Abstract 5442: Microscale methods for preparation and screening of antibody-drug conjugates
Authors: Yoder Nicholas C. , Catcott Kalli C. , McShea Molly A. , Bialucha Carl Uli , Saxena Parmita , Bai Chen , Miller Kathy L. , Gesner Thomas G. , Woldegiorgis Mikias , Hicks Stuart W. , Lewis Megan E. , Fleming Michael S. , Erickson Hans K. , Ettenberg Seth E. , Keating Thomas A. 
Published: August 2015

Abstract
Antibody-drug conjugates (ADCs) are hybrid biotherapeutics that combine the targeting specificity of monoclonal antibodies with chemically conjugated, highly potent small molecule chemotherapeutics. Using established research scale approaches, the amount of antibody material needed to prepare candidate ADCs far exceeds the quantities required for initial in vitro screening. The need to scale up production across many antibodies slows down early lead selection efforts and wastes material. We have therefore developed methods for conjugating multiple antibodies with ADC payloads in parallel at the 50-150 μg scale in 96-well plates. Pilot reactions show that antibodies can be titrated to different final drug:antibody ratios (DARs) with different payloads, and that differences in pH can alter the reaction kinetics with useful effects. We show that 96-well centrifugal ultrafiltration enables highly parallel ADC purification while maintaining the rigorous removal of residual cytotoxic impurities observed with more established methods such as gel filtration chromatography. In addition, we describe characterization of microscale ADCs using a single chromatographic assay requiring ∼5 μg of material. The resulting platform reduces the required input quantity of antibody required for in vitro ADC screening by at least 5-10 fold. It also enables much higher conjugation throughput with concomitant decrease in time needed to generate and characterize ADCs.
To assess the utility of the platform for ranking candidate antibodies, we compare in vitro cytotoxicity results for a panel of ADCs produced by both microscale and research scale methods. We also present three example screens in which antibody libraries of 10-85 members against different targets were conjugated using microscale methods and the resulting ADCs ranked by in vitro potency. For each antibody library, the screen size, conjugation conditions, and target DAR range were adjusted to suit the target biology, antibody type,and payload class. For example, we present evidence suggesting that, for targets in which functional antibody activity is not observed, normalization of DAR to the 2-6 range is adequate for screening. Across the screens, the success rates for producing ADC in quantity and quality suitable for screening were in the 75-90% range, using 200-600 μg of input antibody. Cytotoxic potencies ranging over 2-3 orders of magnitude were observed in the resulting ADC libraries, suggesting that microscale conjugation can rapidly focus ADC discoverycampaigns on high potency molecules.
For early stage antibody and ADC screening efforts, we find that microscale conjugation methods yield ADCs that can substitute for traditionally prepared conjugates. We expect these methods will be applicable across many different ADC targets and payloads, and possibly applicable more generally to conjugated macromolecule therapeutic or diagnostic reagents.
Citation Format: Nicholas C. Yoder, Kalli C. Catcott, Molly A. McShea, Carl Uli Bialucha, Parmita Saxena, Chen Bai, Kathy L. Miller, Thomas G. Gesner, Mikias Woldegiorgis, Stuart W. Hicks, Megan E. Lewis, Michael S. Fleming, Hans K. Erickson, Seth E. Ettenberg, Thomas A. Keating. Microscale methods for preparation and screening of 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 5442. doi:10.1158/1538-7445.AM2015-5442