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.
The cytokine interleukin (IL)‐11 has been shown to play a role in promoting fibrosis and cancer, including lung adenocarcinoma, garnering interest as an attractive target for therapeutic intervention. We used combinatorial methods to engineer an IL‐11 variant that binds with higher affinity to the IL‐11 receptor and stimulates enhanced receptor‐mediated cell signaling. Introduction of two additional point mutations ablates IL‐11 ligand/receptor association with the gp130 coreceptor signaling complex, resulting in a high‐affinity receptor antagonist. Unlike wild‐type IL‐11, this engineered variant potently blocks IL‐11‐mediated cell signaling and slows tumor growth in a mouse model of lung cancer. Our approach highlights a strategy where native ligands can be engineered and exploited to create potent receptor antagonists.
more » « less- NSF-PAR ID:
- 10474614
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Bioengineering & Translational Medicine
- Volume:
- 8
- Issue:
- 6
- ISSN:
- 2380-6761
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Background Alternative RNA splicing is widely dysregulated in cancers including lung adenocarcinoma, where aberrant splicing events are frequently caused by somatic splice site mutations or somatic mutations of splicing factor genes. However, the majority of mis-splicing in cancers is unexplained by these known mechanisms. We hypothesize that the aberrant Ras signaling characteristic of lung cancers plays a role in promoting the alternative splicing observed in tumors.
Methods We recently performed transcriptome and proteome profiling of human lung epithelial cells ectopically expressing oncogenic KRAS and another cancer-associated Ras GTPase, RIT1. Unbiased analysis of phosphoproteome data identified altered splicing factor phosphorylation in KRAS-mutant cells, so we performed differential alternative splicing analysis using rMATS to identify significantly altered isoforms in lung epithelial cells. To determine whether these isoforms were uniquely regulated by KRAS, we performed a large-scale splicing screen in which we generated over 300 unique RNA sequencing profiles of isogenic A549 lung adenocarcinoma cells ectopically expressing 75 different wild-type or variant alleles across 28 genes implicated in lung cancer.
Results Mass spectrometry data showed widespread downregulation of splicing factor phosphorylation in lung epithelial cells expressing mutant KRAS compared to cells expressing wild-type KRAS. We observed alternative splicing in the same cells, with 2196 and 2416 skipped exon events in KRASG12Vand KRASQ61Hcells, respectively, 997 of which were shared (
p < 0.001 by hypergeometric test). In the high-throughput splicing screen, mutant KRAS induced the greatest number of differential alternative splicing events, second only to the RNA binding protein RBM45 and its variant RBM45M126I. We identified ten high confidence cassette exon events across multiple KRAS variants and cell lines. These included differential splicing of the Myc Associated Zinc Finger (MAZ). As MAZ regulates expression of KRAS, this splice variant may be a mechanism for the cell to modulate wild-type KRAS levels in the presence of oncogenic KRAS.Conclusion Proteomic and transcriptomic profiling of lung epithelial cells uncovered splicing factor phosphorylation and mRNA splicing events regulated by oncogenic KRAS. These data suggest that in addition to widespread transcriptional changes, the Ras signaling pathway can promote post-transcriptional splicing changes that may contribute to oncogenic processes.
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