Intact quantitation of cysteine-conjugated antibody-drug conjugates using native mass spectrometry.

RATIONALE: A common strategy for antibody-drug conjugate (ADC) quantitation from in vivo study samples involves measurement of total antibody, conjugated ADC, and free payload concentrations using multiple reaction monitoring (MRM) mass spectrometry. This not only provides a limited picture of biotransformation but can also involve lengthy method development. Quantitation of ADCs directly at the intact protein level in native conditions using high-resolution mass spectrometers presents the advantage of measuring exposure readout as well as monitoring the change in average drug-to-antibody ratio (DAR) and in vivo stability of new linker payloads with minimal method development. Furthermore, site-specific cysteine-conjugated ADCs often rely on non-covalent association to retain their quaternary structure, which highlights the unique capabilities of native mass spectrometry (nMS) for intact ADC quantitation. METHODS: We developed an intact quantitation workflow involving three stages: automated affinity purification, nMS analysis, and data processing in batch fashion. The sample preparation method was modified to include only volatile ion-pairing reagents in the buffer systems. A capillary size-exclusion chromatography (SEC) column was coupled to a quadrupole time-of-flight high-resolution mass spectrometer for high-throughput nMS analysis. Samples from two mouse pharmacokinetic (PK) studies were analyzed using both intact quantitation workflow and the conventional MRM-based approach. RESULTS: A linear dynamic range of 5-100 µg/mL was achieved using 20 µL of serum sample volume. The results of mouse in vivo PK measurement using the intact quantitation workflow and the MRM-based approach were compared, revealing excellent method agreement. CONCLUSIONS: We demonstrated the feasibility of utilizing nMS for the quantitation of ADCs at the intact protein level in preclinical PK studies. Our results indicate that this intact quantitation workflow can serve as an alternative generic method for high-throughput analysis, enabling an in-depth understanding of ADC stability and safety in vivo.

Discovery of ABBV-154, an anti-TNF Glucocorticoid Receptor Modulator Immunology Antibody-Drug Conjugate (iADC).

Stable attachment of drug-linkers to the antibody is a critical requirement, and for maleimide conjugation to cysteine, it is achieved by ring hydrolysis of the succinimide ring. During ADC profiling in our in-house property screening funnel, we discovered that the succinimide ring open form is in equilibrium with the ring closed succinimide. Bromoacetamide (BrAc) was identified as the optimal replacement, as it affords stable attachment of the drug-linker to the antibody while completely removing the undesired ring open-closed equilibrium. Additionally, BrAc also offers multiple benefits over maleimide, especially with respect to homogeneity of the ADC structure. In combination with a short, hydrophilic linker and phosphate prodrug on the payload, this afforded a stable ADC (ABBV-154) with the desired properties to enable long-term stability to facilitate subcutaneous self-administration.

Chemoproteomic Identification of Serine Hydrolase RBBP9 as a Valacyclovir-Activating Enzyme.

Prodrug discovery and development in the pharmaceutical industry have been hampered by a lack of knowledge of prodrug activation pathways. Such knowledge would minimize the risks of prodrug failure by enabling proper selection of preclinical animal models, prediction of pharmacogenomic variability, and identification of drug-drug interactions. Technologies for annotation of activating enzymes have not kept pace with the growing need. Activity-based protein profiling (ABPP) has matured considerably in recent decades, leading to widespread use in the pharmaceutical industry. Here, we report the extension of competitive ABPP (cABPP) to prodrug-activating enzyme identification in stable isotope-labeled cell lysates using a modified fluorophosphonate probe. Focusing on the antiviral ester prodrug valacyclovir (VACV), we identified serine hydrolase RBBP9 as an activating enzyme in Caco-2 cells via shotgun proteomics, validating the activity via the selective inhibitor emetine (EME). Kinetic characterization of RBBP9 revealed a catalytic efficiency (kcat·KM-1 = 104 mM-1·s-1) comparable to that of BPHL, the only known VACV-activating enzyme prior to this work. EME incubation in wild-type and Bphl-knockout jejunum and liver lysates demonstrated the near-exclusivity of VACV activation by RBBP9 in the intestine. Additionally, these studies showed that RBBP9 and BPHL are the two major and coequal VACV-activating enzymes in the liver. Single-pass intestinal perfusions of VACV ± EME in mice showed EME coperfusion significantly inhibited the intestinal activation of VACV, implying the in vivo relevance of RBBP9-mediated VACV activation. We envision that others might use the cABPP approach in the future for global, rapid, and efficient discovery of prodrug-activating enzymes.