Abstract 7183: Liver toxicity of amanitin-based antibody drug conjugates (ATACs) is caused by unspecific uptake of the ATAC into liver cells
Authors: Orlik Christian , Decker Kristin , Vranic Marija , Schmitt Marisa , Pahl Andreas , Kulke Michael , Hechler Torsten 
Published: March 2024

Abstract
Background: Antibody drug conjugates (ADCs) were developed to increase the therapeutic window (TW) of cytotoxins by combining their efficacy with the precision of antibodies (Ab). Nevertheless, adverse events, still limit the use of ADCs. The tox profile of an ADC is determined by its on- and off-target effects. On-target tox is caused by specific binding of the ADC to healthy target-positive cells; off-target tox is caused by target-independent binding of the ADC, e.g. via the Ab backbone or by payload release in circulation. In this study, we unravel the off-target tox mechanisms of ADCs that use amatoxins (RNA polymerase II inhibitor) as payload (ATACs) and use this knowledge to improve the TW of ATACs. Amatoxins are distinct from other ADC payloads as they are hydrophilic and thus require active transport to pass the cell membrane. The primary transporter of amatoxins in humans is the OATP1B3 transporter, which is exclusively expressed on hepatocytes. Thus, unspecific ATAC tox can be caused by free amatoxin, premature released in circulation and uptake by liver cells via OATP1B3, or by uptake of the ATAC via a yet unknown mechanism.
Material and Methods: ATACs are based on cysteine-reactive and site-specific amatoxin-linker constructs by HDP. In vitro: Binding of ATACs to FcγRI by BLI; Cytotoxicity of ATACs on HEKwt, HEK-OATP1B3 and THP1 cells. In vivo: Single i.v. dose of ATAC; Tolerability in mice; Efficacy in s.c. tumor models; Blood sampling for PK analysis; ADC/total Ab detection by ELISA; DAR-loss analysis and toxin measurement by (intact) LC-MS.
Results: Liver tox caused by premature release of amatoxin in circulation was investigated thoroughly in vivo. DAR-loss, survival analysis, ADC and Ab measurements, indicated that the release of amatoxin in circulation cannot be the root-cause for off-target ATAC toxicity. Further, it was ruled out that the ATAC is taken up by OATP1B3 in vitro and in vivo. Thus, uptake of the intact ATAC via yet unknown mechanisms is responsible for liver tox. As liver tox is also known for other ADCs, several uptake mechanisms are described, one of which being FcγR-mediated uptake. Indeed, ATACs bind to the FcγR and trigger target-independent cytotoxicity on FcγR+ cells. Point mutations at L234 and L235 (LALA mutation), the amino acids in the Fc part of the Ab responsible for FcγR binding, were shown to reduce FcγR binding. The LALA mutation significantly increased the tolerability of ATACs in mice and monkeys, whereas the anti-tumor efficacy was not impaired, leading to an improved TW.
Conclusion: Our data show that liver toxicity of ATACs is caused by unspecific uptake of the ATAC into liver cells at least partially mediated by FcγR. This knowledge was used to optimize the Ab backbone to minimize unspecific uptake into liver cells. Insertion of a LALA mutation to avoid uptake via FcγR reduced off-target toxicity of ATACs leading to better tolerability and an increased TW.
Citation Format: Christian Orlik, Kristin Decker, Marija Vranic, Marisa Schmitt, Andreas Pahl, Michael Kulke, Torsten Hechler. Liver toxicity of amanitin-based antibody drug conjugates (ATACs) is caused by unspecific uptake of the ATAC into liver cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7183.