Large Molecule Therapeutics Modulating Therapeutic Activity and Toxicity of Pyrrolobenzodiazepine Antibody–Drug Conjugates with Self-Immolative Disulfide Linkers Thomas H. Pillow 1 , Melissa Schutten 1 , Shang-Fan Yu 1 , Rachana Ohri 1 , Jack Sadowsky 1 , Kirsten Achilles Poon 1 , Willy Solis 1 , Fiona Zhong 1 , Geoffrey Del Rosario 1 , Mary Ann T. Go 1 , Jeffrey Lau 1 , Sharon Yee 1 , Jintang He 1 , Luna Liu 1 , Carl Ng 1 , Keyang Xu 1 , Douglas D. Leipold 1 , Amrita V. Kamath 1 , Donglu Zhang 1 , Luke Masterson 2 , Stephen J. Gregson 2 , Philip W. Howard 2 , Fan Fang 3 , Jinhua Chen 3 , Janet Gunzner-Toste 1 , Katherine K. Kozak 1 , Susan Spencer 1 , Paul Polakis 1 , Andrew G. Polson 1 , John A. Flygare 1 , and Jagath R. Junutula 1 Abstract A novel disulfide linker was designed to enable a direct con- nection between cytotoxic pyrrolobenzodiazepine (PBD) drugs and the cysteine on a targeting antibody for use in antibody–drug conjugates (ADCs). ADCs composed of a cysteine-engineered antibody were armed with a PBD using a self-immolative disulfide linker. Both the chemical linker and the antibody site were optimized for this new bioconjugation strategy to provide a highly stable and efficacious ADC. This novel disulfide ADC was compared with a conjugate containing the same PBD drug, but attached to the antibody via a peptide linker. Both ADCs had similar efficacy in mice bearing human tumor xenografts. Safety studies in rats revealed that the disulfide-linked ADC had a higher MTD than the peptide-linked ADC. Overall, these data suggest that the novel self-immolative disulfide linker represents a valu- able way to construct ADCs with equivalent efficacy and improved safety. Mol Cancer Ther; 16(5); 871–8. Ó2017 AACR. Introduction Antibody–drug conjugates (ADCs) have proven to be an effec- tive method of selectively delivering a small cytotoxic payload to a targeted cell. There are over 55 ADCs currently in human clinical testing and the approval of ADCETRIS (brentuximab vendotin) and KADCYLA (ado-trastuzumab emtansine) has spurred interest in expanding the utility and scope of these powerful agents (1–3). The antibody, linker, and payload of an ADC all play a large and synergistic role in modulating the efficacy and toxicity of the conjugate. A variety of different cytotoxic payloads have been effectively attached to an antibody to produce potent conjugates. These include microtubule-disrupting drugs such as maytansines (4) and auristatins (5), as well as DNA damaging agents such as duocarmycins (6), calicheamicins (7), pyrrolobenzodiazepines (PBDs) (8), and indolinobenzodiazepines (IGNs) (9). Although the above-referenced ADCs are quite efficacious in preclinical studies, many have found limited use clinically due to a low therapeutic index, supporting a need to discover ADCs with an improved efficacy and safety profile. Although the linker connecting these cytotoxic drugs to the antibody plays a critical role in the stability of the ADC and release of the payload (10), few studies have looked systemat- ically at the impact of linker on efficacy and safety. Most work has focused on site-specific and stable conjugation chemistry rather than changing the mechanism by which the linker is cleaved and payload is released (11–13). Although there exist several ADC linker types (hydrazone, disulfide, peptide, glucuro- nide, noncleavable), each with distinct mechanisms of release [acid, glutathione (GSH), protease, glucuronidase, antibody cat- abolism], these linkers typically release unique metabolites, there- by obscuring the impact of the linker release mechanism itself on efficacy and toxicity (14–17). Although the b-glucuronide and peptide linkers were designed to release the same metabolite in two reported ADCs, and efficacy differences were observed, phar- macokinetic data were not provided and the only data around tolerability reported was body weight loss in mice (18). The general dogma is that ADCs with non-cleavable linkers are often safer but less broadly efficacious. In support of this, a noncleavable linker gave an improved preclinical safety profile over a cleavable disulfide linker with maytansinoid ADCs (19). Yet in lymphoma models, the same ADC with a noncleavable linker was inferior in therapeutic activity to the ADC with a cleavable disulfide (20). The safety improvement is likely an effect of the increased stability of the noncleavable linker, requiring 1 Genentech, Inc., South San Francisco, California. 2 Spirogen Ltd., QMB Innovation Centre, London, United Kingdom. 3 WuXi AppTec Co., Ltd., Shanghai, P.R. China. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Current address for K. Poon: Denali Therapeutics, South San Francisco, Cali- fornia; current address for W. Solis, Bristol Myers Squibb, Princeton, New Jersey; current address for J.R. Junutula, Cellerant Therapeutics, San Carlos, California. Corresponding Author: Thomas H. Pillow, Genentech, Inc., South San Francisco, CA 94030. Phone: 650-225-1652; Fax: 650-467-5155; E-mail: thomashp@gene.com doi: 10.1158/1535-7163.MCT-16-0641 Ó2017 American Association for Cancer Research. Molecular Cancer Therapeutics www.aacrjournals.org 871 Downloaded from http://aacrjournals.org/mct/article-pdf/16/5/871/1853922/871.pdf by guest on 24 March 2023