TGFβ Blockade by Inhibition of Enolase-1-Mediated Plasmin Targets Tumor-Associated Macrophages in Pancreatic Ductal Adenocarcinoma.

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an immunosuppressive and metabolically rewired tumor microenvironment (TME). Although α-enolase (ENO1) is frequently overexpressed in PDAC and associated with poor prognosis, its functional role in TME remodeling remains unclear. This study investigated the role of ENO1 in plasmin-dependent transforming growth factor β (TGFβ) activation and metabolic adaptation in PDAC and evaluated the therapeutic potential of HuL001, a first-in-class humanized anti-ENO1 monoclonal antibody. Methods: ENO1 expression and clinical relevance were evaluated in PDAC tissues by immunohistochemistry. Mechanistic studies were performed using PDAC-monocyte co-culture systems, reverse transcription-quantitative PCR (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), flow cytometry, and cell viability assays. The therapeutic effects of HuL001 were further assessed in multiple PDAC xenograft models. Results: Surface ENO1 expression was elevated in advanced PDAC and was associated with poorer overall survival. HuL001 suppressed monocyte-driven PDAC proliferation and reduced factors associated with M2-like macrophages (alternatively activated macrophages), including TGFB1, IL10, and VEGFA. Mechanistically, HuL001 inhibited ENO1-dependent plasmin-mediated activation of latent TGFβ and attenuated TGFβ-induced ENO1 expression, plasmin activity, hexokinase 2 (HK2) expression, and lactate production. In vivo, HuL001 inhibited PDAC growth and enhanced the antitumor efficacy of gemcitabine in three xenograft models, with greater suppression of tumor growth, intratumoral lactate, and active TGFβ than gemcitabine alone. Conclusion: These findings identify ENO1 as a functional driver of plasmin-dependent TGFβ activation and metabolic adaptation in PDAC and support HuL001 as a promising therapeutic strategy, particularly in combination with gemcitabine.