Discovery of Potent and Selective Antibody–Drug Conjugates with Eg5 Inhibitors through Linker and Payload Optimization

Abstract

Targeted antimitotic agents are a promising class of anticancer therapies. Herein, we describe the development of a potent and selective antimitotic Eg5 inhibitor based antibody–drug conjugate (ADC). Preliminary studies were performed using proprietary Eg5 inhibitors which were conjugated onto a HER2-targeting antibody using maleimido caproyl valine-citrulline para-amino benzocarbamate, or MC-VC-PABC cleavable linker. However, the resulting ADCs lacked antigen-specificity in vivo, probably from premature release of the payload. Second-generation ADCs were then developed, using noncleavable linkers, and the resulting conjugates (ADC-4 and ADC-10) led to in vivo efficacy in an HER-2 expressing (SK-OV-3ip) mouse xenograft model while ADC-11 led to in vivo efficacy in an anti-c-KIT (NCI-H526) mouse xenograft model in a target-dependent manner.

The Chemistry Behind ADCs

“…In a similar approach, researchers at Novartis started from imidazole containing KSP inhibitors ( Figure 18 ) as starting points in their Eg5 ADC effort. Using the primary alcohol or the secondary amide moieties they installed non cleavable linkers with a maleimide end group [ 32 ]. When coupled with antibodies targeting HER2 and c-KIT the resulting ADCs demonstrated superior in vivo efficacy compared to ado-trastuzumab emtansine (Kadcyla, 3 ).…”

Tailored Linker Chemistries for the Efficient and Selective Activation of ADCs with KSPi Payloads

“…To expand the spectrum of therapeutic options, we recently described the utilization of a highly potent pyrrole subclass of kinesin spindle protein inhibitors (KSPis) as a versatile payload class for ADCs with a novel mode of action . However, highly potent and selective ADCs with KSPi payloads could only be obtained with noncleavable linker chemistries so far. , Currently, ADCs with cleavable linkers are in most cases designed for a proteolytic cleavage by the lysosomal protease cathepsin B . For an efficient drug release, a lipophilic self-immolating spacer unit is required at the cleavage site, resulting in the formation of chinoid entities, which is often associated with aggregation .…”

“…However, highly potent and selective ADCs with KSPi payloads could only be obtained with noncleavable linker chemistries so far. , Currently, ADCs with cleavable linkers are in most cases designed for a proteolytic cleavage by the lysosomal protease cathepsin B . For an efficient drug release, a lipophilic self-immolating spacer unit is required at the cleavage site, resulting in the formation of chinoid entities, which is often associated with aggregation . Furthermore, peptide sequences employed in the linker have shown only moderate specificity for cathepsin B, and premature linker cleavage, e.g., by neutrophil elastase, was observed and has been associated with side effects such as neutropenia in the clinic .…”

“…13 However, highly potent and selective ADCs with KSPi payloads could only be obtained with noncleavable linker chemistries so far. 13,14 Currently, ADCs with cleavable linkers are in most cases designed for a proteolytic cleavage by the lysosomal protease cathepsin B. 15 For an efficient drug release, a lipophilic selfimmolating spacer unit is required at the cleavage site, resulting in the formation of chinoid entities, which is often associated with aggregation.…”

“…15 For an efficient drug release, a lipophilic selfimmolating spacer unit is required at the cleavage site, resulting in the formation of chinoid entities, which is often associated with aggregation. 14 Furthermore, peptide sequences employed in the linker have shown only moderate specificity for cathepsin B, and premature linker cleavage, e.g., by neutrophil elastase, was observed and has been associated with side effects such as neutropenia in the clinic. 16 Taken together, there is an urgent need for novel cleavable linkers with improved tumor specificity.…”