Abstract 645: Stability and efficacy comparison of site-specific and lysine-linked maytansinoid antibody-drug conjugates
Authors: Yoder Nicholas C. , Bai Chen , Tavares Daniel , Widdison Wayne C. , Ab Olga , Whiteman Kathleen R. , Wilhelm Alan , Maloney Erin K. , Erickson Hans K. , Keating Thomas A. 
Published: August 2015

Abstract
As with any therapeutic molecule, antibody-drug conjugates (ADCs) exhibit structure-activity relationships, and medicinal chemistry efforts in this field strive to optimize structure to give the maximum therapeutic index. Recent interest in ADCs as cancer therapy has led to a number of different combinations of linker, payload, and conjugation chemistry.In particular, site-specific methods of payload conjugation have been suggested to generally improve therapeutic properties as compared with more established approaches directed toward lysines or endogenous cysteines.We have investigated the preparation, stability, and activity of anti-folate receptor alpha (FRα) ADCs carrying the microtubule inhibitor, DM1, and conjugated to engineered cysteine mutants utilizing different sites, and compared these ADCs with lysine-directed heterogeneous conjugates. In both embodiments, the DM1 is linked with a protease-cleavable linker. We show that highly homogeneous DM1 ADCs can be produced using engineered cysteine chemistry, enabling assessment of the effects of site-specific conjugation in cells and in animal models. We find that in vitro potency of both lysine-linked and engineered cysteine-linked ADCs against FRα-positive KB cells scales with the total DM1 delivered to cells. Buffer stability experiments in the presence of excess thiol suggest that most engineered cysteine conjugates are comparable in stability to the lysine-linked ADC. A notable exception shows about twice as much fractional DM1 loss upon 3 days of incubation as the other conjugates. Comparison of in vivo activity of two site-specific DM1 ADCs in a KB xenograft model shows measurable activity differences between different conjugation sites. However, a lysine-linked conjugate using almost identical linker chemistry shows approximately 2-fold superior activity to either site-specific construct on a molar DM1 basis. We conclude that, while site-specific conjugation of ADCs may provide a benefit in certain contexts, in other contexts, it may lead to decreased activity, such as in the anti-FRα/KB model examined here. We also observe that different conjugation sites may offer significant differences in activity. It is therefore advisable to evaluate each unique combination of payload, linker, drug:antibody ratio, conjugation site(s), and antibody to the maximum extent possible.
Citation Format: Nicholas C. Yoder, Chen Bai, Daniel Tavares, Wayne C. Widdison, Olga Ab, Kathleen R. Whiteman, Alan Wilhelm, Erin K. Maloney, Hans K. Erickson, Thomas A. Keating. Stability and efficacy comparison of site-specific and lysine-linked maytansinoid antibody-drug conjugates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 645. doi:10.1158/1538-7445.AM2015-645