Abstract 5757: Exatecan payload-based antibody-drug conjugates with a short hydrophilic beta-glucuronidase cleavable linker

Abstract Antibody-drug conjugates (ADCs) enable the specific targeting of a potent cytotoxic drug to cancer cells expressing a select antigen via the release of the cytotoxin covalently linked to a target-specific antibody. A classic ADC comprises three elements: (a) a target-specific antibody that enables the binding of the ADC to cells expressing the target antigen, (b) a potent cytotoxic drug (payload) that kills cells when released from the ADC, and (c) a linker that covalently links the payload to the antibody until pre-defined conditions cause release of the payload. One of the factors limiting the in vivo biological activity of an ADC is high hydrophobicity, which stems partly from the linker or spacer and partly from the payload. In the body, high hydrophobicity often leads to aggregation of the ADC molecules; these aggregates are then rapidly cleared from circulation by the liver. Several approaches investigated in the past to reduce the overall hydrophobicity of ADCs include (a) reducing the hydrophobicity of the payload itself, (b) positioning the (hydrophobic) payload in a shielded pocket of the antibody, and (c) modifying the linker structure by introduction of hydrophilic spacers such as polyethylene glycol (PEG), polysarcosine (PSAR), hydrophilic macrocycles or long hydrophilic XTEN peptides. However, all these linker modifications involve bulky spacers, which can impact permeability into tumor tissue. Our goal was to develop short, streamlined hydrophilic linkers that boost ADC solubility without adding significant bulk. We successfully synthesized and screened several linker-payloads, and validated a novel hydrophilic, lysosomal β-glucuronidase cleavable linker combined with a short sugar-based spacer (AV-L03). We synthesized an ADC comprising trastuzumab conjugated to an exatecan payload via this novel, short, stable, hydrophilic linker AV-L03 at a drug-to-antibody ratio (DAR) of 8 and compared it to Enhertu®, which comprises trastuzumab conjugated to a DXd payload via a (hydrophobic) tetrapeptide linker. The ADC with the novel AV-L03 linker showed superior hydrophilicity, in vitro cytotoxicity, improved plasma stability, and in vivo efficacy in a mouse xenograft model compared to Enhertu®. The AV-L03 linker makes possible the use of exatecan as a payload for ADCs, which is promising given that exatecan is a more potent inhibitor of TOP1 isomerase compared to DXd, with higher permeability and bystander activity, and is a poorer substrate for some MDR pumps. Moreover, the AV-L03 linker, the shortest hydrophilic linker reported to date, is compatible with diverse payloads, and thus opens up several avenues for the design of superior ADCs. Citation Format: Vasu Jammalamadaka, Jiang Liu, Sunil Bhakta, Vidya S. Jonnalagadda, Jagath R. Junutula. Exatecan payload-based antibody-drug conjugates with a short hydrophilic beta-glucuronidase cleavable linker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5757.