Small-molecule inhibitors of PD-L1 are postulated to control immune evasion in tumors similar to antibodies that target the PD-L1/PD-1 immune checkpoint axis. However, the identity of targetable PD-L1 inducers is required to develop small-molecule PD-L1 inhibitors. In this study, using chromatin immunoprecipitation (ChIP) assay and siRNA, we demonstrate that vitamin D/VDR regulates PD-L1 expression in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) cells. We have examined whether a VDR antagonist, MeTC7, can inhibit PD-L1. To ensure that MeTC7 inhibits VDR/PD-L1 without off-target effects, we examined competitive inhibition of VDR by MeTC7, utilizing ligand-dependent dimerization of VDR-RXR, RXR-RXR, and VDR-coactivators in a mammalian 2-hybrid (M2H) assay. MeTC7 inhibits VDR selectively, suppresses PD-L1 expression sparing PD-L2, and inhibits the cell viability, clonogenicity, and xenograft growth of AML cells. MeTC7 blocks AML/mesenchymal stem cells (MSCs) adhesion and increases the efferocytotic efficiency of THP-1 AML cells. Additionally, utilizing a syngeneic colorectal cancer model in which VDR/PD-L1 co-upregulation occurs in vivo under radiation therapy (RT), MeTC7 inhibits PD-L1 and enhances intra-tumoral CD8+T cells expressing lymphoid activation antigen-CD69. Taken together, MeTC7 is a promising small-molecule inhibitor of PD-L1 with clinical potential.
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Design of a protease‐activated PD‐L1 inhibitor
Immune checkpoint inhibitors that bind to the cell surface receptor PD‐L1 are effective anti‐cancer agents but suffer from immune‐related adverse events as PD‐L1 is expressed on both healthy and cancer cells. To mitigate toxicity, researchers are testing prodrugs that have low affinity for checkpoint targets until activated with proteases enriched in the tumor microenvironment. Here, we engineer a prodrug form of a PD‐L1 inhibitor. The inhibitor is a soluble PD‐1 mimetic that was previously engineered to have high affinity for PD‐L1. In the basal state, the binding surface of the PD‐1 mimetic is masked by fusing it to a soluble variant of its natural ligand, PD‐L1. Proteolytic cleavage of the linker that connects the mask to the inhibitor activates the molecule. To optimize the mask so that it effectively blocks binding to PD‐L1 but releases upon cleavage, we tested a set of mutants with varied affinity for the inhibitor. The top‐performing mask reduces the affinity of the prodrug for PD‐L1 120‐fold, and binding is nearly fully recovered upon cleavage. In a cell‐based assay measuring inhibition of the PD‐1:PD‐L1 interaction on the surface of cells, the IC50s of the masked inhibitors were up to 40‐fold higher than their protease‐treated counterparts. The changes in activity we observe upon protease treatment are comparable to systems currently tested in the clinic and provide evidence that natural binding partners are an excellent starting point for creating a prodrug.
more » « less- NSF-PAR ID:
- 10397068
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Protein Science
- Volume:
- 32
- Issue:
- 3
- ISSN:
- 0961-8368
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Checkpoint blockade immunotherapies harness the host's own immune system to fight cancer, but only work against tumors infiltrated by swarms of preexisting T cells. Unfortunately, most cancers to date are immune‐deserted. Here, a polymer‐assisted combination of immunogenic chemotherapy and PD‐L1 degradation is reported for efficacious treatment in originally nonimmunogenic cancer. “Priming” tumors with backbone‐degradable polymer‐epirubicin conjugates elicits immunogenic cell death and fosters tumor‐specific CD8+ T cell response. Sequential treatment with a multivalent polymer‐peptide antagonist to PD‐L1 overcomes adaptive PD‐L1 enrichment following chemotherapy, biases the recycling of PD‐L1 to lysosome degradation via surface receptor crosslinking, and produces prolonged elimination of PD‐L1 rather than the transient blocking afforded by standard anti‐PD‐L1 antibodies. Together, these findings establish the polymer‐facilitated tumor targeting of immunogenic drugs and surface crosslinking of PD‐L1 as a potential new therapeutic strategy to propagate long‐term antitumor immunity, which might broaden the application of immunotherapy to immunosuppressive cancers.
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