# Claudin-1 is a mediator and therapeutic target in primary sclerosing cholangitis

Fabio del Zompo, Emilie Crouchet, Tessa Ostyn, Zeina Nehme, Mélissa Messé, Frank Juehling, Romain Désert, Angelica Vieira, Julien Moehlin, Diana Nakib, et al.

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## To cite this version:

Fabio del Zompo, Emilie Crouchet, Tessa Ostyn, Zeina Nehme, Mélissa Messé, et al. Claudin-1 is a mediator and therapeutic target in primary sclerosing cholangitis. Journal of Hepatology, 2025, 10.1016/j.jhep.2025.08.005. hal-05225527

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# Claudin-1 is a mediator and therapeutic target in primary sclerosing cholangitis

## Authors

Fabio Del Zompo, Emilie Crouchet, Tessa Ostyn, ..., Olivier Govaere, Catherine Schuster, Thomas F. Baumert

## Correspondence

thomas.baumert@unistra.fr (T.F. Baumert).

## Graphical abstract

CLDN1 expression is increased in PSC in cholangiocytes, progenitor cells and hepatocytes

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Therapeutic effect of CLDN1 mAb in vivo

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## Highlights

* Claudin-1 is overexpressed in PSC cholangiocytes and its expression correlates with prognosis in patients.

* Spatial transcriptomics, proteomics, and loss-of-function studies unravel Claudin-1 as a disease driver.

* Treatment with a Claudin-1-specific monoclonal antibody improves survival, fibrosis, inflammation and cholestasis in PSC mouse models.

* Claudin-1 antibodies inhibit profibrotic and proinflammatory signaling in cholangiocytes.

* Preclinical proof-of-concept supports further development of Claudin-1-specific monoclonal antibodies for patients with PSC.

## Impact and implications

Primary sclerosing cholangitis (PSC) is a chronic fibrosing cholangiopathy with limited therapeutic options. We identified the cell surface protein Claudin-1 as a key mediator and potential therapeutic target for PSC. In patients, Claudin-1 expression correlates with disease stage and clinical outcomes. Conditional liver epithelial-specific Claudin-1 knockout in mice reduced liver injury, fibrosis, and cholestasis. Monoclonal antibodies targeting Claudin-1 inhibited fibrosis, cholestasis, and the ductular reaction across advanced PSC mouse models by suppressing pro-inflammatory and fibrogenic signaling. These preclinical findings support the clinical development of Claudin-1-specific antibodies for PSC treatment and have significant implications for physicians, researchers, and drug developers in the field of biliary disease.
# Claudin-1 is a mediator and therapeutic target in primary sclerosing cholangitis

Fabio Del Zompo¹, Emilie Crouchet¹, Tessa Ostyn², Zeina Nehme¹, Mélissa Messé¹, Frank Jühling¹, Romain Désert¹, Angelica T. Vieira¹, Julien Moehlin¹, Diana Nakib³,⁴, Tallulah Andrews³,⁴, Catia Perciani³,⁴, Sai Chung³,⁴, Gary D. Bader<fnref n="5" />, Ian McGilvray⁴, Chiara Caime⁶,⁷, Miki Scaravaglio⁶,⁷, Marco Carbone⁷,⁸, Pietro Invernizzi⁶,⁷, Sheraz Yaqub⁹, Trine Folseraas¹⁰, Tom H. Karlsen¹⁰, Gautam Shankar²,¹¹, Mark Primeaux¹², Punita Dhawan¹², Jesus M. Banales¹³,¹⁴, Natascha Roehlen¹,<fnref n="5" />, Roberto Iacone¹⁵, Geoffrey Teixeira¹⁵, Mathias Heikenwälder¹⁶,¹⁷, Laurent Mailly¹, Sonya MacParland³,⁴, Tania Roskams², Olivier Govaere², Catherine Schuster¹, Thomas F. Baumert¹,¹⁸,¹⁹,²⁰,*

Journal of Hepatology **2025**. vol. 83 | 1305–1319

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Check for updates

**Background & Aims:** Primary sclerosing cholangitis (PSC) is a cholangiopathy associated with a high risk of progression to end-stage liver disease and hepatobiliary cancer. Its pathogenesis remains poorly understood, and current clinical management offers limited therapeutic options, primarily liver transplantation. Claudin-1 (CLDN1), a transmembrane protein highly expressed in liver epithelial cells, plays a critical role in cell-cell communication and signaling. We aimed to investigate the functional role of CLDN1 as both a mediator and potential therapeutic target for PSC using patient cohorts alongside murine and patient-derived intervention models.

**Methods:** CLDN1 expression patterns and associated cellular phenotypes were analyzed in liver tissues from five PSC patient cohorts using single-cell RNA sequencing, spatial transcriptomics, and multiplex proteomics. Proof-of-concept studies employing CLDN1-specific monoclonal antibodies (mAbs) and genetic loss-of-function approaches were performed in state-of-the-art mouse models of PSC and cholangiopathies. Perturbation studies in human cell-based models were conducted to explore underlying mechanisms.

**Results:** In liver tissues from patients with PSC, CLDN1 expression was markedly upregulated and correlated with disease progression. Spatial transcriptomics and proteomics revealed elevated CLDN1 expression in diseased cholangiocytes and cholestatic periportal hepatocytes, accompanied by activation of pro-inflammatory and pro-fibrotic signaling pathways. Therapeutic administration of CLDN1-specific mAbs or genetic knockout improved liver function in PSC mouse models by reducing hepatobiliary fibrosis and cholestasis. Mechanistic studies indicated that mAb treatment inhibited pro-inflammatory and pro-fibrotic signaling in cholangiocytes and hepatocytes perturbed in PSC liver tissues.

**Conclusions:** These findings demonstrate a functional role for CLDN1 in the pathogenesis of PSC and biliary fibrosis. *In vivo* proof-of-concept studies, combined with expression analyses in patients with PSC, support the clinical development of CLDN1-specific mAbs as a therapeutic strategy for PSC.

© 2025 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 license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

Primary sclerosing cholangitis (PSC) is a progressive cholestatic liver disease of unknown origin. Genetic and autoimmune mechanisms have been suggested as predisposing factors, although the exact pathogenesis remains elusive.¹,² The natural course of PSC leads to biliary fibrosis and strictures, resulting in chronic cholestasis and progressing to cirrhosis and liver failure. Liver cancer is a major complication

of PSC at any stage. The risk of cholangiocellular carcinoma (CCA) is estimated at 20%.³ Liver transplantation is the only therapeutic option available for patients with advanced disease, limited by recurrence in up to 25% of recipients.³ Histological hallmarks of the disease are inflammation, fibrosis, cholestasis, and the ductular reaction, which is considered a mediator of disease progression.⁴,<fnref n="5" /> While a large series of compounds have been investigated in clinical trials, none has been shown to alter the natural progression of PSC.¹,⁶,⁷ The

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<footnote>* Corresponding author. Address: Inserm U1110, ITM, University of Strasbourg, 3 Rue Koeberlé, 67000 Strasbourg, France, Tel.: (+33) 3365583703. E-mail address: thomas.baumert@unistra.fr (T.F. Baumert).</footnote>

<footnote n="5">⁵ Present address: Department of Medicine II, Gastroenterology, Hepatology, Endocrinology and Infectious Diseases, Freiburg University Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany; Berta-Ottenstein-Programme, Faculty of Medicine, University of Freiburg, Freiburg, Germany.
https://doi.org/10.1016/j.jhep.2025.08.005</footnote>
CLDN1 and primary sclerosing cholangitis

lack of approved disease-modifying drugs shows the high unmet clinical need for new therapeutic strategies.

