Abstract P062: The CL2A-SN38 linker-payload system conjugated to trastuzumab results in improved cellular cytotoxicity over time relative to T-DM1
Authors: Singh Hardeep , Leyton Victor Jeffrey 
Published: December 2021

Abstract
Background The approved antibody-drug conjugate (ADC) sacituzumab-govitecan (SG) that exploits the topoisomerase I inhibitor SN38, represents a substantial advance in the ADC field. SN38 is the first payload with low nanomolar cytotoxicity. This deviates from current design principles, which utilize ultracytotoxic payloads. The CL2A linker has a short non-cleavable seven polyethylene glycol segment that enables a higher number of hydrophobic SN38 molecules to be attached to reduced cysteines. CL2A is also designed to permit the release of SN38 in an acidic environment via a pH-sensitive benzyl carbonate bond to SN38’s lactone ring. There is no cathepsin B cleavage site. Key to the performance of SG is the SN38 release half-life of 1-2 days. This also deviates from very stable linker systems that require full ADC internalization and lysosomal processing for payload release. As a result of these novel ADC properties, SG is given in higher and repeated doses, which results in slow release at the tumor site and enhanced uptake by target cancer cells and reduced toxicity on healthy cells due to low potency SN38 alone. Our goal is to further evaluate the CL2A-SN38 system when conjugated to trastuzumab. Material and methods Trastuzumab was conjugated to CL2A-SN38 to reduced cysteines (T-SN38). Monitoring the changes in DAR over time was performed to determine the stability of T-SN38 in PBS at pH values of 7.4, 5.72, and 4.58, to mimic circulating blood, early endosomes, and lysosomes, respectively. Cytotoxicity assays for 72 h on HER2-positive ovarian cancer SKOV3.ip and HER2-negative CHO cells were performed and compared to the approved anti-HER2 ADC trastuzumab-emtansine (T-DM1). Cytotoxicity assays were also performed between T-SN38 and T-DM1 at their respective IC50 values and the percent of cell survival determined daily from 24-96 h. Results T-SN38 was generated with a DAR of 6.97. Surprisingly, the half-life of SN38 release in acidic pH was extremely slow (t1/2=~120 h) and at the final evaluated time point (120 h) 69% of SN38 remained conjugated to trastuzumab. As anticipated, as a function of concentration T-DM1 was significantly more potent than T-SN38 (IC50=0.0013 vs 0.0832 mg/mL). However, when cytotoxicity was evaluated as a function of time T-SN38 switched to the more potent ADC at the late time points. T-SN38 killed SKOV3.ip cells 36% more effectively than T-DM1 at 96 h. Conclusions Our data from SN38 release experiments in acidic buffer suggest that pH-induced cleavage of the CL2A linker is not a contributable release mechanism of SN38 when bound to trastuzumab and is counterintuitive for what is currently described for SG. However, traceless release of hydrophobic SN38 capable of traversing cell membranes is only possible by hydrolysis of the benzyl carbonate bond. Our cytotoxicity studies over time suggest that pH-dependent release does occur. Further investigations are ongoing to more precisely determine the contribution of the carbonate bond for SN38 release and cytotoxic potency.
Citation Format: Hardeep Singh, Victor Jeffrey Leyton. The CL2A-SN38 linker-payload system conjugated to trastuzumab results in improved cellular cytotoxicity over time relative to T-DM1 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P062.