to-normal organ ratios for iso-[At]SAGMB-5F7 were greater than 10:1 by 2 h, and significantly higher than those for [At]SAGMB-5F7.

Conclusion: These At-labeled sdAb conjugates, particularly iso-[At]SAGMB-5F7, warrant further evaluation for targeted α-particle radiotherapy of HER2-expressing cancers.