CLDN1 is a transmembrane protein highly expressed in epithelial cells mediating cell-cell communication and signaling.⁸ CLDN1 has been shown to play a functional role in the disease biology of inflammation, fibrosis and cancer.⁸⁻¹¹ In the liver, it is expressed in both junctional and non-junctional forms, exposed on the basolateral membrane of polarized hepatocytes, where it contributes to liver fibrosis and progression to hepatocellular carcinoma (HCC).⁸,⁹,¹² We have previously developed monoclonal antibodies (mAbs) targeting a conformational epitope in the CLDN1 extracellular loop 1 comprising motif W(30)-GLW(51)-C(54)-C(64). The mAbs are highly specific for non-junctional CLDN1 without cross-reactivity to other Claudins.¹³ In a metabolic dysfunction-associated steatohepatitis (MASH)-driven HCC mouse model, mAb treatment inhibited liver fibrosis and progression to HCC, showing an excellent safety profile in non-human

primates and healthy volunteers.⁸,¹²,¹⁴ While patients with genetic CLDN1 mutations can present with sclerosing cholangitis,¹⁵ the functional role of CLDN1 in PSC disease biology is unknown. Here, we investigated the functional role of CLDN1 as a mediator and therapeutic target in PSC.

Materials and methods

Patient selection

CLDN1 expression was investigated in five cohorts of patients with cholangiopathies. Liver transcriptomic datasets of patients with PSC and their respective controls were retrieved from ArrayExpress (EMBL-EBI), accession number E-GEOD-61260¹⁶, and Gene Expression Omnibus (NIH), accession numbers GSE118373,⁴ GSE243981.¹⁷ FFPE liver samples of patients with PSC were retrospectively obtained from the biobanks of the Norwegian PSC Research Center of Oslo, Norway (Table S1), the Department of Medicine and Surgery of

<figure><img src="image_7.png" /><figcaption>**Fig. 1.** CLDN1 is upregulated in liver tissues from patients with PSC and correlates with disease progression. (A) CLDN1 gene expression in whole-liver tissues (Mann-Whitney, *p* = 0.0175)(E-GEOD-61260)¹⁶ and laser micro-dissected ductular reaction areas of patients with PSC (Mann-Whitney, *p* = 0.0043)(GSE118373).⁴ (B) Quantification of CLDN1 expression in CLDN1-stained liver biopsies from two independent cohorts of patients with PSC, showing marked CLDN1 upregulation in patients, associated with fibrosis stage (Milan cohort: Mann-Whitney, *p* = 0.009; Oslo cohort, continuous line indicates Kruskal-Wallis *p* = 0.0036; dashed lines indicate pairwise Mann-Whitney: control vs. Early-ALP<sup>high</sup> *p* = 0.0286; control vs. cirrhosis *p* = 0.002; Early-ALP<sup>low</sup> vs. cirrhosis *p* = 0.008. (C) CLDN1 protein expression correlates with clinical prognostic scores, including the magnitude of the ductular reaction (Spearman's correlation, *p* = 0.019, *p* = 0.004, *p* = 0.004, *p* = 0.004, *p* = 0.004, *p* = 0.004, *p* = 0.04, *p* = 0.04 top to bottom). (D) Immunohistochemical staining on a PSC liver explant reveals strong CLDN1 expression in damaged bile ducts, ductular reaction, and cholestatic peri-portal hepatocytes. (E) Immunofluorescent staining showing robust CLDN1 expression in CK19-positive cells in PSC samples. Scale bars: 50 µm. (F) CLDN1 IHC staining of PSC samples at different stages, showing CLDN1 expression increasing along with disease stage, ductular reaction, and fibrotic content. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001. ALP, alkaline phosphatase; CK19, cytokeratin 19; CLDN1, Claudin-1; IHC, immunohistochemistry; PSC, primary sclerosing cholangitis. (This figure appears in color on the web.)</figcaption></figure>
A

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B

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C

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D

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E

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F

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G

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**Fig. 2.** *CLDN1* expression co-localizes with known PSC disease drivers in patients. (A) In a published PSC scRNA-seq atlas,¹⁷ *CLDN1* is upregulated in cholangiocytes of patients with PSC. (B) DEG analysis in PSC-derived *CLDN1*High vs. *CLDN1*Low biliary epithelial cells. Analysis of a spatial transcriptomic atlas of PSC.¹⁷ (C) Pathway enrichment analyses revealed that *CLDN1*High biliary cells are characterized by distinct signaling, phenotype, and plasticity features compared to *CLDN1*Low counterparts. (D) Analysis of a spatial transcriptomic atlas of PSC.¹⁸ *CLDN1* expression co-localizing with *CDKN1A*, *RELA*, and *CXCL8* at the interface of scar lesions. Insets show the interface region between a peribiliary scar and surrounding non-fibrotic liver tissue. Dashed lines delineate a fibrotic scar. (E) Whole PSC liver unbiased analysis of top 40 genes significantly correlating with *CLDN1* expression. (F) Top 20 genes significantly correlating with *CLDN1* expression in the 'Cholangiocyte' cluster. (G) Genes significantly correlating with *CLDN1* expression in a neighboring transcriptomic spot. CLDN1, Claudin-1; DEG, differentially expressed gene; PSC, primary sclerosing cholangitis; scRNA-seq, single-cell RNA sequencing. (This figure appears in color on the web.)
CLDN1 and primary sclerosing cholangitis

<figure><img src="image_15.png" /><figcaption>**Fig. 3.** Spatial multiplex proteomics reveals an increase in CLDN1-expressing liver epithelial cells in PSC liver tissues at the single-cell level. (A) UMAP clustering of spatial proteomics-phenotyped liver tissue cells. (B) Quantitative cytometry shows increased numbers of cholangiocytes and intermediate epithelial cells in PSC compared to non-diseased livers (Mann-Whitney, *p* <0.0001). Expression of CK19+CK7+ was used to identify cholangiocytes including ductular reactive cells, staining of CK18+CK7+CK19- cells were used to identify intermediate epithelial cells, such as dedifferentiating hepatocytes. (C) Quantitative cytometry shows</figcaption></figure>
the University of Milano-Bicocca, Italy (Table S2), and the University Hospital Leuven, Belgium (Table S3). Biopsies were assessed by an expert liver pathologist. The use of human samples was approved by the respective local ethical committees with informed patient consent.

## Computational analyses of patient samples

Raw count matrices from microarray studies were pre-processed and normalized using the *oligo* package in R. Single-cell RNA sequencing (scRNA-seq) PSC expression data¹⁷ were analyzed using *Seurat*, *rstatix*, and *fgsea* packages in R. PSC spatial transcriptomics dataset¹⁷ was analyzed using *Seurat* package in R. Detailed technical information is described in the supplementary material and methods.

## Immunohistochemistry, immunofluorescence, and multiple iterative labelling by antibody neodeposition

Detailed technical information is described in the supplementary material and methods.

