Facile Gram-Scale Solution Phase Synthesis of a Universal Cathepsin B-Cleavable Antibody-Drug Conjugate Linker

Antibody drug conjugates (ADCs) are a key therapy in the cancer treatment arsenal. Critical to the success of the ADC is the release of the drug cargo at the desired site, and thus, enzymatically or chemically responsive linkers, connecting the drug to the antibody, play a central role. Linkers containing the peptide sequence Glu-Val-Cit are particularly important as cleavage of this peptide occurs by both murine and human cathepsin B enzymes. Additional linker design considerations include components for free drug release via the self-immolative moiety para-aminobenzyl alcohol (PABOH), copper-free click conjugation to an azide-functionalized antibody using dibenzocyclooctyne (DBCO), and aqueous solubility via a polyethylene glycol spacer (PEG). To facilitate widespread use and translation, we describe the 9-step gram-scale synthesis of the linker DBCO-PEG4-Glu-Val-Cit-PABOH conjugated to monomethyl auristatin E (MMAE). Using only liquid-phase synthesis protocols and chromatography-free, precipitation-based intermediate isolations gives the drug-linker conjugate in 36% overall yield, and this method is an improvement over prior methods (yield < 1%). Finally, we prepare the corresponding MMAE-linked ADC and validate its target-dependent potency across ErbB2/Her2-high (MDA-MB-453) and ErbB2/Her2-low (MDA-MB-231) human breast cancer cell lines.