Abstract 5730: Discovery of a novel antibody-drug conjugate linker-payload with a distinct killing mechanism via prolonged unfolded protein response activation
Authors: Rui Liu, Yushi Chi, Yongqiang Shan, Chenqiang Jia, Yingying Zhang, Xiayan Zhang, Wentao Zhang, Xuemei Tian, Yifan Zhang, Fan Yang, Dongmei Han, Simon Hsu, Jiamin Su, Jijun Yuan, Wan-Jen Yang, Xiaoling Yuan, Zhiyu Ma, Chen Hu
Journal: Cancer Research (2025)

Abstract Introduction: A specific class of compounds featuring a 3-(4-hydroxyphenyl) indoline-2 scaffold demonstrated potent antitumor activity against cancer cells with elevated TRPM4 expression or estrogen receptor positivity through the sustained activation of unfolded protein response. This mechanism is distinct from existing toxins. Generally, these compounds exhibit low toxicity to most healthy tissues, with gastrointestinal toxicity being the primary adverse effect. Notable examples of this class include ErSO and laxative oxyphenisatin acetate. This study aimed to optimize this scaffold for use as antibody-drug conjugate (ADC) payloads. By selecting antigens with low gastrointestinal expression, this strategy could potentially overcome ADC resistance or synergize with existing ADCs, leading to durable patient responses and improved survival outcomes. Methods: The cytotoxicity of lead toxin, HLX91-048, and its related ADCs were evaluated in immortalized cell lines and treatment-resistant patient-derived organoids (PDOs) using the CellTiter-Glo® Assay. To create the HER2-targeting ADC, HLX91-048 was linked to a HER2 antibody via a cleavable GGFG linker, resulting in drug-to-antibody ratio (DAR) of 4 or 8 as needed. Flow cytometry was employed to analyze the binding and internalization of the ADCs. Bystander killing assays were performed on the HER2- MCF-7 cells using conditioned media from HER2+ BT474 cell cultures. In vivo efficacy was tested in the BT474 breast cancer model and the DS-8201-resistant NCI-N87R gastric cancer model. The tolerability of HLX91-048 and the ADC was compared in parallel to DXd and DS-8201 in rats. Results: HLX91-048 demonstrates &amp;gt;10-fold increase in cytotoxicity compared to ErSO, exhibiting sub-nanomolar to nanomolar IC50 values across various tumor cell types. When compared to DS-8201 analog the HLX91-048-based ADC showed enhanced cytotoxicity in multiple cell lines, stronger bystander killing, and greater stability in human and monkey plasma. As expected, the HLX91-048-based ADC significantly activates the UPR. The DAR4 HER2 ADC derived from HLX91-048 exhibited dose-dependent antitumor efficacy in both BT474 and NCI-N87 models, leading to substantial tumor regression at higher doses (10-12 mpk). Notably, 5 out of 8 PDOs from patients resistant to various standard treatments remained sensitive to the HLX91-048-based ADC, while showing reduced sensitivity to DS-8201. Lastly, in a pre-toxicology evaluation in rats, both the DS-8201 analog and the HLX91-048-based ADC were well-tolerated at a dose of 60 mpk once weekly for three weeks. Conclusion: We reported a first-in-class ADC linker-payload featuring a highly differentiated killing mechanism that demonstrated exceptional efficacy and safety in preclinical evaluations. Preliminary toxicology studies in non-human primates have been scheduled. Citation Format: Rui Liu, Yushi Chi, Yongqiang Shan, Chenqiang Jia, Yingying Zhang, Xiayan Zhang, Wentao Zhang, Xuemei Tian, Yifan Zhang, Fan Yang, Dongmei Han, Simon Hsu, Jiamin Su, Jijun Yuan, Wan-Jen Yang, Xiaoling Yuan, Zhiyu Ma, Chen Hu. Discovery of a novel antibody-drug conjugate linker-payload with a distinct killing mechanism via prolonged unfolded protein response activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5730.