## Antibodies

Monoclonal anti-CLDN1 and IgG isotype control antibodies have been described.⁸,¹⁸ Staining antibodies are described in Tables S5-S6.

## Animal experiments

The bile duct ligation (BDL), 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and *Mdr2*⁻/⁻ mouse models,¹⁹ expressing a human/mouse chimeric CLDN1 as a knock-in, were used to study the efficacy and safety of CLDN1 mAbs *in vivo*. Details are described in the supplementary materials and methods.

## Bioinformatic and statistical analyses

Bioinformatic procedures are described in supplementary data. Continuous data were compared using Student's *t* test when normally distributed (Shapiro-Wilk test) or non-parametric tests (Mann-Whitney *U* test and Kruskal-Wallis test) when non-normally distributed. Correlation was assessed by Spearman correlation test. Categorical data were analyzed using Fisher's exact test. Outlier identification was carried out using the ROUT method (Q = 1%). *p* values <0.05 were considered statistically significant. Statistical analyses were performed using GraphPad Prism 9 and R.

## Results

### Claudin-1 expression is upregulated in the livers of patients with PSC and correlates with disease progression

To investigate the role of CLDN1 in clinical disease biology, CLDN1 expression was analyzed in PSC cohorts by immunohistochemistry (IHC) and quantitative proteomics at the single-

cell level. Furthermore, publicly available single-cell,¹⁷ spatial,¹⁷ and bulk RNA transcriptomic⁴,¹⁶ data sets of patients with PSC were analyzed (Figs 1-3).

CLDN1 gene expression was markedly and significantly upregulated in liver tissues of patients with PSC, including the pro-fibrogenic ductular reaction (Fig. 1A). IHC staining of PSC samples from two well characterized cohorts (Milan, Oslo, Tables S1–2) revealed that CLDN1 protein upregulation was robustly associated with disease progression (Fig. 1B, Tables S1–2), as shown by markedly increased CLDN1 expression with progressing liver fibrosis stage (Fig. 1B), independent of comorbid inflammatory bowel disease (IBD) (Fig. S1A). CLDN1 expression in patient liver tissues correlated with clinically validated prognostic scores including the Amsterdam-Oxford PSC score, the Mayo risk score for PSC, and the PREsTO score²⁰,²¹ (Fig. 1C and S1B). CLDN1 expression also correlated with the magnitude of the ductular reaction (Fig. 1C), which is associated with poor prognosis in PSC.⁵ Immunohistopathology analyses revealed that CLDN1 is robustly expressed in cholangiocytes lining damaged bile ducts, as well as ductular reactive cells (Fig. 1D). Hepatocytes close to portal spaces showed elevated CLDN1 expression with a membranous pattern, likely in association with a cholestatic metaplastic phenotype (Fig. 1D). Multi-color fluorescent staining validated high CLDN1 protein expression in virtually all cytokeratin (CK) 19+ ductular cells in PSC liver tissues (Fig. 1E). Consistently, CLDN1 protein expression increased with disease progression from early to advanced fibrosis stages in non-cirrhotic PSC liver samples, along with ductular reactive cells and liver fibrotic content in immunohistochemistry analyses (Fig. 1F).

The marked upregulation of CLDN1 in PSC tissues, its expression in PSC-driving cells, and its association with disease progression suggest that CLDN1 plays a pathogenic role in PSC and represents a potential therapeutic target.

## Spatial transcriptomics and multi-plex proteomics in PSC liver tissues reveals co-localization of CLDN1 with known drivers of inflammation, fibrogenesis and stemness

To investigate the biological role of CLDN1 in PSC progression, its expression in liver samples of PSC was investigated by single cell-resolved and spatial transcriptomics. At the single-cell level, the highest CLDN1 expression levels were found in cells expressing markers of the biliary lineage, including cholangiocytes and biliary epithelial cells (Fig. 2A). An unbiased analysis of marker genes in CLDN1High cholangiocytes (Fig. 2B) revealed that top four differentially expressed genes included TNF-related weak inducer of apoptosis receptor (TWEAK receptor, *TNFRSF12A*), cytokeratin 7 (*KRT7*), chemokine (C-X-C motif) ligand 6 (*CXCL6*), and SRY-box transcription factor 4 (*SOX4*) (Fig. 2B and S2A). Confirmatory studies at the protein level revealed that CLDN1+ CK19+ biliary epithelial cells were the major source of TNFα in PSC liver tissues (Fig. S2B). Gene set-enrichment analysis of

<footnote>increased numbers of CLDN1+ cells in PSC compared to non-diseased livers (Mann-Whitney, *p* = 0.0002). (D) Quantitative cytometry showing increased CLDN1 staining intensity in CLDN1+ cells in PSC compared to non-diseased livers (Mann-Whitney, *p* = 0.0034). (E) Protein expression of CLDN1, TNFα, and SPP1/osteopontin in the cluster of cholangiocytes and intermediate epithelial cells. (F) Digital reconstruction of representative TMA cores, phenotyped by spatial proteomics. (G) Individual panels showing representative TMA cores stained for CLDN1, CK19, SPP1, CLDN1+CK19+SPP1, and TNFα (upper to lower). \*\*\*p < 0.001; \*\*\*\*p < 0.0001. CLDN1, Claudin-1; PSC, primary sclerosing cholangitis; TMA, tissue microarray; UMAP, uniform manifold approximation and projection. (This figure appears in color on the web.)</footnote>
CLDN1 and primary sclerosing cholangitis

A

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B

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<table><thead><tr><th colspan="7">No. at risk</th></tr></thead><tbody><tr><td>Control</td><td>20</td><td>20</td><td>18</td><td>15</td><td>15</td><td>12</td><td>10</td></tr><tr><td>CLDN1 mAb</td><td>20</td><td>20</td><td>20</td><td>19</td><td>19</td><td>16</td><td>15</td></tr></tbody></table>

C

Liver injury

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D

Cholestasis

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E

Liver function

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F

Fibrosis

G

H

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Fibrosis

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Cell fate, plasticity and ductular reaction

I

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J

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K

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Inflammation

**Fig. 4. CLDN1 mAb treatment improves survival, liver function, cholestasis, and liver fibrosis in the bile duct ligation mouse model.** (A) Illustration of the experimental approach. (B) Survival analysis of bile duct-ligated mice revealed that CLDN1 mAb improved survival at 7 days (Log-rank test, *p* = 0.08). (C-E) CLDN1 mAb treatment significantly ameliorated liver function tests, included ALT (Mann-Whitney, *p* = 0.0163) and AST (Mann-Whitney, *p* = 0.0185) (C), total bilirubin (Mann-Whitney, *p* = 0.0118) and ALP (Mann-Whitney, *p* = 0.0168) as markers of cholestasis (D), and albumin (Mann-Whitney, *p* = 0.0006) as a marker of liver biosynthetic function (E). (F) CLDN1 mAb treatment reduced fibrosis levels as measured by Sirius red CPA (Mann-Whitney, *p* <0.0001). (G) Representative images of Sirius red-
CLDN1<sup>High</sup> vs. CLDN1<sup>Low</sup> differentially expressed genes revealed that high CLDN1 expression was associated with gene sets of bile duct proliferation, cholangitis, and senescence (Fig. 2C, left). Signaling pathways associated with CLDN1 expression included KRAS, NF-κB, epithelial-to-mesenchymal transition, STAT3, and AKT (Fig. 2C, middle). Additionally, stemness-related gene sets were enriched in CLDN1<sup>high</sup> cholangiocytes (Fig. 2C, right). Analysis of a published spatial transcriptomics dataset<sup>17</sup> revealed that CLDN1 gene expression co-localized with the expression of known drivers of PSC, including CDKN1A (p21), NF-κB effector RELA (p65), and CXCL8 (IL-8), at the edges of PSC scar lesions (Fig. 2D). Interestingly, CLDN1 expression correlated with the expression of pro-inflammatory and pro-fibrogenic genes, including CXCL8 (Fig. 2E,F and S2C,D) and CXCL6 and MMP7 in transcriptomic regions neighboring high CLDN1 expression (Fig. 2G and S2E). These findings suggest that high CLDN1 expression is associated with expression of pro-inflammatory and pro-fibrogenic pathways.

