A Multi-Analyte Hplc-Ms/Ms Approach To Assessing Exposure Of A Probody Drug Conjugate In Preclinical Studies

CX-2029 is a protease-activatable antibody prodrug (Probody TM Therapeutic) targeted against CD71 (transferrin receptor) and conjugated to a vcMMAE cytotoxic payload with a purified Drug to Probody Ratio (DPR) of 2. In the intact, prodrug form, each light chain of CX-2029 contains an N-terminal prodomain that masks the target-binding region of the parental antibody and decreases antigen binding. In vivo proteolytic cleavage of the prodomain in the tumor microenvironment exposes the target-binding region, yielding the active antibody. In this way, Probody therapeutics are designed to avoid on-target toxicity in normal tissues while preserving antitumor activity. In vivo, CX-2029 may be present in several forms as a result of activation of the antibody prodrug, as well as deconjugation of the cytotoxic payload. We have developed a multi-analyte HPLC-MS/MS approach to monitor levels of four analytes (Intact Probody Therapeutic, Total Probody Therapeutic, Probody-Conjugated MMAE, and Unconjugated MMAE) in cynomolgus monkey plasma to evaluate the exposure of both the intact CX-2029 and activated antibody prodrug, as well as to monitor changes in the DPR over time. To understand the impact of antidrug antibodies (ADA) on exposure, a bridging assay was developed to monitor the formation of ADA. These assays were used to assess exposure of CX-2029 in an ascending dose toxicity study (6, 12, and 18 mg/kg/dose) in cynomolgus monkeys, in support of dose selection for an IND-enabling study. CX-2029 was administered as an intravenous bolus dose to groups of 3 monkeys (2 males, 1 female) once every three weeks for a total of two doses. Samples were collected for pharmacokinetic analysis at time points spanning 21 days after the first dose and 7 days after the second dose. Samples were collected for ADA analysis prestudy and 7 days after the second dose. Dose-dependent increases in Cmax were observed between 6 and 18 mg/kg for the analytes measured. Half-life estimates were similar for Intact Probody Therapeutic, Total Probody Therapeutic, and Probody-Conjugated MMAE and ranged from 2.5 to 6.3 days. The ratio of Intact Probody Therapeutic to Total Probody Therapeutic was used to assess stability of the cleavable prodomain in the cynomolgus monkey over time. By 7 days post-dose, approximately 80% of CX-2029 in plasma was in the intact, prodrug form. The average DPR was evaluated over three weeks in vivo. Average DPR decreased from 2 shortly after dosing to approximately 0.5 by 21 days post-dose. ADA were detected in 3 of 9 animals dosed with CX-2029. In cynomolgus monkeys, CX-2029 exposure is maintained throughout the 21-day dosing interval, and the majority of CX-2029 in circulation is intact. CX-2029 is currently under development, with an IND filing expected in 2018. PROBODY is a trademark of CytomX Therapeutics, Inc. Citation Format: Laura Serwer, Shweta Singh, Claus Krebber, Shouchun Liu, Niharika Chauhan, Robert Leanna, Hong Lu, Ilaria Badagnani, Tracy Henriques, Susan Morgan-Lappe, William Mylott, Sridhar Viswanathan, Jennifer Richardson, Michael Kavanaugh. A multi-analyte HPLC-MS/MS approach to assessing exposure of a Probody drug conjugate in preclinical studies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B103.