P147 In Vitro Investigation of Catabolism and Metabolism of Antibody Drug Conjugates (ADCs) 

Mithat Gunduz , Analytical Sciences & Imaging Metabolite Identification Group, Novartis Institutes for Biomedical Research Inc., Cambridge, MA
Amanda L Cirello , Analytical Sciences & Imaging, Metabolite Identification Group, Tarveda Therapeutics, Watretown, MA
Jennifer L Dumouchel , Novartis Institutes for Biomedical Research Inc., Cambridge, MA
Dominik Hainzl , Novartis Institutes for Biomedical Research Inc., Cambridge, MA
Tony D’Alessio , Novartis Institutes for Biomedical Research Inc., Cambridge, MA
Carl Bialucha , Novartis Institutes for Biomedical Research Inc., Cambridge, MA
Vladimir Capka , Novartis Institutes for Biomedical Research Inc., Cambridge, MA
Upendra A Argikar , Novartis Institutes for Biomedical Research Inc., Cambridge, MA
Objective: To establish an in vitro system suitable to investigate the catabolism of the antibody drug conjugates (ADCs). A successful system must provide information on the fate of the ADCs catabolism by releasing the expected catabolite.

Background: ADCs are used as targeted chemotherapies and ideally should only internalize in tumor cells with limited interaction with healthy cells. An ADC must be stable enough in circulation and behave like non-specific naked antibody. The ADC must internalize in tumor cells and sufficiently release the toxin. The released toxin as a therapeutic agent concentration must be potent at low levels; hence it provides a substantial analytical challenge in investigating the metabolic fate of the ADC and its released toxin.

Methods: The ADC catabolism studies are usually investigated in antigen expressing cell line which provides a complete biotransformation pathway. In target antigen expressing cell line experiments, the typical dose of total ADC was10 nM. At 6, 24 and 48 hours post-dose, time points were directly taken from the seeded cells (5x106 cells/well). The experiment included antigen negative cell line and naked antibody arms as controls.

The in vitro sub-cellular methodology included liver lysosomal fractions, di-sulfide bond reducing agent and the ADC. Time points at 0, 1, 2 and 4 hours were directly taken from the incubate and the reaction was quenched by adding equal amount of MeCN (acetonitrile). The incubate/MeCN mixture was vortex-mixed and centrifuged. The resulting supernatants were transferred to a clean 96 deep well plate and analyzed by Thermo Orbitrap Elite® with accurate mass measurement and MSn capabilities.

Results: At the end of lysosomal incubation, all expected toxins were released and responded well to a time course. The assay was optimum at a concentration of 0.25 mg of lysosomal protein/mL. Increasing the lysosomal protein concentration did not result in increased toxin formation. In addition to the expected catabolite(s), combinations of the accurate masses corresponding to sequences up to three neighboring amino acids next to the linker and toxin were explored, but not observed. This may suggest that the ADC underwent complete hydrolysis in the lysosomal fractions.

Conclusion: In this study, commercially available liver lysosomal fractions were sufficient in hydrolyzing the ADC to release the toxin within four hours. Following the incubation of ADC in liver lysosomal fractions, the product (toxin) formation was monitored. As expected, the lysosomal fraction did not provide any additional biotransformation reactions on the released toxins. As a result, the biotransformation of the released toxins should be investigated in more established in vitro systems such as microsomes, S9 etc. In this methodology, biotransformation reactions from lysosomal and S9 fractions provided similar findings as the target antigen expressing cell lines.