Cholangiocarcinoma (CCA) is an adenocarcinoma of the hepatobiliary system that has recently risen in incidence and mortality with unsatisfactory treatment options. Claudin-1 (CLDN1) is a transmembrane protein expressed in tight junctions and exposed on the cell surface in liver fibrosis and cancer. Using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics of tissues of patients with CCA, we show that CLDN1 expression is up-regulated in cancer cells and is associated with stemness and cell fate. Genetic gain-of-function studies in CCA orthotopic in vivo mouse models showed decreased survival and enhanced tumor growth, unraveling a functional role of CLDN1 as an oncogenic driver. Targeting exposed nonjunctional CLDN1 using highly specific CLDN1 monoclonal antibodies (mAbs) inhibited tumor growth across CCA CDX and PDX mouse models and patient-derived CCA organoids, including tumors with medium or low CLDN1 expression. Moreover, antibody treatment inhibited tumor cell migration, invasion, and extrahepatic metastasis. Mechanistically, targeting exposed cell surface CLDN1 on CCA tumors using mAbs inhibited Notch1 and TROP2/STAT3 signaling pathways, resulting in decreased cancer cell stemness and epithelial-to-mesenchymal transition. Loss-of-function studies using CRISPR-Cas9 and RNAi combined with rescue and confocal imaging studies confirmed the functional and mechanistic role of these pathways. In conclusion, these results uncover CLDN1 as a previously undiscovered CCA driver and therapeutic target, paving the way for the clinical development of CLDN1 mAbs to improve the dismal outcome of patients with advanced CCA.

Abstract Introduction: Cholangiocarcinoma (CCA) is a highly aggressive adenocarcinoma of the hepatobiliary system showing an alarming rise in incidence and mortality with unsatisfactory treatment options. Claudin-1 (CLDN1) is a transmembrane protein expressed in tight junctions, but exposed at the cell surface on cancer epithelial cells. Using highly specific monoclonal antibodies (mAbs) targeting the extracellular loop 1 of exposed CLDN1 with an excellent safety profile (Roehlen, Saviano et al. Science Transl Med 2022), we aimed to investigate the role of CLDN1 as therapeutic target for CCA. Methods: Integrative CCA patient CLDN1 expression analyses, spatial transcriptomics and mouse models were used to evaluate the role of CLDN1 as an oncogenic driver for CCA. Proof-of-concept studies were performed in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse as well as patient CCA organoid models using humanized CLDN1 mAbs. Results: Integrative expression analyses of CLDN1 in CCA patient tissues revealed robust CLDN1 upregulation across several cohorts and patients with well-characterized driver mutations. scRNASeq and spatial transcriptomics of patient CCA showed that CLDN1 expression in cancer cells is associated with stemness, oncogenic signaling and EMT. Gain-of-function studies using an orthotopic HDTVi and syngeneic mouse model revealed a decrease in survival and an enhanced tumor growth, unraveling a functional role of CLDN1 as an oncogenic driver in CCA. Targeting exposed CLDN1 using highly CLDN1-specific mAb demonstrated a robust anti-tumoral effect across intra- and extra-hepatic CCA mouse models, with a significant inhibition of metastatic disease including models with medium or low CLDN1 expression. Functional studies in patient-derived CCA organoids demonstrated that CLDN1 mAb decreased cellular viability and altered cancer cell plasticity and fate. Mechanistically, CLDN1 mAb treatment suppressed gene expression of pathways mediating proliferation, stemness and EMT by inhibition of Notch1, SRC-FAK, and Hippo-YAP signaling. Conclusion: These results demonstrate that CLDN1 is a CCA driver and therapeutic target. The proof-of-concept studies in patient-derived models pave the way for the clinical development of CLDN1 mAbs to improve the outcomes of patients with advanced CCA. Citation Format: Zeina Nehme, Marion Muller, Emilie Crouchet, Frank Juehling, Julien Moehlin, Romain Désert, Jade Brochon, Fabio Del Zompo, Natascha Roehlen, Christine Thumann, Patrick Pessaux, Emanuele Felli, Aïna Venkatasamy, Patrice Marchand, Mihaela Alina Onea, Roberto Iacone, Markus Meyer, Alberto Toso, Nabeel Bardeesy, Lipika Goyal, Vikas Prakash Ranvir, Mirian Fernández-Vaquero, Mathias Heikenwälder, Tessa Ostyn, Tania Roskams, Patrice Laquerriere, Catherine Schuster, Laurent Mailly, Thomas F. Baumert. Treatment of cholangiocarcinoma using humanized monoclonal antibodies targeting claudin-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5981.