To validate the key single-cell gene expression findings at the protein level, 291,283 cells were phenotyped across samples of an independent cohort of patients with PSC (Fig. 3A, Table S3) using multiplex spatial proteomic analysis based on multiple iterative labelling by antibody neodeposition.<sup>22</sup> Quantitative cytometry in PSC vs. non-diseased tissues revealed a marked increase of CK19+CK7+ cholangiocytes including ductular reactive cells and CK18+CK7+CK19- intermediate epithelial cells, such as dedifferentiating hepatocytes (Fig. 3B). A robust and significant increase of the total number of CLDN1+ cells in PSC compared to non-diseased liver tissues was observed (Fig. 3C), along with the increase of CLDN1 signal intensity per cell (Fig. 3D), validating the results obtained by IHC (Figs 1 and 2) at single-cell resolution in an independent cohort. Protein expression analysis indicated co-localization of CLDN1 with mediators of biliary inflammation and fibrosis, such as the pro-inflammatory cytokine TNFα, and immune and fibrosis modulator secreted phosphoprotein 1 (SPP1, osteopontin) in cholangiocytes and intermediate epithelial cells (Fig. 3E). CLDN1+TNFα+SPP1+ cells were observed surrounding peribiliary fibrotic lesions (Fig. 3F,G and S3A,B), suggesting a potential role for CLDN1 in the biology of diseased cholangiocytes.

## Treatment with CLDN1-specific mAbs improves liver function and survival by reducing fibrosis and cholestasis in state-of-the-art mouse models of PSC

To study the functional role of CLDN1 in the disease biology of PSC and to investigate the role of CLDN1 as a therapeutic target, proof-of-concept studies were performed in three complementary PSC animal models using highly specific CLDN1-specific antibodies.

Since the mAbs partially cross-react with mouse CLDN1,<sup>8</sup> we engineered a mouse model expressing a human/mouse (h/ m) hybrid CLDN1 in all organs and cells where native CLDN1 is expressed. This was achieved by exchanging three amino acids in the mouse CLDN1 EL1-coding region using homologous recombination. The BDL model was applied first as it recapitulates cholestasis-driven fibrosis, as well as cholangiocyte reactivity and ductular reaction.<sup>19,23</sup> Forty 8-10 week-old male mice underwent surgical ligation of the common bile duct. Mice received 25 mg/kg CLDN1 mAb (n = 20) or vehicle control (n = 20) i.p. immediately after surgery and again on day 4 (Fig. 4A). Survival analysis of BDL mice showed that CLDN1 mAb treatment improved survival at day 7 (Fig. 4B). Liver function tests revealed an improvement of markers of liver injury, liver function, and cholestasis in CLDN1 mAb-vs. control-treated mice as shown by reduced levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) (Fig. 4C), total bilirubin and alkaline phosphatase (ALP) (Fig. 4D). Bile acids remained unchanged (Fig. S4A). Furthermore, significantly increased levels of albumin (Fig. 4E) indicated liver function improvement. Automated analysis of the collagen proportionate area (CPA) (Fig. 4F,G and S4B) of Sirius red-stained livers revealed a significant reduction of liver fibrosis in CLDN1 mAb-vs. control-treated mice. Transcriptomic analyses revealed that CLDN1 mAb treatment modulated gene expression of fibrosis-related markers Col1a1, Tgfb1, Acta2, and Timp1 in both RNA-seq (Fig. 4H) and qPCR (Fig. S4C) analyses. Moreover, a robust reduction of the expression of markers of the ductular reaction including Epcam, Krt19, Spp1 (Fig. 4I) and cytokeratin-7 (Fig. 4J) was observed. Additionally, CLDN1 mAb-treated mice exhibited reduced expression of pro-inflammatory cytokines (Fig. 4K).

Next, the DDC mouse model was applied, a chemical model for PSC recapitulating key features of sclerosing cholangitis and peribiliary fibrosis.<sup>24</sup> Forty 8-week-old male mice were fed with a 0.1% DDC-supplemented diet for 4 weeks. Following establishment of peribiliary fibrosis in week 1,<sup>24</sup> mice were assigned 1:1 to receive weekly i.p. injections of 25 mg/kg CLDN1 mAb or vehicle control for 3 weeks (Fig. 5A). CLDN1 mAb treatment did not change survival (Fig. S5A) and decreased liver-to-body weight ratio (Fig. 5B). Analysis of liver function tests revealed significant decreases of plasma ALT (Fig. 5C) and bile acids (Fig. 5D), but not ALP (Fig. S5B). Treatment with CLDN1 mAb resulted in a significant and robust reduction of liver fibrosis as shown by CPA analysis (Fig. 5E). CLDN1 treatment also resulted in inhibition of portal bridging fibrosis – a key marker of disease progression in patients (Fig. 5F and S5C). The decreased expression of cytokeratin-19 (Krt19) and cytokeratin-7 indicated that CLDN1 mAb treatment reduced the ductular reaction (Fig. 5G). Analysis of differentially expressed genes by RNA-seq and qPCR (Fig. S5D) revealed the downregulation of several pro-inflammatory mediators in CLDN1 mAb-treated mice (Fig. 5H

stained livers of control and CLDN1 mAb-treated mice. Scale bars: 500 µM. (H–K) Expression of fibrosis (H), cell fate and ductular reaction (I–J) and inflammation (K) markers in the livers of control and CLDN1 mAb-treated mice (Col1a1: *p* = 0.0079; Tgfb1: *p* = 0.0079; Acta2: *p* = 0.0317; Timp1: *p* = 0.0079, Krt19: *p* = 0.0056; Spp1: *p* = 0.0011; Ck7: *p* = 0.0357; Ccl24: *p* = 0.0079; Tnf: *p* = 0.0079; Il1b: *p* = 0.0079; Mann-Whitney). Scale bars: 250 µM *p* < 0.05; ***p* < 0.01; ** *p* < 0.001; **** *p* < 0.0001. AST (C, right) and bilirubin (D, left) panels show n = 7 control and n = 10 CLDN1 mAb-treated mice. The plasma of eight control and five treated mice could not be analyzed due to hemolysis interfering with analyte measurements. Data in (H) and (K) were obtained from RNA-seq analyses of a subset of five representative liver tissues (n = 5 vs. n = 5). ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CLDN1, Claudin-1; CPA, collagen proportionate area; mAb, monoclonal antibody; PSC, primary sclerosing cholangitis. (This figure appears in color on the web.)
CLDN1 and primary sclerosing cholangitis

