Abstract 5814: EPriL macrocycles as a platform for the rapid generation of effective kinase degrader antibody conjugates (DACs)
Authors: Uitdehaag Joost C. , e Man Jos , Muller Michelle , an Cauter Freek , an Gemert Sander , Hoffmann Milan , an Mil Yvonne G. , Mulder Winfried R. , Prinsen Martine B. , Sterrenburg Jan Gerard , Vu Diep , e Wit Joeri , Ensing Erik , Buijsman Rogier C. 
Published: March 2024

The therapeutic success of antibody drug conjugates (ADCs) drives a continuous search for novel payloads that can increase therapeutic window and thereby widen the applications for ADCs. Recently, heterobifunctional degraders have gained great interest as payloads, and degrader antibody conjugates (DACs) are seen as a novel therapeutic modality. Heterobifunctional degraders consist of a small molecule ligand that binds a target (protein of interest or POI), a spacer and an E3 ligase ligand, which can catalyze target degradation. Owing to their catalytic activity, degraders can have better potency than the equivalent inhibitors, making them suitable as ADC payload [1]. Degrader payloads can target a wider variety of mechanisms than classic payloads, which use the same antitumor strategies as chemotherapy, such as tubulin binding or topoisomerase inhibition.

Since many FDA-approved targeted therapies are based on the inhibition of protein kinases, we investigated degraders of these kinases as DAC payloads. Kinase DACs could bring enhanced targeting and therefore better therapeutic window to a field where classic ADC payloads have often shown substantial toxicities. As many heterobifunctional degraders show poor cell membrane penetration, their inhibitory potential could be increased as part of a DAC, where endocytic uptake is followed by intracellular release of the degrader payload.

To identify kinase degrader payloads, we present a workflow based on a platform called Energetically Privileged Ligands (EPriLs). EPriLs are macrocycle scaffolds that bind non-covalently in the kinase ATP pocket. Their unique binding mode avoids contacts with amino acid positions where resistance to kinase inhibitors frequently occurs. EPriL macrocycles can be decorated appropriately to rationally design specific inhibitors for many therapeutically relevant kinases, and provide synthetic handles to couple them to VHL or CRBN ligands to generate effective kinase degraders.

Here we describe how EPriL kinase degraders can be developed into effective DACs, using consecutive libraries of EPriL ligands, spacers, E3 ligase ligands and linkers. First, suitable degraders are identified, based on rapid and deep target degradation and potent antiproliferative activity on target cell lines. Degraders are then transformed into maleimide-linked degraders using convenient attachment of enzymatically cleavable linkers. In a medium throughput fashion, these maleimides are coupled to antibodies to generate DACs, which are tested for stability and biological potency. Applying this workflow to various well-validated kinase targets in oncology resulted in a promising kinase targeting DAC with favorable ADME properties, clear potentiation compared to the parent degrader, and increased selectivity for tumor cell lines.

1] Dragovich et al., Chem. Soc. Rev. (2022) 51, 3886-3897.

Citation Format: Joost C. Uitdehaag, Jos e Man, Michelle Muller, Freek an Cauter, Sander an Gemert, Milan Hoffmann, Yvonne G. an Mil, Winfried R. Mulder, Martine B. Prinsen, Jan Gerard Sterrenburg, Diep Vu, Joeri e Wit, Erik Ensing, Rogier C. Buijsman. EPriL macrocycles as a platform for the rapid generation of effective kinase degrader antibody conjugates (DACs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5814.