Since the introduction of Antibody-Drug Conjugates (ADCs) nearly three decades ago, suitable linker technology at the payload attachment site is a proven requirement for safe and efficacious ADCs. These cleavable linkers allow for controlled-release of payloads from the antibody once at the target site. Herein, we report the discovery of a “click”-ready pH-tunable phosphoramidate linker that can be utilized for intracellular endosomal (low pH environment) release of Monomethyl Auristatin E (MMAE), a cytotoxic payload. The choice of the linker was determined from a series of three phosphoramidates coupled to a synthetic MMAE model we will report in this presentation. As shown in the scheme below, the chosen linker has a homoserine moiety and self-immolative amine spacer. Key to the pH-triggered release of the amine from this linker is a proximal carboxylic acid (on the homoserine moiety) that promotes hydrolysis of the phosphoramidate P−N bond, usually via an intramolecular general acid-type mechanism. The released amine undergoes a self-immolative cascade to release the resulting payload. pH-decay of P−N bond was monitored by 31P NMR and payload release was monitored by UV-VIS Spectrophotometry and HPLC. In addition, buffers (pH 4.5 – 7.4) were used to mimic plasma, endosomal and lysosomal pHs. The PhosAm linker showed minimal to no release of payload at pH 7.4, suggesting potential for stability in plasma. Furthermore, t1/2 of the PhosAm linker was 30 minutes, which implies a rapid release of cargo once at the lysosome. We will also demonstrate the diverse utility of this scaffold to release various payloads (paclitaxel, doxorubicin, phenol- and aniline-based dyes). In conclusion, most ADC technologies have found it difficult to incorporate acid-labile cleavable linkers in their scaffold because of the inherent difficulty to strictly discriminate between plasma and endosomal/lysosomal pHs. However, the ability of PhosAm linker to differentiate between plasma and intracellular lysosomal environment hints at a promise of ADCs and Small Molecule Drug Conjugates (SMDCs) that will be potent with higher tumor selectivity compared to the acid-cleavable linkers reported in the past.

Novel "click"-ready pH-triggered phosphoramidate linker for controlled-release of cytotoxic payloads

ACS Fall 2020 Virtual Meeting

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