Background and Aims: Claudin-1 (CLDN1), a transmembrane protein involved in epithelial tight junctions, can, in idiopathic pulmonary fibrosis (IPF), be exposed outside the tight junctions of proliferative airway epithelial cells. ALE.F02 is, a first-in-class monoclonal antibody (mAb) highly specific in targeting exposed CLDN1, currently in development for the treatment of liver and kidney fibrosis. By blocking CLDN1 on injured lung epithelial cells, ALE.F02 inhibits pro-fibrotic pathways and modulates extracellular matrix remodelling. This study investigated the functional role of CLDN1 as a therapeutic target for IPF. Methods: CLDN1 expression in lung tissues from patients with different IPF stages was analyzed using immunohistochemistry. Using preclinical animal models of IPF and precision-cut lung slices (PCLS) generated from IPF lung explants, we investigated the individual and combined effects of ALE.F02 and Nintedanib or Pirfenidone on parameters related to the pathogenesis of IPF. Results: In tissues of IPF patients, exposed CLDN1 increased during disease progression in the bronchiolar and the alveolar epithelia and around fibrotic areas. Preclinical studies in aged bleomycin mouse model showed that therapeutic treatment with anti-CLDN1 mAb reduced fibrosis and improved lung function. Treatment of IPF PCLS with ALE.F02 reduced fibrosis and disease biomarkers (MMP7, TIMP1, Col1a1, MCP-1). Conclusion: Our results suggest a functional role for CLDN1 in the pathogenesis of IPF, providing preclinical proof-of-concept for ALE.F02 as a novel airway-centric therapeutic approach in patients with pulmonary fibrosis.

Introduction: Cholangiocarcinoma (CCA) shows an alarming rise in incidence and mortality with unsatisfactory treatment options. Claudin-1 (CLDN1), a member of the tight junction family, is a transmembrane protein mediating cell stemness, plasticity and signaling. The functional role of CLDN1 as a therapeutic target for CCA is unknown. We have previously developed highly specific monoclonal antibodies (mAb) targeting exposed non-junctional CLDN1 exhibiting an excellent safety profile in non-human primates (Roehlen, Saviano et al. Science Translational Medicine 2022). Here, we aimed to explore the role of CLDN1 as an oncogenic driver and therapeutic target for CCA. Methods: Comprehensive CLDN1 expression analyses in patient tissues were performed to evaluate CLDN1 as a therapeutic target. Proof-of-concept studies using CLDN1 mAbs were performed in state-of-the-art mouse CDX and PDX models including models for advanced metastatic disease. Single-cell RNA sequencing and proteomics were applied to investigate tumor cell fate and signaling in vivo and ex vivo models. Results: Comprehensive analyses of CLDN1 protein and RNA expression in CCA patient tissues revealed a marked and significant upregulation of CLDN1 in CCA. Single-cell RNA sequencing of the CCA microenvironment revealed strong expression in tumor cells showing EMT, cell cycle and interferon response signature, uncovering CLDN1 as a therapeutic target. Targeting exposed CLDN1 by highly specific mAbs resulted in a significant and robust antitumoral effect in vivo across CDX and PDX models for intra- and extrahepatic CCA including advanced metastatic disease. Functional studies in cell-based models of CCA showed that CLDN1 mAbs markedly and significantly suppressed migration and invasion of tumor cells. Mechanistically, treatment with CLDN1 mAb suppressed Notch1, Src, and Hippo-YAP signaling - key signal transduction pathways implicated in CCA development and progression. Conclusion: Collectively, these results support an important functional role for CLDN1 in CCA pathogenesis and provide robust pre-clinical proof-of-concept for CLDN1-specific mAbs to treat CCA, setting the stage for its clinical development. Citation Format: Marion Muller, Zeina Nehme, Emilie Crouchet, Frank Jühling, Natascha Röhlen, Patrick Pessaux, Emanuele Felli, Lipika Goyal, Markus Meyer, Roberto Iacone, Alberto Toso, Luigi Manenti, Patrice Laquerriere, Aïna Venkatasamy, Nabeel Bardeesy, Catherine Schuster, Laurent Mailly, Thomas F. Baumert. Claudin-1 is a driver and therapeutic target for cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB012.

