Stability of ADC toxins in sub-cellular fractions

04th Jun 2024

Measurement of toxin and metabolites of common and proposed ADCs after incubation in lysosomes and S9 fraction

Antibody drug conjugates (ADCs) represent a growing class of anti-cancer drugs, combining the specific targeting nature of monoclonal antibodies with the highly cytotoxic nature of small molecule oncology drugs. However, ADCs are generally trafficked into cells via the endocytic pathway resulting in their processing in lysosomes, whereas small molecules commonly use other pathways . Whereas many ADCs with cleavable linkers take advantage of the chemical conditions within these vesicles, the toxin itself must be able to withstand their digestive enzymes. Toxic payloads containing peptide moieties may be metabolised before they can reach their cell killing targets.

Here, we outline an analysis strategy to determine the stability of three cytotoxic compounds (MMAE, Exetecan (Exe), and Carfilzomib) in human liver S9 fraction and human lysosome in line with a paper by Zhang et al. The samples were incubated in human liver lysosomes, or S9 fraction (Xenotech), at a concentration of 0.05 mg/ml for time periods up to 24 hours. Sub-cellular fractions were also bolstered with catbolic buffer and NADPH RapidStart (Xenotech) to ensure metabolic activity. The samples were heat inactivated after incubation for five minutes at 95°C before removal of the protein via solvent crash. All data was collected on a SCIEX X500B Mass Spectrometer with an EXION HPLC. The instrument was operated in IDA mode to confirm species via fragmentation. The chromatography was performed with a Kinetix C18 column (1.7 μm, 50×2.1mm) on a five minute gradient of water and acetonitrile both with 0.1% formic acid.

In trials, all the toxins were stable at up to 24 hours in catabolic buffers lacking the sub-cellular fractions. Figure 1 shows the XIC of MMAE and Exe after 24 hours with human lysosomal extract. There is a decrease in the peak area due to small metabolites appearing, which we characterised as hydration and oxidation events, but the major peak remained constant. Similar data was observed in the S9 fraction.

Figure 1: XIC of MMAE and Exetecan in human liver lysosome. MMAE (717 m/z) and Exe (436 m/z) were monitored over a 24 hour period and saw no significant drop in intensity nor the appearance of new peaks.

Carfilzomib, however, showed a decline in peak intensity from the first time point of 15 minutes. Figure 2 shows the XIC of Carfilzomib and its known metabolites over the time course. The area declined over time, firstly into the metabolite described as M1 (with loss of C18H24N2O2). After 24 hours, the dominant peak was the metabolite described as M2 (loss of C24H35N3O3) with a loss of over 80% of the Carfilzomib. It was observed that metabolism occurred more rapidly in lysosomes than S9, with relative intensity loss at 120 minutes, with ~60% is S9 and 80% in lysosomes.

Figure 2: XIC of Carfilzomib and its metabolites in human liver S9 fraction. Carfilzomib peak area drops steadily over the time course with two major metabolites. Blue T0 Purple T15 Red T30 Orange T60 Green T90 Dark green T120 Cyan T1440 (minutes).

Figure 3: Relative intensity of Carfilzomib and its metabolites over time in S9 fraction. Over the first few hours of incubation, Carfilzomib breaks down into metabolite 1, before this then breaks down into metabolite 2, which is dominant after 24 hours.

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