<figure><img src="image_26.png" /><figcaption>**Fig. 5.** CLDN1 mAb treatment ameliorates liver injury and fibrosis in the DDC mouse model. (A) Illustration of the experimental approach. (B) Analysis of the liver index (liver-to-body weight ratio) suggesting reduced cell proliferation in treated mice (Mann-Whitney, *p* = 0.0055). (C) CLDN1 mAb treatment ameliorated liver injury as shown by decreased levels of ALT (Mann-Whitney, *p* = 0.0009). (D) CLDN1 mAb treatment reduced cholestasis as measured by plasma concentrations of bile acids (Mann-Whitney, *p* = 0.0559). (E) CLDN1 mAb treatment reduced fibrosis levels as measured by Sirius red CPA (Mann-Whitney, *p* < 0.0001). (F) Representative images of Sirius red-stained livers of control and CLDN1 mAb-treated mice. Scale bars: 500 µM. (G) CLDN1 mAb treatment reduced the magnitude of ductular reaction, as measured by *Krt19* gene expression (Mann-Whitney, *p* = 0.0777) and cytokeratin-7 immunostaining (Mann-Whitney, *p* = 0.0159). Scale bars: 250 µM. (H) Liver expression (RNA-seq analyses of five liver tissues of each group, n = 5 vs. n = 5) of genes encoding for inflammation and fibrosis markers (*Ccl20*: *p* = 0.0079; *Ccl17*: *p* = 0.0079; *Cxcl5*: *p* = 0.0079; *Fbln2*: *p* = 0.0079; *Itgb6*: *p* = 0.0159; *Mmp7*: *p* = 0.0317; *Mmp9*: *p* = 0.0079; Mann-Whitney). (I) Experimental approach of *Alb.* Cre/Cldn1<sup>fl/fl</sup> mice with conditional Cldn1 knockout in liver epithelial cells and subjected to DDC diet. (J) Liver Cldn1 expression in *Alb.*Cre and *Alb.*Cre/Cldn1<sup>fl/fl</sup> mice,</figcaption></figure>
and S5D). Confirming the histopathology findings, expression of genes involved in fibrogenesis and extracellular matrix remodeling was significantly decreased, including fibulin 2 (Fbln2), integrin subunit beta 6 (Itgb6), matrix metallopeptidase 7 (Mmp7), matrix metallopeptidase 9 (Mmp9) (Fig. 5H), and transforming growth factor beta 2 (Tgfb2) (Fig. S5D).

Since CLDN1 has been shown to be upregulated in the colon of patients with IBD,²⁵ the effect of CLDN1 mAb treatment on the colon was investigated. MAb treatment did not result in significant differences in colon length, colon weight, and intestinal permeability (Fig. S6A-E). Furthermore, no colon histopathological changes were observed, as previously shown for healthy mice across organs.¹⁴ Treatment effects were similar in male and female mice (Figs S6B and S7A-C), suggesting that there is likely no sex-dependency for CLDN1 mAb efficacy. A control group fed standard chow served as a baseline to distinguish the specific effects of diet from other variables (Fig. S7A-D). The effects of CLDN1 mAb were target-specific, since an isotype control did not show therapeutic effects (Fig. S7A-D). Moreover, CLDN1 mAb treatment did not modulate liver function tests in h/mCLDN1 KI mice under non-disease modeling conditions (Fig. S7D).

To further validate the functional role of CLDN1 in the pathogenesis of biliary fibrosis, the generation of an *Alb.Cre/Cldn1fl/fl* mouse model enabled investigation of biliary fibrosis development in mice with *Cldn1* conditional knockout in liver epithelial cells. When challenged with 0.1% DDC feeding (Fig. 5I), *Alb.Cre/Cldn1fl/fl* robustly maintained the *Cldn1* knockout phenotype, as shown by the absence of CLDN1 expression in the liver (Fig. 5J), while exhibiting significantly less liver injury (Fig. 5K), less cholestasis (Fig. 5L), and less collagen deposition (Fig. 5M,N) compared to *Alb.Cre* controls.

The *Mdr2*<sup>-/-</sup> mouse model is a state-of-the-art model for PSC, as it recapitulates chronic disease progression, modeling biliary fibrosis, cholestasis and hepatobiliary cancer, similar to the clinical course of PSC.<sup>26</sup> *Mdr2*<sup>-/-</sup> mice were treated with CLDN1 mAb or control at the age of 6 weeks, when fibrosis and portal PSC-like lesions were already established.<sup>26</sup> After 12 weeks of treatment, mice were sacrificed, and plasma and livers harvested (Fig. 6A). While this model is characterized by low mortality, our data indicate that treatment with CLDN1 mAb improved survival compared to control animals (Fig. 6B). In a per-protocol analysis of relative weight change, CLDN1 mAb treatment significantly increased growth rate (Fig. 6C). CLDN1 mAb-treated *Mdr2*<sup>-/-</sup> mice exhibited a robust improvement of cholestasis as shown by reduced total bilirubin (Fig. 6D and S8A), plasma bile acids (Fig. 6D and S8B), and ALP (Fig. 6D). ALP levels, which are used as endpoints in clinical trials,<sup>1</sup> were normalized in 73% of CLDN1 mAb-treated mice vs. 28% of control-treated mice (Fig. 6D). The improvement of cholestasis was accompanied by reduced liver injury, as shown by decreased AST and ALT levels (Fig. 6E). Importantly, CLDN1 mAb treatment resulted in reduced liver fibrosis, including the inhibition of bridging fibrosis as shown by CPA

analyses (Fig. 6F,G and S8C). The histopathological features of fibrosis reduction were accompanied by reduced gene expression of pro-inflammatory mediators (Fig. 6H). Analysis of cell fate marker expression revealed that CLDN1 mAb treatment downregulated biliary-fate marker Sox9 while upregulating hepatic nuclear factor 4 alpha (Hnf4a) (Fig. 6I), while markers of the ductular reaction remained unchanged (Fig. S8D). The expression of extracellular matrix components collagen type IV alpha-1 chain (Col4a1), collagen type V alpha-2 chain (Col5a2), and laminin subunit beta 1 (Lamb1) was significantly downregulated in CLDN1 mAb-treated mice (Fig. 6J and S8E).

Collectively, proof-of-concept studies in three state-of-the-art PSC mouse models showed improvement of liver function, cholestasis, and fibrosis on CLDN1 mAb treatment.

