CD64+ fibroblast-targeted vilanterol and a STING agonist augment CLDN18.2 BiTEs efficacy against pancreatic cancer by reducing desmoplasia and enriching stem-like CD8+ T cells

Objective The objective of this study is to improve the efficacy of CLDN18.2/CD3 bispecific T-cell engagers (BiTEs) as a promising immunotherapy against pancreatic ductal adenocarcinoma (PDAC). Design Humanised hCD34 + /hCD3e + , Trp53 R172H Kras G12D Pdx1-Cre (KPC), pancreas-specific Cldn18.2 knockout (KO), fibroblast-specific Fcgr1 KO and patient-derived xenograft/organoid mouse models were constructed. Flow cytometry, Masson staining, Cell Titer Glo assay, virtual drug screening, molecular docking and chromatin immunoprecipitation were conducted. Results CLDN18.2 BiTEs effectively inhibited early tumour growth, but late-stage efficacy was significantly diminished. Mechanically, the Fc fragment of BiTEs interacted with CD64 + cancer-associated fibroblasts (CAFs) via activation of the SYK-VAV2-RhoA-ROCK-MLC2-MRTF-A-α-SMA/collagen-I pathway, which enhanced desmoplasia and limited late-stage infiltration of T cells. Molecular docking analysis found that vilanterol suppressed BiTEs-induced phosphorylation of VAV2 (Y172) in CD64 + CAFs and weakened desmoplasia. Additionally, decreased cyclic guanosine-adenosine monophosphate synthase/stimulator of interferon genes (STING) activity reduced proliferation of TCF-1 + PD-1 + stem-like CD8 + T cells, which limited late-stage effects of BiTEs. Finally, vilanterol and the STING agonist synergistically boosted the efficacy of BiTEs by inhibiting the activation of CD64 + CAFs and enriching proliferation of stem-like CD8 + T cells, resulting in sustained anti-tumour activity. Conclusion Vilanterol plus the STING agonist sensitised PDAC to CLDN18.2 BiTEs and augmented efficacy as a potential novel strategy.