Abstract

Background: Selgantolimod (SLGN) is an oral selective small molecule TLR8 agonist with antiviral potential that has been shown to be safe and well-tolerated in individuals with chronic hepatitis B (CHB). SLGN stimulates a robust pharmacodynamic response as measured by the detection of TLR8 pathway cytokines such as interleukin (IL)-12p40 and IL-1RA. We further characterized the molecular impact of SLGN treatment in peripheral blood mononuclear cells (PBMCs) using single cell profiling technologies.

Methods: Viremic chronic hepatitis B (CHB) patients received once weekly 3 mg SLGN for 24 weeks. Blood samples were collected before and 4 hours post dosing at baseline, week 11 and week 23 for four participants. PBMCs were isolated and gene expression was evaluated using the 10X single cell RNA-seq protocol. Analysis was performed using the Seurat R package with cell type identification performed with SingleR. Differences in gene expression between baseline and on- treatment timepoints were determined by a non-parametric Wilcoxon rank sum text.

Results: Seventeen cell clusters were identified by single cell gene expression analysis. Pathway-level analysis using blood transcriptional modules (BTM) indicated type I interferon modulation at on treatment time-points across multiple cell types (FDR <0.01). Particularly, interferon stimulated genes, such as IFI144L, STAT1, and MX1 were upregulated after SLGN treatment in several clusters, including those annotated as B, T, and NK cells. Furthermore, we observed modulation of TLR and inflammatory signaling associated with classical and non-classical monocytes. Of the many differentially expressed genes (DEGs) identified in the classical monocyte cluster (FDR <0.05), MECP2 and FKBP5, among others, were upregulated at all 4 hr postdose timepoints. Their gene products have been reported to act as regulator of chromatin structure and gene expression in immune cells (MECP2), and to be involved in the downregulation of excess interferon responses (FKBP5). Further, in the non-classical monocyte cluster, we observed DEGs at the same on-treatment timepoints, with overall lower fold change differences as compared to classical monocytes (mean log2 fold change 0.66 compared to 0.49). Of interest, different kinetics for DEGs were observed for naïve T cells in comparison to baseline. While several hundred DEGs were observed at 4hr post first administration of SLGN in the naïve T-cell cluster, including STAT3 and NFkB pathway-related genes, subsequent on-treatment timepoints demonstrated overall fewer DEGs.

Conclusion: Single cell PBMC analysis from SLGN-treated CHB viremic patients identified substantive changes in gene expression in multiple cell types. These findings provide greater insight into the cellular changes following SLGN treatment, and may inform complementary mechanisms for a HBV cure combination strategy.