## CLDN1 mAb treatment inhibits pro-inflammatory and pro-fibrogenic signaling in PSC mouse and patient-derived models

To investigate the mechanism of action of CLDN1 mAb treatment, liver gene expression from the three animal models was analyzed using RNA-seq and compared with the perturbed liver transcriptome of patients with PSC.<sup>16</sup> Gene set-enrichment analysis revealed that 1,101 gene sets which were upregulated in their expression in patients with PSC were downregulated following CLDN1 mAb treatment across all mouse models (Fig. 7A). At the same time, 48 gene sets that were downregulated in patients with PSC showed restored expression across all mouse models (Fig. 7A). CLDN1 mAb treatment robustly suppressed the expression of PSC disease drivers and pathogenic signaling pathways (Fig. 7B). These included NF-κB signaling, T cell receptor and macrophage signaling, TGFβ response, collagen formation as well as Notch and KRAS signaling and epithelial-to-mesenchymal transition. Of note, the suppression of bile acid metabolism in patients with PSC was also restored by CLDN1 mAb treatment. The perturbation of key pro-inflammatory and pro-fibrotic signaling pathways was validated in the BDL mouse model on the protein level using IHC and immunoblotting (Fig. 7C and S9A,B). CLDN1 mAb treatment significantly suppressed NF-κB signaling, as shown by decreased p65-positive area in reactive ductules in the BDL model *in vivo* (Fig. 7C), accompanied by marked reduction of nuclear p65 translocation (Fig. S9A). Moreover, CLDN1 treatment resulted in inhibition of pro-fibrotic SRC, AKT and RAS signaling as shown by decreased phosphorylation of SRC, AKT, and decreased RAS protein expression in immunoblot analyses of mouse liver tissues treated with mAb (Fig. 7C and S9B). Since the majority of these proteins have been shown to bind/interact with CLDN1 in the cell membrane<sup>8,27</sup> and the CLDN1 antibody was not internalized following binding to the cholangiocyte cell membrane (Fig. S10A and B), it is likely the mAb inhibits signaling by

validating the loss of CLDN1 expression in DDC-fed KO mice. (K) Plasma ALT (Mann-Whitney, *p* = 0.0039) and (L) ALP (Mann-Whitney, *p* = 0.2799) levels were decreased in *Alb.Cre/Cldn1fl/fl* mice. (M) Cldn1 knockout in liver epithelial cells significantly reduced fibrosis development in the DDC mouse model (Mann-Whitney, *p* = 0.0011). (N) Representative images of Sirius red-stained liver of *Alb.Cre* and *Alb.Cre/Cldn1fl/fl* mice. Scale bars: 500 µM *p* < 0.05; ** *p* < 0.01; *** *p* < 0.001; **** *p* < 0.0001. ALP, alkaline phosphatase; ALT, alanine aminotransferase; CPA, collagen proportionate area; DDC, 3,5-diethoxycarbonyl-1,4-dihydrocollidine; mAb, monoclonal antibody; PSC, primary sclerosing cholangitis. (This figure appears in color on the web.)
CLDN1 and primary sclerosing cholangitis

A

<figure><img src="image_27.png" /></figure>

B

<figure><img src="image_28.png" /></figure>

<table><thead><tr><th colspan="2">No. at risk</th></tr></thead><tbody><tr><td>Control</td><td>23</td></tr><tr><td>CLDN1 mAb</td><td>23</td></tr></tbody></table>

C

<figure><img src="image_29.png" /></figure>

D

Cholestasis

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

E

Liver injury

<figure><img src="image_34.png" /></figure>

F

G

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

H

Inflammation

<figure><img src="image_37.png" /></figure>

I

Cell fate and plasticity

<figure><img src="image_38.png" /></figure>

J

Fibrosis

<figure><img src="image_39.png" /><figcaption>**Fig. 6.** CLDN1 mAb treatment improves survival, liver function, and liver fibrosis in the *Mdr2*<sup>-/-</sup> mouse model. (A) Illustration of the experimental approach. (B) Survival analysis of *Mdr2*<sup>-/-</sup> mice revealing improved survival in CLDN1 mAb-treated group (log-rank test, *p* = 0.0887). (C) Increased growth rate in CLDN1 mAb-treated *Mdr2*<sup>-/-</sup> mice (extra sum-of-squares F-test, *p* < 0.0001). Error bars: mean ± SEM. (D) CLDN1 mAb treatment significantly decreased markers of cholestasis (bilirubin: *p* = 0.0023; bile acids: *p* = 0.0519, Mann-Whitney, and ALP: *p* = 0.077, *t* test), resulting in the normalization of plasma ALP levels in 73% of mice (Fisher's exact test, *p* = 0.01). (E) Plasma levels of AST (Mann-Whitney, *p* = 0.0242) and ALT (Mann-Whitney, *p* = 0.0936) as markers of liver injury were reduced in CLDN1 mAb-treated group. (F) CLDN1 mAb treatment reduced fibrosis levels as measured by Sirius red CPA (Mann-Whitney, *p* < 0.0001). (G) Representative images of Sirius red-stained livers of control and CLDN1 mAb-treated mice. Scale bars: 500 µm. (H-J) Liver gene expression (RNA-seq, n = 5 vs. n = 5) of key inflammation (H), cell fate (I), and fibrosis markers (J). (Cc12: *p* = 0.0317; Ccl12 *p* = 0.0317; *Tlr4*: *p* = 0.0159; Sox9: *p* = 0.0159, *Hnf4a*: *p* = 0.0079; Col4a1: *p* = 0.0317; Col5a2: *p* = 0.0317; *Lamb1*: *p* = 0.0317, Mann-Whitney). \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.0001. Bilirubin (D, left) and AST (E, left) panels show n = 14 control and n = 21 CLDN1 mAb-treated mice. The plasma of four control and one treated mice could not be analyzed due to hemolysis interfering with analyte measurements. ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CPA, collagen proportionate area; mAb, monoclonal antibody; PSC, primary sclerosing cholangitis. (This figure appears in color on the web.)</figcaption></figure>
A

<figure><img src="image_40.png" /></figure>

(A): Upregulated in PSC vs. healthy liver

(B): Downregulated by CLDN1 mAb in DDC mice

(C): Downregulated by CLDN1 mAb in BDL mice

(D): Downregulated by CLDN1 mAb in *Mdr2*<sup>+/+</sup> mice

<figure><img src="image_41.png" /></figure>

(E): Downregulated in PSC vs. healthy liver

(F): Upregulated by CLDN1 mAb in DDC mice

(G): Upregulated by CLDN1 mAb in BDL mice

(H): Upregulated by CLDN1 mAb in *Mdr2*<sup>+/+</sup> mice

C

B

<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /><figcaption>**Fig. 7. CLDN1 mAb treatment suppresses pro-inflammatory, pro-fibrotic and pro-carcinogenic signaling pathways in vivo.** (A) Venn diagrams of pathways differentially enriched in the livers of patients with PSC and in the livers of mouse models. (B) Comparison of PSC liver transcriptome with transcriptomic changes induced by CLDN1 mAb treatment in mouse models. Heatmaps illustrate NES of representative altered gene sets, each condition vs. their respective control. (C) Quantification of IHC and immunoblot signals validating transcriptomic findings on the protein level in the BDL mouse model (NF-κB-p65: *p* = 0.0004; AKT: *p* < 0.0001; SRC: *p* = 0.0475; RAS: *p* = 0.0226, Mann-Whitney). \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001. BDL, bile duct ligation; IHC, immunohistochemistry; mAb, monoclonal antibody; NES, normalized enrichment score; PSC, primary sclerosing cholangitis. (This figure appears in color on the web.)</figcaption></figure>

interfering with protein-protein interactions at the cell membrane.