Background & Aims: Despite recent approvals, the response to treatment and prognosis of patients with advanced hepato-cellular carcinoma (HCC) remain poor. Claudin-1 (CLDN1) is a membrane protein that is expressed at tight junctions, but it can also be exposed non-junctionally, such as on the basolateral membrane of the human hepatocyte. While CLDN1 within tight junctions is well characterized, the role of non-junctional CLDN1 and its role as a therapeutic target in HCC remains unexplored.Methods: Using humanized monoclonal antibodies (mAbs) specifically targeting the extracellular loop of human non-junctional CLDN1 and a large series of patient-derived cell-based and animal model systems we aimed to investigate the role of CLDN1 as a therapeutic target for HCC.Results: Targeting non-junctional CLDN1 markedly suppressed tumor growth and invasion in cell line-based models of HCC and patient-derived 3D ex vivo models. Moreover, the robust effect on tumor growth was confirmed in vivo in a large series of cell line -derived xenograft and patient-derived xenograft mouse models. Mechanistic studies, including single-cell RNA sequencing of multicellular patient HCC tumorspheres, suggested that CLDN1 regulates tumor stemness, metabolism, oncogenic signaling and perturbs the tumor immune microenvironment.Conclusions: Our results provide the rationale for targeting CLDN1 in HCC and pave the way for the clinical development of CLDN1-specific mAbs for the treatment of advanced HCC.(c) 2022 The Authors. Published by Elsevier B.V. on behalf of European Association for the Study of the Liver. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Claudin 1 (CLDN1) is a protein confined within the normal epithelial tight junctions of different tissues. Upon malignant transformation, CLDN1 is overxpressed and epitopes become exposed outside the tight junctions (non-Junctional CLDN1). ALE.C04 is a highly specific humanized monoclonal antibody that recognizes a unique CLDN1 exposed epitope in different solid tumors.

Background: Cholangiocarcinoma (CCA) is an adenocarcinoma of the hepatobiliary system showing an alarming rise in incidence and mortality with unsatisfactory treatment options. Claudin-1 (CLDN1) is a transmembrane protein involved in epithelial tight junctions and is also expressed non-junctionally mediating cell plasticity and signaling. However, its functional role in CCA pathogenesis and progression is unknown. We have previously developed a highly specific monoclonal antibody (mAb) targeting exposed non-junctional CLDN1 exhibiting an excellent safety profile in non-human primates. Here, we aimed to explore the role of CLDN1 as an oncogenic driver and therapeutic target in intra- and extrahepatic CCA. Material and methods: Comprehensive CLDN1 expression analyses were performed to evaluate CLDN1 as a target for treatment of CCA. Proof-of-concept studies using CLDN1 mAbs were performed in state-of-the-art cell line-derived and patient-derived xenograft mouse models, as well as in human CCA cell lines. Single-cell and bulk RNA sequencing, and proteomics were applied to investigate tumor cell fate and signaling in vivo. Results: Comprehensive analyses of CLDN1 protein and RNA expression in CCA patient tissues revealed a marked and significant upregulation of CLDN1. Single-cell RNA sequencing of the CCA microenvironment revealed strong expression in tumor cells showing EMT, cell cycle and interferon response signature, uncovering CLDN1 as a therapeutic target. Targeting exposed non-junctional CLDN1 by highly specific mAbs resulted in a significant and robust antitumoral effect in vivo across cell line-derived and patient-derived xenograft models for intra- and extrahepatic CCA. Functional studies in cell-based models of CCA showed that CLDN1 mAbs markedly and significantly suppressed migration and invasion of tumor cells. Mechanistically, treatment with CLDN1 mAb suppressed Notch1, Src, and Hippo-YAP signaling - key signal transduction pathways implicated in CCA development and progression. Conclusions: Collectively, these results provide robust pre-clinical proof-of-concept for CLDN1-specific mAbs to treat CCA and set the stage for its clinical development. No conflict of interest.

