(1025-B) High-throughput screening of small molecules targeting FoxP3 in regulatory T cells for cancer treatment

Abstract:
FoxP3+ regulatory T cells (Tregs) are a critical subset of CD4+ T cells responsible for regulating immune homeostasis by suppressing excessive anti self-antigen immunity. The suppressive function of FoxP3+ Tregs also facilitates tumor cell proliferation due to inhibition of anti-tumor immunity. Thus, targeting Tregs is a popular therapeutic strategy in drug discovery to block of anti-tumor immune evasion mechanisms. However, most current efforts focus on developing antibodies against surface markers on Tregs which may result in immune-related adverse effects due to overlapping expression profile of these molecules between T cell subsets.

FoxP3 is the key lineage-defining transcription factor in Tregs and regulates expression of genes essential for Treg activation and suppressive functions. It turns out that targeting FoxP3 can be an effective strategy of drug development. We established a high-throughput phenotypic drug screen in primary T cells from healthy donors using flow cytometry-based assays by measuring percentage of FoxP3 in CD4+ T cells. Cells were treated with drugs in 384-well plates, followed by antibody staining using a BioMek automated liquid handling system and high-throughput flow cytometry analysis with the iQue screener, a state-of-art instrument. Having implemented an automated pipeline, we first screened a library of 1522 approved drugs and then successfully expanded to screen a large diversity-oriented library of 28,500 compounds. In addition, as a secondary screen, we also developed biochemical assays using Biacore SPR and AlphaScreen to identify compounds that can directly target FoxP3 or interfere with FoxP3-DNA binding activity.

From screening the library of approved drugs, a few candidates with promising potential to down-regulate FoxP3 were selected for further evaluation. Among these, we identified an anti-malaria drug potently reducing FoxP3 expression. Drug-like analogous compounds were searched by in-silico predictions and screened, revealing SAR information of various chemical groups essential for FoxP3 regulation. Next, the original hit and its more efficient analogues were examined in Treg functional assays, showing inhibition of Treg suppressive function on effector T cells (Teffs) and recovery of Teff activation. Mechanistically, these compounds could selectively inhibit FoxP3+ Tregs, rather than effector T cells, through direct binding to FoxP3 and interfering with FoxP3-DNA binding activity. Furthermore, these compounds demonstrated FoxP3 down-regulation activity and enhanced anti-tumor immunity in vivo in a mouse tumor model. In conclusion, the efficiency of this anti-malaria drug and its analogs on regulation of FoxP3+ Tregs and enhanced immune response was confirmed both in vitro and in vivo. Further investigations will be addressing potential therapeutic anti-tumor applications.