Abstract A016: High drug-to-antibody ratio (DAR) anti-TROP2 degrader-antibody conjugate (DAC) demonstrated poor solubility at high dose level leading to the fast clearance in mice

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Abstract Proteolysis-Targeting Chimeras (PROTACs) targeting BRM (SMARCA2) afford selective in vivo degradation over BRG1 (SMARCA4) and are active in BRG1 mutant xenograft tumor models. To further enhance in vivo performance, a novel BRM degrader was conjugated to an anti-TROP2 antibody to create a degrader-antibody conjugate (DAC) with a drug-to-antibody ratio (DAR) of 6. When the DAR6 DAC was dosed in preclinical species at 30 mg/kg, the observed Cmax values were much lower than expected based on 5 mg/kg PK data. In addition, PBS formulations of the DAR6 DAC were turbid while clear solutions were obtained using a specialized formulation buffer (20mM Histidine Acetate pH 5.5 with 240mM Sucrose). To determine what may cause the low Cmax, we have investigated the solubility of the DAR6 DAC in formulation buffer as well as in plasma from various species. The DAR6 DAC and its unconjugated intermediate mAb were first radiolabeled with iodine-125, then fortified into human, cynomolgus monkey, rat, and mouse plasma, as well as PBS and the specialized formulation buffer. We employed DAC concentrations in these experiments that corresponded with the 5 and 30 mg/kg PK dose levels mentioned above. The samples were analyzed by centrifugation at 3000 rcf to determine the radiolabeled fraction that was able to be spun down from precipitation or complex formation compared to the soluble fraction. Finally, after optimization, a set of second-generation DAR4 BRM-degrader DACs were tested in the solubility assay to see if improved solubilities could be obtained. Using the described solubility assay, we found that the DAR6 DAC recovery at 200 ug/mL (expected Cmax for 5 mg/kg) was around 80% while at a higher concentration of 1 mg/mL (around the Cmax for 30 mg/kg), the recovery dropped to about 40%. The percentage recovered was consistent across the tested species and in PBS control. In contrast, the DAR4 DAC showed a much better solubility at 1 mg/mL with greater than 80% recovery. Additionally, the DAR4 DAC at 1.5 mg/mL (equivalent amount of degrader loading as the DAR6 DAC at 1 mg/mL) still showed a solubility of over 80%, similar to that of DAR4 DAC at 1 mg/mL, demonstrating that DAR has a greater impact than drug-loading in this DAC. Finally, the DAR4 DAC was dosed in-vivo in mouse at 30 mg/kg and showed the expected Cmax value. In conclusion, this study demonstrated that DAR6 ADC at high dose can cause poor solubility both in PBS buffer and plasma, contributing to a lower than expected Cmax. By reducing the DAR and increasing the solubility in the DAR4 DAC, it showed a recovered Cmax, highlighting the importance of solubility testing in the appropriate matrices in ADC development. In addition, the DAR4 DAC showed much improved solubility compared to the DAR6 entity, thus establishing a correlation between favorable DAC plasma solubility properties and low in vivo clearance. Citation Format: Victor Yip, Gillie Roth, Stephanie Monson, Peter Dragovich, Amrita Kamath, Ben-Quan Shen. High drug-to-antibody ratio (DAR) anti-TROP2 degrader-antibody conjugate (DAC) demonstrated poor solubility at high dose level leading to the fast clearance in mice [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr A016.

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