Introduction
Tumour necrosis factor-like ligand 1A (TL1A) and its signalling receptor, death receptor 3 (DR3), have been implicated in the pathogenesis of inflammatory bowel disease (IBD) through effects on both immune and tissue-resident cells.1-5 In TUSCANY-2 (NCT04090411), a double-blind, randomised, phase 2b trial, adults with moderately to severely active ulcerative colitis (UC) treated with the anti-TL1A antibody afimkibart showed a positive benefit-risk profile compared with placebo.6 Here, we assessed cell-specific changes in TL1A pathway gene expression in colonic tissue collected in TUSCANY-2 to define pharmacodynamic effects and provide mechanistic insights into the mode of action of afimkibart at the cellular level.
Aims & Methods
Colonic pinch biopsy samples were measured from patients in the afimkibart 450 mg (n=58) and placebo (n=28) arms of the TUSCANY-2 trial at screening and week 14. Single nucleus RNA sequencing (10X Genomics FLEX snRNAseq) analysis was performed to characterise changes in gene expression, cellular abundance, and pathways across colonic cell populations. A TL1A-responsive CD4 T-cell gene expression signature was used as a pharmacodynamic measure of target engagement.
Results
The snRNAseq dataset captured multiple relevant mucosal cell types. Consistent with prior biology, TL1A was highly expressed in monocytes, macrophages, and fibroblasts; DR3 was expressed in innate lymphoid, T helper 17 (Th17), CD4+, T regulatory, and endothelial cells. The TL1A decoy receptor DcR3 was highly expressed in enterocytes, endothelial cells, and fibroblasts. A TL1A-responsive CD4 T-cell gene expression signature was significantly inhibited (p=0.0000252) in CD4 T-cells from screening to week 14 with afimkibart treatment, but not placebo. In addition, samples from afimkibart-treated patients showed broad transcriptomic changes across multiple cellular lineages, particularly epithelial, myeloid, fibroblast, and T-cell lineages, whereas very few changes in gene expression were observed in the placebo group, indicating that TL1A blockade influences gene expression beyond immune cell populations, including epithelial and stromal compartments. Pathway analysis within CD4 T-cells showed decreases in cellular division post-treatment, consistent with reductions observed in T-cell lineage abundance at week 14 in the afimkibart arm.
Conclusion
This is the first interventional trial in this class to assess TL1A blockade at single-cell resolution in colonic tissue. Afimkibart induced complete target inhibition in the tissue and broad transcriptional changes across immune and tissue-resident mucosal populations, including cell types without prominent DR3 expression. These findings support a mode of action that extends beyond suppression of inflammation to modulation of dysregulated epithelial and stromal/fibrotic tissue programmes after TL1A blockade.
References
1Bamias G, et al. Gut. 2025;74(4):652–668.
2Xu W-D, et al. Front Immunol. 2022;13:891328.
3Pappu BP, et al. J Exp Med. 2008;205(5):1049–1062.
4Wang S, et al. Front Immunol. 2024;15:1340467.
5Solitano V, et al. Med. 2024;5(5):386–400.
6Danese S, et al. Lancet Gastroenterol Hepatol. 2025;10:882-95.
Disclosure
Funded by F. Hoffmann-La Roche, Ltd