Liver scRNA-seq analysis of BDL mice revealed additional mechanistic events induced by antibody treatment at single-cell resolution (Fig. 8A). All the major liver cell types were captured, hepatocytes, and macrophages being the most abundant cell types (Fig. S9C). Given the CLDN1 expression profile in scRNA-seq and spatial transcriptomics analyses in patients (Fig. 1, 2), we first focused on epithelial cell biology. CLDN1 mAb treatment induced a significant downregulation of PSC-associated cholangiocyte and hepatocyte marker genes (Fig. 8B). Single-cell gene set variation analysis revealed that CLDN1 mAb treatment reduced the expression of TNFα-NFκB,

NOTCH1, AKT, and SRC signaling pathways in both hepatocytes and cholangiocytes (Fig. 8C). The inhibition of pro-inflammatory and pro-fibrogenic signaling was validated on the protein level where CLDN1 mAb treatment modulated SRC, IKBα, and p65 phosphorylation (Fig. S11A and B) in primary human cholangiocytes. The inhibition of epithelial cell signaling resulted in a modulation of macrophage and myofibroblast functions, the effector cell types involved in PSC and biliary fibrosis.<sup>17,28</sup> Single cell-resolved gene expression analysis of the pro-fibrotic niche of the BDL mouse model revealed that CLDN1 mAb treatment suppressed the expression of key pro-inflammatory and pro-fibrotic cytokines in nonparenchymal cells (Fig. 8D), resulting in the suppression of
CLDN1 and primary sclerosing cholangitis

<figure><img src="image_44.png" /><figcaption>**Fig. 8.** CLDN1 mAb treatment suppresses pro-inflammatory and pro-fibrotic signaling in liver epithelial cells with inhibition of macrophage and fibroblast activation. (A) scRNA-seq clustering of livers from the BDL mouse model. (B) scGSVA enrichment analysis of gene signatures of PSC cholangiocytes and PSC hepatocytes (Mann-Whitney, both *p* <0.0001). (C) scGSVA enrichment analysis of TNF-NF-κB, SRC, NOTCH1, and AKT signaling pathways (Mann-Whitney, all *p* <0.0001). (D) Cell type-specific gene expression of key mediators of the pro-fibrotic niche in biliary fibrosis, showing the ultimate effect of CLDN1 mAb on collagen</figcaption></figure>
the expression of major ECM components in fibroblasts, including Col1a1 (Fig. 8D).

Collectively, these results identify the targeted cell types and mechanistic events through which CLDN1 mAb treatment improves cholestatic liver disease.

Discussion

In this study, we identify CLDN1 as a previously unrecognized driver and therapeutic target for PSC. This discovery is based on the following key findings: (1) CLDN1 is overexpressed in liver tissues of patients with PSC and its level of expression correlates with disease progression (Fig. 1); (2) CLDN1 expression co-localizes with disease drivers and pathways in the diseased livers of patients with PSC (Figs 2 and 3); (3) a mAb targeting exposed CLDN1 on cholangiocytes and hepatocytes reduces fibrosis, inflammation and cholestasis - hallmarks of PSC - in three state-of-the-art mouse models (Figs 4-6); (4) a loss-of-function study using a liver-specific CLDN1 knockout mouse model supports a functional role of CLDN1 in PSC disease biology (Fig. 5I-N).

Mechanistically, our data are consistent with a model that CLDN1 overexpression in cholangiocytes and hepatocytes induces pro-inflammatory and pro-fibrogenic signaling (Figs 7 and 8E), resulting in the perturbation of epithelial cell fate and induction of the ductular reaction. Subsequent macrophage and fibroblast activation mediates inflammation, cholestasis and fibrosis (Figs 7 and 8F). Since the pathogenic role of the ductular reaction and these signaling pathways have been well described in PSC disease biology,⁵,⁸,²⁹ it is likely that their inhibition mediates the effects of CLDN1 mAb treatment.

Our study has some limitations: first, we cannot exclude the involvement of other CLDN1-related signaling pathways or additional mechanistic events in mediating the effects of CLDN1 mAb. Second, although we used a large panel of complementary model systems for PSC disease biology, these model systems only partially recapitulate the complex pathogenesis of fibrosing cholangiopathies in patients (e.g. absence of IBD or intestinal biology). Third, analysis of fibrosis was

limited to Sirius red staining and collagen gene expression. Fourth, further studies will be needed to study whether CLDN1 mAb treatment will reduce the development of CCA in PSC-CCA models.

The overexpression of CLDN1 in PSC tissues across several patient cohorts combined with the robust effect of CLDN1 mAb treatment across three state-of-the-art in vivo models without detectable adverse events identify CLDN1 as a previously unrecognized therapeutic target in PSC. The correlation of CLDN1 expression with disease biology (Fig. 1) identifies CLDN1 as a candidate biomarker for patient stratification. The modulation of secretory proteins, including TIMP1, metalloproteinases (Figs 4 and 5), and CCL20 - a cytokine associated with PSC³⁰ and suppressed by antibody treatment in all models (Figs 4-6) - provides opportunities for non-invasive target engagement markers in patients.

Given the absence of approved therapeutic options and the limited success of compounds in clinical development, treatment with CLDN1 mAb could provide a new opportunity to improve the dismal prognosis of patients with PSC.

Interestingly, we observed that the therapeutic effect of CLDN1 treatment on cholestasis, fibrosis and survival was most pronounced in the BDL model, suggesting that BDL best models the pathways targeted by the antibody. Whether this finding has clinical relevance for patients, e.g. large duct vs. small duct disease or major strictures, remains to be determined.

A clinical challenge in PSC is the high risk of CCA and HCC and the lack of effective surveillance. Since CLDN1 is over-expressed in CCA and HCC and CLDN1 mAbs have been shown to potently inhibit the development and growth of hepatobiliary cancers in patient-derived tumor models,⁸,¹²,³¹ it is likely that treatment with mAb will also reduce the risk of CCA and HCC, which are key determinants of outcome and survival in patients with PSC. Furthermore, CLDN1 mAbs have been shown to be safe, including in non-human primates¹² and healthy volunteers.³² Collectively, the results of this study pave the way for clinical development of CLDN1 mAbs as a first-in-class candidate treatment for PSC.

Affiliations

¹Inserm U1110, Institute of Translational Medicine and Liver Diseases (ITM), University of Strasbourg, Strasbourg, France; ²Department of Imaging and Pathology, KU Leuven and University Hospitals Leuven, Leuven, Belgium; ³Department of Immunology, University of Toronto, Toronto, Canada; ⁴Ajmera Transplant Center, University Health Network, Toronto, Canada; ⁵Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Canada; ⁶Division of Gastroenterology, Center for Autoimmune Liver Diseases, European Reference Network on Hepatological Diseases (ERN RARE-LIVER), Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy; ⁷Department of Medicine and Surgery, University of Milano-Bicocca, Milan, Italy; ⁸Hepatology and Gastroenterology Unit, ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy; ⁹Department of Hepatobiliary Surgery, Oslo University Hospital and Institute of Clinical Medicine, University of Oslo, Norway; ¹⁰Norwegian PSC Research Center (NoPSC), Oslo, Norway; ¹¹KU Leuven Institute for Single Cell Omics (LISCO), KU Leuven, Leuven, Belgium; ¹²Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA; ¹³Department of Liver and Gastrointestinal Diseases, Biogipuzkoa Health Research Institute - Donostia University Hospital, University of the Basque Country (UPV/EHU), CIBERehd, Ikerbasque, 20014, San Sebastian, Spain; ¹⁴Department of Biochemistry and Genetics, School of Sciences, University of Navarra, Pamplona, Spain; ¹⁵Alentis Therapeutics, Allschwil, Switzerland; ¹⁶Division of Chronic Inflammation and Cancer, German Cancer Research Center Heidelberg, Heidelberg, Germany; ¹⁷M3 Research Institute, Medical Faculty Tuebingen (MFT), Tuebingen, Germany; ¹⁸HU Strasbourg, France; ¹⁹Gastroenterology and Hepatology Service, Strasbourg University Hospitals, Strasbourg, France; ²⁰Institut Universitaire de France, Paris, France

