Quantitative Conjugated Payload Measurement Using Enzymatic Release of Antibody–Drug Conjugate with Cleavable Linker

Abstract

As antibody-drug conjugate (ADC) design is evolving with novel payload, linker, and conjugation chemistry, the need for sensitive and precise quantitative measurement of conjugated payload to support pharmacokinetics (PK) is in high demand. Compared to ADCs containing noncleavable linkers, a strategy specific to linkers which are liable to pH, chemical reduction, or enzymatic cleavage has gained popularity in recent years. One bioanalytical approach to take advantage of this type of linker design is the development of a PK assay measuring released conjugated payload. For the ADC utilizing a dipeptide ValCit linker studied in this report, the release of payload PF-06380101 was achieved with high efficiency using a purified cathepsin B enzyme. The subsequent liquid chromatography mass spectrometry (LC/MS) quantitation leads to the PK profile of the conjugated payload. For this particular linker using a maleimide-based conjugation chemistry, one potential route of payload loss would result in an albumin adduct of the linker-payload. While this adduct's formation has been previously reported, here, for the first time, we have shown that payload from a source other than ADC contributes only up to 4% of total conjugated payload while it accounts for approximately 35% of payload lost from the ADC at 48 h after dosing to rats.

Recent Developments in ADC Technology: Preclinical Studies Signal Future Clinical Trends

“…When a maleimide-conjugated vc-MMAE-bearing ADC is dosed in vivo, the retro-Michael reaction can result in formation of an albumin-vc-MMAE conjugate. This phenomenon had been observed previously [ 20 , 21 ], but a group from Pfizer quantified it for the first time in 2017 [ 22 ]. At 48 h post-dose, albumin-vc-MMAE represented 4% of total circulating payload and ~ 35% of all MMAE that was deconjugated from the antibody [ 22 ].…”

“…This phenomenon had been observed previously [ 20 , 21 ], but a group from Pfizer quantified it for the first time in 2017 [ 22 ]. At 48 h post-dose, albumin-vc-MMAE represented 4% of total circulating payload and ~ 35% of all MMAE that was deconjugated from the antibody [ 22 ]. Slowing or stopping this linker/payload transfer to albumin represents a good opportunity for mitigating off-target toxicities, as the distribution and disposition profiles of albumin conjugates will likely differ from those of antibody conjugates.…”

Challenges and new frontiers in analytical characterization of antibody-drug conjugates

“…54 Based on a similar concept, a novel approach using cathepsin B enzyme has been reported for the quantification of DAR for random lysine-conjugated ADCs. 55,56 This method is applicable to either random or sitespecific ADCs conjugated using hydrolytically stable cathepsin-B cleavable linker. 55 It involves a series of treatments with enzymes and reductant to allow complete release of the conjugated drugs followed by chromatographic separation.…”

Quantification of an Antibody-Conjugated Drug in Fat Plasma by an Affinity Capture LC-MS/MS Method for a Novel Prenyl Transferase-Mediated Site-Specific Antibody–Drug Conjugate

“…Several bioanalytical approaches for the development of ADCs have been discussed by many researchers [10][11][12]. The bioanalysis of ADCs has many challenges with absorption, distribution, metabolism, and elimination (ADME) due to their complexity derived from three distinct components-the cytotoxic chemical drug, the linker, and the antibody [13]. There are several major analytes that can be used for the evaluation of ADCs' pharmacokinetic (PK) profile, including free drug and its metabolites, antibody-conjugated drug (acDrug), total antibody (tAb), drug-conjugated antibody, and naked antibody [12].…”