Abstract View

Objectives: Recently, there has been great interest in developing GPC3-targeted single domain antibody fragments (sdAbs) for the treatment of hepatocellular carcinoma. Specifically, site-selective conjugation is believed to offer improved reproducibility and homogeneity over “random” techniques. Herein, we examined *I and ²¹¹At conjugated A345 monomers and evaluated them in GPC3-expressing cell lines and xenografts.

Methods: A345 was radiohalogenated using N-succinimidyl 5-guanidinomethyl-3-[*I]iodobenzoate (iso-SGMIB), a random conjugation precursor. A corresponding A345 analogue bearing a C-terminus GGC tail was also labeled with a pheynloxadiazolyl methylsulfone (PODS) derivative, a site-specific residualizing prosthetic agent. Binding affinity and internalization of labeled sdAbs were evaluated in GPC3-positive HepG2 cells, followed by animal studies in NSG/athymic nude mice bearing H660 xenografts.

Results: A345 constructs were successfully conjugated with 16% RCY for iso-[¹²⁵I]SGMIB and 23% RCY for [¹³¹I]GMIB-PODS. Immunoreactivity assays retained high specific binding of iso-[¹²⁵I]SGMIB and [¹³¹I]GMIB-PODS to human GPC3 at 83.8 ± 0.3 % and 78.4 ± 0.7% of input counts, respectively. Paired-label cell internalization assays in HepG2 cells revealed total uptake peaked at 4 h and declined by 24 h, with iso-[¹²⁵I]SGMIB-A345 and [¹³¹I]GMIB-PODS-A345-GGC measuring at 18.6 ± 0.3% and 21.6 ± 0.9%, respectively at the latter time point. Both conjugates had low nonspecific binding (< 5%). Preliminary biodistribution studies of iso-[¹³¹I]SGMIB-A345 in NSG mice showed measurable tumor uptake: 3.96 ± 1.00% ID/g at 1 h, 2.68 ± 0.68% ID/g at 4 h, 1.04 ± 0.25% ID/g at 21 h. Although similar for 1 h and 4 h time points, tumor accumulation of iso-[²¹¹At]SAGMB-A345, was greater at the 21 h time point (p < 0.05). Minimal thyroid and stomach uptake (< 0.5% ID/organ) was seen for iso-[¹³¹I]SGMIB-A345 whereas its ²¹¹At-labeled analogue demonstrated greater thyroid uptake (~0.6% ID/organ) and stomach accumulation (~3-6% ID/organ). Biodistribution with iso-[¹³¹I]SGMIB-A345 in athymic nude mice yielded comparable results to NSG mice.

Conclusions: PODS-labeled A345-GGC exhibited favorable cellular internalization in GPC3-positive cells compared to the iso-SGMIB construct, while retaining high specific binding. These findings support further in vivo evaluation of [¹³¹I]GMIB-PODS-A345-GGC and investigation of A345-multimers for developing targeted radiotherapeutics, particularly when labeled with alpha-particle emitter ²¹¹At.

Acknowledgements: I would like to thank the Radiopharmaceutical Chemistry Lab for their help, guidance, and support on this research.

References: (1) Feng, Y.; Sarrett, S.; Meshaw, R.; Vaidyanathan, G.; Cornejo, M.; Zeglis, B.; Zalutsky, M. Site-Specific Radiohalogenation of a HER2-Targeted Single-Domain Antibody Fragment Using a Novel Residualizing Prosthetic Agent. J. Med. Chem. 2022, 65,15358-15373.