Self-Immolative Carbamate Linkers for CD19-Budesonide Antibody–Drug Conjugates

Abstract

Antibody–drug conjugates consist of potent small-molecule payloads linked to a targeting antibody. Payloads must possess a viable functional group by which a linker for conjugation can be attached. Linker-attachment options remain limited for the connection to payloads via hydroxyl groups. A releasing group based on 2-aminopyridine was developed to enable stable attachment of para-aminobenzyl carbamate (PABC) linkers to the C21-hydroxyl group of budesonide, a glucocorticoid receptor agonist. Payload release involves a cascade of two self-immolative events that are initiated by the protease-mediated cleavage of the dipeptide–PABC bond. Budesonide release rates were determined for a series of payload–linker intermediates in buffered solution at pH 7.4 and 5.4, leading to the identification of 2-aminopyridine as the preferred releasing group. Addition of a poly­(ethylene glycol) group improved linker hydrophilicity, thereby providing CD19-budesonide ADCs with suitable properties. ADC23 demonstrated targeted delivery of budesonide to CD19-expressing cells and inhibited B-cell activation in mice.

The medicinal chemistry evolution of antibody–drug conjugates

“…Payload release is a 3-step process with a) protease cleavage of the dipeptide, b) self-immolation of the PABC group, c) self-immolation of the ABA group to afford the payload and the cyclised ABA, 1-methyl-3,4-dihydroquinazolin-2(1 H )-one (Scheme 1). 15…”

“…3) had the desired effect of lowering the levels of aggregation for ADCs on an anti-CD19 antibody though they were still considered too high at around 10%. 15 As a result our focus moved to designing a GRM for the specific purpose of an ADC payload.…”

“…Payload release is a 3-step process with a) protease cleavage of the dipeptide, b) self-immolation of the PABC group, c) self-immolation of the ABA group to afford the payload and the cyclised ABA, 1-methyl-3,4dihydroquinazolin-2(1H)-one (Scheme 1). 15 Drug-linkers with the PABC-ABA dual self-immolative group were conjugated to an anti-CD19 antibody and profiled for both potency and stability. Using a K562 CD19 GRE assay efficient release of payload was shown while in both PBS buffer and plasma there was no change observed by mass spectrometry over 7 days.…”