Abstract A106: Disrupting ribosome assembly to block protein translation: A novel ADC payload with strong antitumor activity in mono- and dual-payload formats

Abstract Background: Antibody-drug conjugates (ADCs) have shown clinical success, but resistance, often driven by the payload, can limit long-term efficacy. To address this, Hexagon Bio is mining its natural product library to discover new payloads suitable for mono- or dual-payload ADCs. We report on a ribosome assembly inhibitor (RAI) with strong antitumor activity when conjugated to the anti-TROP2 antibody sacituzumab, both as a mono payload and in combination with the topoisomerase inhibitor Dxd (dual payload). The RAI also shows promise when conjugated to other antibodies, including those targeting low-abundance antigens. Materials and methods: By mining small molecules produced by Hexagon Bio’s 120,000-strain microbial library, several potent cytotoxic compounds were identified. The lead RAI was evaluated as a free agent in cell-free translation assays and as an ADC. It was conjugated to sacituzumab at drug-to-antibody ratios (DARs) of 4 and 8 and tested in:In vitro cytotoxicity (CellTiter-Glo, 120 hrs) and immunogenic cell death (ICD) assays (detection of hallmarks of ICD: extracellular ATP, secreted high mobility group box 1 and cell surface calreticulin, 120-168 hrs)In vivo efficacy models (KPL4, NCI-N87, HT1376)Pharmacokinetics/pharmacodynamic (PK/PD) studiesRodent tolerability studiesTo assess broader applicability, the RAI was also conjugated to other antibodies (anti-HER3, -Nectin4, -ITGB6, -B7H3, -B7H4), and cytotoxicity was evaluated. A dual-payload ADC combining the RAI on sacituzumab was also generated and tested. Results: As a free agent, the RAI potently inhibited protein translation in cell-free assays (EC50∼ 15 nM). When conjugated to sacituzumab, the ADC exhibited strong in vitro cytotoxicity (sub- to single-digit nM) across TROP2-expressing cell lines, including those that have low antigen levels and are resistant to Dxd. The ADC also induced hallmarks of ICD, suggesting it may combine well with checkpoint inhibitors. In vivo, RAI-based ADCs delivered complete responses at ≥ 5 mg/kg (DAR 4) in both Dxd-sensitive (KPL4, NCI-N87) and Dxd-resistant (HT1376) models, outperforming sacituzumab-Dxd (DAR 8). PK profiles were favorable, with circulating t1/2 in line with that of naked sacituzumab. In preliminary tolerability studies, the DAR 4 ADC was well tolerated in mice and rats, suggesting a reasonable therapeutic index.RAI-conjugated ADCs remained cytotoxic when paired with other antibodies, including those targeting antigens expressed as low as 20 K receptors per cell, supporting its translatability. The dual-payload ADC (RAI + Dxd) outperformed the Dxd-only ADC in vitro, including in Dxd-resistant cell lines, demonstrating its potential to broaden efficacy and overcome resistance. Conclusions: Our findings highlight the promise of a novel RAI as a versatile ADC payload, demonstrating potent antitumor activity alone or in combination with Dxd. Its compatibility with diverse antibodies, and favorable PK/tolerability profiles support its further evaluation as an approach to overcome resistance and improve patient outcomes. Citation Format: Tara L. Arvedson. Disrupting ribosome assembly to block protein translation: A novel ADC payload with strong antitumor activity in mono- and dual-payload formats [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A106.