Spop-binding bifunctional degraders: a novel approach for cancer immunotherapy.

[INTRODUCTION] Current PD-L1 degraders, whether antibody-based or small-molecule-mediated, are hindered by limitations in pharmacokinetics (e.g., poor tissue penetration) or pharmacodynamics (e.g., suboptimal degradation efficacy, immunogenicity concerns). These drawbacks highlight the necessity for novel PD-L1 degradation platforms using innovative technologies.

[OBJECTIVES] This study aims to design and synthesize bifunctional small molecules as PD-L1 degraders by leveraging the unexplored E3 ligase SPOP, aiming to overcome the limitations of existing degraders and evaluate their potential in cancer immunotherapy.

[METHODS] A series of SPOP-based bifunctional small molecules were designed and synthesized. Their PD-L1 inhibitory and degradation activities were assessed using HTRF and western blot assays, respectively. Mechanistic studies (His pull-down, bio-layer interferometry, western blot) were performed to verify ternary complex formation with PD-L1 and SPOP. In vivo pharmacokinetic properties and antitumor efficacy were evaluated in a B16-F10 tumor model, with analysis of tumor-infiltrating lymphocytes (TILs) to explore immune microenvironment effects.

[RESULTS] Compound SPOP9 exhibited potent PD-L1 inhibition (IC = 357.2 nM) and degradation (DC = 1.0 μM). Mechanistic studies confirmed its assembly into a stable ternary complex with PD-L1 and SPOP. SPOP9 showed favorable in vivo bioavailability (F = 74.8 %) and, at 10 mg/kg (i.p.), reduced tumor weight by 44 % in B16-F10 mice, superior to anti-PD-L1 antibody (TGI = 34.4 %). TIL analysis indicated SPOP9 activated the tumor immune microenvironment and downregulated PD-L1.

[CONCLUSION] SPOP9, as the first SPOP-binding bifunctional PD-L1 degrader, demonstrates promising preclinical efficacy and pharmacokinetic properties, addressing key limitations of existing degraders. It merits further investigation as a potential agent for cancer immunotherapy.