Abstract B130: Correlation of in vitro cytotoxicity and catabolism of antibody-drug conjugates (ADCs) in target cancer cells
Authors: Singh Rajeeva , Salomon Paulin , Setiady Yulius , Ponte Jose 
Published: May 2016

Abstract
A sensitive and convenient ELISA method has been developed to quantify catabolites generated upon treatment of target cancer cells with an antibody-drug conjugate (ADC). The levels of catabolites generated by different ADCs in target cells were correlated with their in vitro cytotoxic potencies. The ELISA method, exemplified for maytansinoid ADCs, measures catabolite levels as low as 0.03 pmol, requiring only 1 million cells, and is suitable as a high-throughput assay to compare processing efficiency of multiple conjugates with different antibodies or linkers.
An anti-epidermal growth factor receptor antibody conjugate, anti-EGFR-CX-DM1, using our maytansinoid payload and a new, triglycyl (CX) peptide linker was highly active in vitro and in vivo against xenografts of the HSC-2 cell line. In contrast, an anti-EGFR-SMCC-DM1 conjugate using the non-cleavable SMCC linker had little activity. The CX-DM1 conjugate generated 1.7-fold greater amount of total maytansinoid catabolite compared to SMCC-DM1 (3.5 and 2.0 pmol per million cells in one day, respectively), with an 11-fold higher level of catabolite in the media for CX-DM1 (2.8 and 0.25 pmol per million cells, respectively), suggesting trapping of SMCC-DM1 catabolite in the lysosomes of this cell line. The carboxylic acid catabolite of CX-DM1 is presumably effluxed as an uncharged molecule out of acidic lysosomes into the cytoplasm, where it binds to and inhibits microtubules, and is also effluxed out of cells into the media. In contrast, the SMCC-DM1 conjugate generates a charged Lys-SMCC-DM1 catabolite that is not effectively transported out of the lysosome. In another cell line, HCC827, where both anti-EGFR CX-DM1 and SMCC-DM1 conjugates were equally cytotoxic, high levels of total catabolites were generated (2.7 and 2.0 pmol/million cells, respectively) and relatively high levels detected in the media (1.7 and 0.9 pmol/million cells, respectively), suggesting efficient processing and catabolite transport for both CX-DM1 and SMCC-DM1 conjugates in this cell line.
The activity and processing of maytansinoid conjugates of an anti-epithelial cell adhesion molecule antibody (anti-EpCAM) with a hindered-disulfide linker (SPDB-DM4) and a charged sulfonate-bearing hindered-disulfide linker (sulfo-SPDB-DM4) were compared in a multi-drug resistant (MDR) and a non-MDR cell line. The sulfo-SPDB-DM4 conjugate was cytotoxic against the MDR cell line, HCT-15, while the SPDB-DM4 conjugate was inactive. A greater amount of total catabolite was generated for sulfo-SPDB-DM4 than for SPDB-DM4 (0.7 and 0.4 pmol/million cells, respectively), with lower MDR-mediated efflux for sulfo-SPDB-DM4 compared to SPDB-DM4 (intracellular amounts of 0.5 vs 0.1 pmol/million cells, respectively). In a non-MDR cell line, COLO 205, both sulfo-SPDB-DM4 and SPDB-DM4 conjugates were cytotoxic and generated high levels of total catabolites (2.6 and 2.0 pmol/million cells, respectively) and similar levels in the media (0.6 and 0.8 pmol/million cells, respectively), suggesting efficient catabolism and catabolite transport for both conjugates in this cell line.
The ELISA method is a convenient and sensitive tool to measure the intra- and extracellular levels of maytansinoid catabolites generated by processing of ADCs. In several cell lines and ADCs, the method gave good correlation with in vitro cytotoxicity.
Citation Format: Rajeeva Singh, Paulin Salomon, Yulius Setiady, Jose Ponte. Correlation of in vitro cytotoxicity and catabolism of antibody-drug conjugates (ADCs) in target cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B130.