Optimization of a PEGylated Glucuronide-Monomethylauristatin E Linker for Antibody–Drug Conjugates

Abstract

The emergence of antibody-drug conjugates (ADC), such as brentuximab vedotin and ado-trastuzumab emtansine, has led to increased efforts to identify new payloads and develop improved drug-linker technologies. Most antibody payloads impart significant hydrophobicity to the ADC, resulting in accelerated plasma clearance and suboptimal in vivo activity, particularly for conjugates with high drug-to-antibody ratios (DAR). We recently reported on the incorporation of a discrete PEG 24 polymer as a side chain in a b-glucuronidase-cleavable monomethylauristatin E (MMAE) linker to provide homogeneous DAR 8 conjugates with decreased plasma clearance and increased antitumor activity in xenograft models relative to a non-PEGylated control. In this work, we optimized the drug-linker by minimizing the size of the PEG side chain and incorporating a self-stabilizing maleimide to prevent payload de-conjugation in vivo. Multiple PEG-glucuronide-MMAE linkers were prepared with PEG size up to 24 ethylene oxide units, and homogeneous DAR 8 ADCs were evaluated. A clear relationship was observed between PEG length and conjugate pharmacology when tested in vivo. Longer PEG chains resulted in slower clearance, with a threshold length of PEG 8 beyond which clearance was not impacted. Conjugates bearing PEG of sufficient length to minimize plasma clearance provided a wider therapeutic window relative to faster clearing conjugates bearing shorter PEGs. A lead PEGylated glucuronide-MMAE linker was identified incorporating a self-stabilizing maleimide and a PEG 12 side chain emerged from these efforts, enabling highly potent, homogeneous DAR 8 conjugates and is under consideration for future ADC programs.

N‐Hydroxysuccinimide‐Modified Ethynylphosphonamidates Enable the Synthesis of Configurationally Defined Protein Conjugates

“…[29] This peak was clearly shifted towards shorter retention times for more polar trastuzumab-5-DM1 relative to those of the other two tested linker systems (Figure S8). Because it is known that more hydrophilic ADCs are, in general, less prone to undesired aggregation behavior, [30] we believe that compound 5 is able to address current issues associated with the hydrophobic linker used in trastuzumabemtansine. [31] Finally, we also wanted to apply our phosphonamidate building blocks to a more challenging thiol-containing molecule and aimed for the synthesis of an antibody-protein conjugate.…”

Design and characterization of homogenous antibody-drug conjugates with a drug-to-antibody ratio of one prepared using an engineered antibody and a dual-maleimide pyrrolobenzodiazepine dimer

“…The second PEG 8 linker decreases the overall hydrophobicity of the SG3710 payload, a key parameter that has significant effects on ADC pharmacokinetics, biodistribution, and tumor efficacy. [25][26][27][28][29] In addition, the symmetrical structure of SG3710 expands the utility of dual-linker PBDs and opens the door to developing other ADC warhead classes with a drug-to-antibody ratio (DAR) of one based on a sitespecific disulfide re-bridging.…”

Ethynylphosphonamidates for the Rapid and Cysteine‐Selective Generation of Efficacious Antibody–Drug Conjugates

“…[5] Nevertheless,challenges remain, especially in improving the linkage between drug and antibody. [7] Maleimides have become the prime linker reagents for the generation of ADCs,i ncluding two approved ADCs:t rastuzumab emtansine (Kadcyla) and brentuximab vedotin (Adcetris). [7] Maleimides have become the prime linker reagents for the generation of ADCs,i ncluding two approved ADCs:t rastuzumab emtansine (Kadcyla) and brentuximab vedotin (Adcetris).…”