Abbreviations

ACTA2, actin alpha 2; ALB, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AKT, protein kinase B; AST, aspartate aminotransferase; BDL,

bile duct ligation; CCA, cholangiocellular carcinoma; CCL20, C-C motif chemokine ligand 20; CK, cytokeratin; CLDN1, claudin-1; Col1a1, collagen type I alpha 1 chain; Col4a1, collagen type IV alpha 1 chain; Col5a2, collagen type V

production by myofibroblasts (Mann-Whitney, all *p* <0.0001). (E) Mechanistic model of CLDN1 mAb-mediated inhibition of liver and biliary signaling based on transcriptomic and proteomic analyses. (F) Mechanistic model of CLDN1 mAb-mediated anti-fibrotic and anti-inflammatory efficacy in preclinical models for PSC based on scRNA-Seq analyses. *p* <0.05; **p** <0.01; ***p** <0.001; ****p** <0.0001. BDL, bile duct ligation; CLDN1, claudin-1; mAb, monoclonal antibody; PSC, primary sclerosing cholangitis; scGSVA, single-cell gene set variation analysis; scRNA-seq, single-cell RNA sequencing. (This figure appears in color on the web.)
# CLDN1 and primary sclerosing cholangitis

alpha 2 chain; CXCL6, C-X-C motif chemokine ligand 6; DDC, 3,5-diothoxycarbonyl-1,4-dihydrocollidine; ECM, extracellular matrix; Epcam, epithelial cell adhesion molecule; FBLN2, fibulin 2; HCC, hepatocellular carcinoma; HNF4A, hepatic nuclear factor 4 alpha; IBD, inflammatory bowel disease; IKBα, inhibitor of NF-κB alpha; KRT7, cytokeratin 7; KRT19, cytokeratin 19; Lamb1, laminin subunit beta 1; MASH, metabolic dysfunction-associated steatohepatitis; MMP7, matrix metallopeptidase 7; MMP9, matrix metallopeptidase 9; NF-κB, nuclear factor kappa B; PSC, primary sclerosing cholangitis; RELA, v-rel avian reticuloendotheliosis viral oncogene homolog A; SPP1, secreted phosphoprotein 1 (osteopontin); SOX4, SRY-box transcription factor 4; Tgfb, transforming growth factor beta; TIMP1, tissue inhibitor of metalloproteinases 1; TNFα, tumor necrosis factor alpha; TNFRSF12A, TNF-related weak inducer of apoptosis receptor.

## Financial support

The authors acknowledge the following financial support: European Research Council Grant ERC-AdG-2020 FIBCAN (T.F.B.); ARC Grant TheraHCC2.0 IHUARC, IHU201301187 (T.F.B.); French National Research Agency RHU DELIVER (ANR-21-RHUS-0001) and LABEX ANR-10-LABX-0028_HEPSYS (T.F. B.); the University of Strasbourg Foundation, the Institut Universitaire de France (IUF), Paris, France (TFB), the Alsace Cancer Foundation (T. F. B), the German Research Foundation (DFG) RO 5983/1-1 (N.R.). This work of the Interdisciplinary Thematic Institute IMCBio, as part of the ITI 2021-2028 program of the University of Strasbourg, CNRS and Inserm, was further supported by IdEx Unistra (ANR-10-IDEX-0002), and by SFRI-STRAT'US project (ANR 20-SFRI-0012) and EUR IMCBio (ANR-17-EURE-0023) under the framework of the French Investments for the Future Program and the France 2030 program. JMB was funded by Spanish Carlos III Health Institute (ISCIII) [FIS PI18/01075, PI21/00922 and Miguel Servet Program CPII19/00008 to JMB) co-financed by the European Commission, and the European Union's Horizon 2020 Research and Innovation Program [ESCALON; grant number 825510]. ÆPIC was supported by University of Strasbourg IdEX 2024 "Dispositifs plateformes" (RDGGPJ2403M).

## Conflict of interest

Inserm, the University of Strasbourg, the Strasbourg University Hospitals and Alentis Therapeutics have filed a patent application for the use of anti-claudin-1 antibodies to treat cholangiopathies (PCT/IB2023/055666). TFB is founder, shareholder, consultant of Alentis. CS is shareholder of Alentis. GT and RI are employees of Alentis.

Please refer to the accompanying ICMJE disclosure forms for further details.

## Authors' contributions

T.F.B. initiated and coordinated the study. F.D.Z. and T.F.B. designed or performed experiments and analyzed data. T.O., O.G., T.R., C.C., M.S., M.C., P.I., F. D.Z. performed CLDN1 expression analyses of human PSC tissues by IHC or immunofluorescence. T.O., G.S., O.G., T.R. performed multiplex iterative labeling by antibody neodeposition (MILAN) studies. F.D.Z., Z.N., M.M., R.D., M.P., A. T.V., L.M. performed animal experiments and analyzed data. E.C. contributed to the design and analyses of cell-based experiments and prepared the libraries for murine scRNA-seq. F.D.Z., F.J., J.M. performed computational analyses on murine tissues. F.D.Z., F.J., J.M., D.N., and S.M. performed computational analyses on PSC patient tissues. T.F., S.Y., and T.H.K. provided liver tissues of the Oslo cohort. M.H.'s laboratory performed IHC staining of mouse liver tissues. J. M.B., N.R., P.D., and C. S. provided important input into the MS. F.D.Z. and T.F. B. designed the figures and wrote the manuscript with edits from all the authors.

## Acknowledgements

The authors acknowledge Marine Oudot, Sarah Durand, Cloé Gadenne, Nicolas Brignon, Romain Martin (all Inserm U1110, Strasbourg, France) and Danijela Heide (DKFZ Heidelberg, Germany) for excellent technical assistance, and Prof. A. Kramer (Universitätsspital Zürich) and Palak Trivedi (University of Birmingham) for helpful discussions. The authors acknowledge the Animal Experimentation Platform Infection and Cancer (ÆPIC) (University of Strasbourg, ITM Inserm UMR_S1110, Strasbourg, France) for the management of animal experiments.

## Data availability

Transcriptomic data reported in this paper have been deposited at the GEO database with accession numbers GSE261990, GSE261991, and GSE261995.

## Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jhep.2025.08.005.

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### Author names in bold designate shared co-first authorship

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Keywords: antibody therapy; biliary fibrosis; cholangiopathies; signaling; proof-of-concept.

Received 13 September 2024; received in revised form 9 July 2025; accepted 5 August 2025; available online 20 August 2025