Tissue fibrosis is a key driver of end-stage organ failure and cancer, overall accounting for up to 45% of deaths in developed countries. There is a large unmet medical need for antifibrotic therapies. Claudin-1 (CLDN1) is a member of the tight junction protein family. Although the role of CLDN1 incorporated in tight junctions is well established, the function of nonjunctional CLDN1 (njCLDN1) is largely unknown. Using highly specific monoclonal antibodies targeting a conformation-dependent epitope of exposed njCLDN1, we show in patient-derived liver three-dimensional fibrosis and human liver chimeric mouse models that CLDN1 is a mediator and target for liver fibrosis. Targeting CLDN1 reverted inflammation-induced hepatocyte profibrogenic signaling and cell fate and suppressed the myofibroblast differentiation of hepatic stellate cells. Safety studies of a fully humanized antibody in nonhuman primates did not reveal any serious adverse events even at high steady-state concentrations. Our results provide preclinical proof of concept for CLDN1-specific monoclonal antibodies for the treatment of advanced liver fibrosis and cancer prevention. Antifibrotic effects in lung and kidney fibrosis models further indicate a role of CLDN1 as a therapeutic target for tissue fibrosis across organs. In conclusion, our data pave the way for further therapeutic exploration of CLDN1-targeting therapies for fibrotic diseases in patients.

Abstract Introduction: Hepatocellular carcinoma (HCC) is the fastest rising and fourth leading cause of cancer death worldwide. While new therapeutic modalities have been recently approved, treatment response and survival in patients remain poor. Claudin-1 (CLDN1) is a cell membrane protein mediating cell-cell adhesion, fate and differentiation. While the function of CLDN1 within tight junctions is well characterized, the role of non-junctional CLDN1 in HCC remains unexplored. Aim and methods: Using humanized monoclonal antibodies (mAbs) targeting specifically the extracellular loop of human non-junctional CLDN1 and a large series of patient-derived cell-based and animal model systems we aimed to investigate the role of CLDN1 as therapeutic target for treatment of HCC. Results: Targeting non-junctional CLDN1 robustly suppressed tumor growth in a large series of patient-derived model systems, including multicellular tumorspheres and patient-derived xenograft (PDX) mouse models. In vivo and ex vivo studies further suggested synergistic efficacy in combination with sorafenib. Mechanistic studies revealed that CLDN1 mAbs suppressed tumor cell proliferation, invasion and stemness by interfering with Src, Wnt-β-catenin and STAT3 signaling. Treatment with humanized anti-CLDN1 mAbs is considered to be safe, as administration in non-human primates and mouse models did not reveal any major toxicity even when high doses largely exceeding the therapeutic need were repeatedly applied. Conclusion: Our results provide robust pre-clinical proof-of-concept for humanized CLDN1-specific mAbs for treatment of HCC. The unique and differentiated mechanism of action has the potential to break the plateau of limited response and survival offered by currently approved therapies. Citation Format: Natascha Roehlen, Marion Muller, Sara Cherradi, Frank Juehling, Francois H.T. Duong, Nuno Almeida, Fabio Del Zompo, Mirian Fernandez, Tobias Riedl, Hussein El Saghire, Antonio Saviano, Sarah Durand, Clara Ponsolles, Marine Oudot, Emanuele Felli, Patrick Pessaux, Irwin Davidson, Emilie Crouchet, Patrick Laquerriere, Mathias Heikenwälder, Roberto Iacone, Markus Meyer, Greg Elson, Tamas Schweighoffer, Catherine Schuster, Laurent Mailly, Joachim Lupberger, Thomas F Baumert. Claudin-1 is a therapeutic target for hepatocellular carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P153.