Abstract 1529: Characterizing antibody internalization for rational selection of ADC linker design

Abstract Antibody-drug conjugates (ADC), which target a highly potent cytotoxin to a specific protein overexpressed on tumor cells, are a promising class of anticancer therapeutics. In addition to the antibody characteristics, intracellular reactivity of the antibody-cytotoxin linker (which affects drug release) is a key determinant of ADC efficacy. While many different linkers have been used in approved ADCs, the intracellular trafficking profile of an antibody will likely impact the effectiveness of different linkers differently. Here, we determined the cellular internalization of two antibodies, anti-perlecan antibody (‘AM6’), and anti-EGFR antibody (cetuximab; ‘Ctx’), in MDA-MB-231-LM2 cells (a lung-metastatic TNBC cell line). Using live-cell confocal microscopy, we investigated the effect of endocytosis inhibitors on intracellular trafficking of AM6 and Ctx. While AM6 was internalized quicker compared to Ctx, the internalization of both antibodies was reduced significantly by 5-(N-Ethyl-N-isopropyl)-amiloride, a macropinocytosis inhibitor and, to a lesser extent, by chlorpromazine, an inhibitor of clathrin-mediated-endocytosis. The results suggest both AM6 and Ctx were internalized by both macropinocytosis and clathrin-mediated endocytosis. We then investigated whether acidic pH in the lysosomes and the reductive environment in the endosomal vesicles would facilitate drug release from ADC. We conjugated thiol-reactive doxorubicin to reduced interchain disulfides of Ctx via either the acid and glutathione (GSH)-labile pyridinyldithio-hydrazide linker or the non-cleavable C6-maleimide linker. Intracellular doxorubicin release for the two ADCs was evaluated by live-cell confocal microscopy. Doxorubicin was successfully conjugated to Ctx, with a DAR of 4.8 and 8.6 for the cleavable and non-cleavable linkers, respectively. Conjugation of doxorubicin to the antibody was confirmed by fluorescent gel imaging after SDS-PAGE and by using hydrophobic interaction chromatography. Confocal microscopic studies showed that the ADC with cleavable linker resulted in greater doxorubicin accumulation in the nucleus compared to the ADC with non-cleavable linker, which was predominantly sequestered in the lysosomes. These results suggest that the cleavable linker results in better drug release compared to the non-cleavable linker for the Ctx-doxorubicin ADC. Studies examining the effect of linker chemistry on drug release from the anti-perlecan ADC and on the anticancer efficacy of the two ADCs are ongoing. Citation Format: Zekun Shao, Jayanth Panyam. Characterizing antibody internalization for rational selection of ADC linker design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1529.