Improved translation of stability for conjugated antibodies using an in vitro whole blood assay

Abstract

For antibody-drug conjugates to be efficacious and safe, they must be stable in circulation to carry the payload to the site of the targeted cell. Several components of a drug-conjugated antibody are known to influence stability: 1) the site of drug attachment on the antibody, 2) the linker used to attach the payload to the antibody, and 3) the payload itself. In order to support the design and optimization of a high volume of drug conjugates and avoid unstable conjugates prior to testing in animal models, we wanted to proactively identify these potential liabilities. Therefore, we sought to establish an in vitro screening method that best correlated with in vivo stability. While traditionally plasma has been used to assess in vitro stability, our evaluation using a variety of THIOMAB TM antibody-drug conjugates revealed several disconnects between the stability assessed in vitro and the in vivo outcomes when using plasma. When drug conjugates were incubated in vitro for 24 h in mouse whole blood rather than plasma and then analyzed by affinity capture LC-MS, we found an improved correlation to in vivo stability with whole blood (R 2 = 0.87, coefficient of determination) compared to unfrozen or frozen mouse plasma (R 2 = 0.34, 0.01, respectively). We further showed that this whole blood assay was also able to predict in vivo stability of other preclinical species such as rat and cynomolgus monkey, as well as in human. The screening method utilized short (24 h) incubation times, as well as a custom analysis software, allowing increased throughput and in-depth biotransformation characterization. While some instabilities that were more challenging to identify remain, the method greatly enhanced the process of screening, optimizing, and lead candidate selection, resulting in the substantial reduction of animal studies.

Antibody-Mediated Delivery of Chimeric BRD4 Degraders. Part 2: Improvement of In Vitro Antiproliferation Activity and In Vivo Antitumor Efficacy

“…Conjugate CLL1– 9d also exhibited excellent stability properties when exposed to whole blood from various species for 24 h (Table S5). These results suggested that the conjugate and other ADCs derived from the 9d pyrophosphate-containing linker-drug would display favorable stability outcomes if/when assessed in in vivo settings . Accordingly, we continued our exploration of the BRD4 degraders described in this work in an effort to identify additional biologically active ADCs that could be considered for in vivo efficacy assessments.…”

Antibody-Mediated Delivery of Chimeric BRD4 Degraders. Part 1: Exploration of Antibody Linker, Payload Loading, and Payload Molecular Properties

“…In addition, and in agreement with our previous studies, a second control DAR6 conjugate related to STEAP1– 3a -D6 (HER2– 3a -D6; see Figure S1c for evidence of low HER2 expression in PC3-S1 cells) exhibited excellent in vitro stability properties in independent experiments conducted using whole blood from mice, rats, cynomolgus monkeys, and humans (Table S2). These data suggested that conjugates such as STEAP1– 3a -D6 were likely to exhibit acceptable in vivo stability properties in the various species since our previous ADC studies established (1) a reasonable in vitro to in vivo correlation, and (2) the antigen recognized by a given conjugate did not significantly impact the whole-blood stability outcomes . As a result of the above findings, we focused our subsequent efforts on DAR6 ADCs derived from the engineered LC-K149, HC-L174, and HC-Y373 triple mutant STEAP1 monoclonal antibody.…”

Intact Protein Mass Spectrometry for Therapeutic Protein Quantitation, Pharmacokinetics, and Biotransformation in Preclinical and Clinical Studies: An Industry Perspective

“…Several considerations should be factored in, particularly for matrix incubations in ex vivo studies. For example, there may be differences in blood, serum, or plasma incubations or the translation from animal to human matrices . Furthermore, it may be necessary to study injection site stability .…”