Abstract 4483: Novel small-molecule antagonists of the PD-1/PD-L1 axis that mediate cell surface PD-L1 dimerization and internalization
Authors: Liu Phillip C.C. , Wynn Richard , Wu Liangxing , Volgina Alla , Zolotarjova Nina , Lin Luping , Thekkat Pramod , Margulis Alex , Klabe Ronald , Yao Wenqing , Xiao Kaijiong , Li Jingwei , He Xin , Rupar Mark , Chang Hong , Waeltz Paul , Li Yanlong , Scherle Peggy , Huber Reid , Hollis Gregory 
Published: August 2019

Blocking the PD-(L)1 immune checkpoint axis with therapeutic antibodies against either the receptor or the ligand has proven to be an effective treatment modality for multiple cancer histologies. We describe the identification and characterization of novel small molecule antagonists of the PD-(L)1 axis that function by inducing dimerization and subsequent internalization of the PD-L1 protein, effectively depleting the ligand from the cell membrane and preventing PD-1 activation on T cells. Compound-dependent PD-L1 dimerization was characterized using several biophysical techniques including fluorescence resonance energy transfer (FRET) measurements, size exclusion chromatography and thermal shift analysis. Experimental evidence demonstrates compound-dependent dimer conformation with slow dissociation kinetics and significantly enhanced thermal stability. Many of the PD-L1-directed small molecules blocked binding of soluble PD-1 to either native PD-L1 expressed on cancer cell lines or PD-L1 expressed in CHO cells with low nanomolar potency. However, only a subset of the small molecules caused loss of cell surface PD-L1 in a time- and concentration-dependent manner. Importantly, there was a strict correlation between the promotion of PD-L1 internalization secondary to dimerization and the induction of an NFAT response element-luciferase reporter gene. Strikingly, only those small molecules that could produce a specific dimeric PD-L1 conformation as measured using FRET were associated with functional activity in cells, suggesting that PD-L1 dimerization was necessary but not sufficient for internalization and cellular activity. A cell-active tool compound (cell binding IC50 &lt;5 nM, internalization EC50 &lt;10 nM) was fluorescently labeled to enable direct visualization of intracellular trafficking. Confocal microscopy with this PD-L1 antagonist showed time-dependent increases in intracellular fluorescence in PD-L1 expressing, but not PD-L1 deleted, cells. The internalized antagonist showed punctate staining coincident with markers of the early endosome, and independent studies confirmed that the internalized PD-L1 also trafficked to the early endosome. By disrupting the suppressive activity of PD-L1 on PD-1, these inhibitors result in functional activation of T cells in ex vivo cellular assays in a manner equivalent to antibodies directed against either PD-1 or PD-L1. In summary, we have identified a series of potent, small molecule PD-L1 antagonists that induce dimerization of the protein; inhibitors that trigger an appropriate dimeric conformation can also induce PD-L1 internalization thereby alleviating PD-L1-induced suppression of T cell activation.

Citation Format: Phillip C.C. Liu, Richard Wynn, Liangxing Wu, Alla Volgina, Nina Zolotarjova, Luping Lin, Pramod Thekkat, Alex Margulis, Ronald Klabe, Wenqing Yao, Kaijiong Xiao, Jingwei Li, Xin He, Mark Rupar, Hong Chang, Paul Waeltz, Yanlong Li, Peggy Scherle, Reid Huber, Gregory Hollis. Novel small-molecule antagonists of the PD-1/PD-L1 axis that mediate cell surface PD-L1 dimerization and internalization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4483.