Treatment of HCC with claudin-1-specific antibodies suppresses carcinogenic signaling and reprograms the tumor microenvironment.

BACKGROUND & AIMS: Despite recent approvals, the response to treatment and prognosis of patients with advanced hepatocellular 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. IMPACT AND IMPLICATIONS: Hepatocellular carcinoma (HCC) is associated with high mortality and unsatisfactory treatment options. Herein, we identified the cell surface protein Claudin-1 as a treatment target for advanced HCC. Monoclonal antibodies targeting Claudin-1 inhibit tumor growth in patient-derived ex vivo and in vivo models by modulating signaling, cell stemness and the tumor immune microenvironment. Given the differentiated mechanism of action, the identification of Claudin-1 as a novel therapeutic target for HCC provides an opportunity to break the plateau of limited treatment response. The results of this preclinical study pave the way for the clinical development of Claudin-1-specific antibodies for the treatment of advanced HCC. It is therefore of key impact for physicians, scientists and drug developers in the field of liver cancer and gastrointestinal oncology.

In vivo combination of human anti-envelope glycoprotein E2 and -Claudin-1 monoclonal antibodies for prevention of hepatitis C virus infection.

Despite the development of direct-acting antivirals (DAAs), hepatitis C virus (HCV) infection remains a major cause for liver disease and cancer worldwide. Entry inhibitors block virus host cell entry and, therefore, prevent establishment of chronic infection and liver disease. Due to their unique mechanism of action, entry inhibitors provide an attractive antiviral strategy in organ transplantation. In this study, we developed an innovative approach in preventing HCV infection using a synergistic combination of a broadly neutralizing human monoclonal antibody (HMAb) targeting the HCV E2 protein and a host-targeting anti-claudin 1 (CLDN1) humanized monoclonal antibody. An in vivo proof-of-concept study in human liver-chimeric FRG-NOD mice proved the efficacy of the combination therapy at preventing infection by an HCV genotype 1b infectious serum. While administration of individual antibodies at lower doses only showed a delay in HCV infection, the combination therapy was highly protective. Furthermore, the combination proved to be effective in preventing infection of primary human hepatocytes by neutralization-resistant HCV escape variants selected during liver transplantation, suggesting that a combination therapy is suited for the neutralization of difficult-to-treat variants. In conclusion, our findings suggest that the combination of two HMAbs targeting different steps of virus entry improves treatment efficacy while simultaneously reducing treatment duration and costs. Our approach not only provides a clinical perspective to employ HMAb combination therapies to prevent graft re-infection and its associated liver disease but may also help to alleviate the urgent demand for organ transplants by allowing the transplantation of organs from HCV-positive donors.

In vivo proof of concept of adoptive immunotherapy for hepatocellular carcinoma using allogeneic suicide gene-modified killer cells.

Cell therapy based on alloreactivity has completed clinical proof of concept against hematological malignancies. However, the efficacy of alloreactivity as a therapeutic approach to treat solid tumors is unknown. Using cell culture and animal models, we aimed to investigate the efficacy and safety of allogeneic suicide gene-modified killer cells as a cell-based therapy for hepatocellular carcinoma (HCC), for which treatment options are limited. Allogeneic killer cells from healthy donors were isolated, expanded, and phenotypically characterized. Antitumor cytotoxic activity and safety were studied using a panel of human or murine HCC cell lines engrafted in immunodeficient or immunocompetent mouse models. Human allogeneic suicide gene-modified killer cells (aSGMKCs) exhibit a high, rapid, interleukin-2-dependent, and non-major histocompatibility complex class I-restricted in vitro cytotoxicity toward human hepatoma cells, mainly mediated by natural killer (NK) and NK-like T cells. In vivo evaluation of this cell therapy product demonstrates a marked, rapid, and sustained regression of HCC. Preferential liver homing of effector cells contributed to its marked efficacy. Calcineurin inhibitors allowed preventing rejection of allogeneic lymphocytes by the host immune system without impairing their antitumor activity. Our results demonstrate proof of concept for aSGMKCs as immunotherapy for HCC and open perspectives for the clinical development of this approach.

Frequent compartmentalization of hepatitis C virus with leukocyte-related amino acids in the setting of liver transplantation.

BACKGROUND: Nonrandom distribution of hepatitis C virus (HCV) quasispecies (compartmentalization between blood plasma and leukocytes) suggests the presence of HCV leukotropic variants. HCV compartmentalization in the setting of liver transplantation (LT) is poorly understood. To study HCV leukotropic variants, we investigated the evolution of HCV compartmentalization after immunosuppression in liver transplant recipients. METHODS: Plasma and peripheral blood mononuclear cell (PBMC) samples were collected from 5 liver transplant recipients before and after LT. We used clone sequencing to analyze the hypervariable region 1 (HVR1)-E2(384-419) region, which plays a key role in HCV entry and the induction of neutralizing responses, and assessed compartmentalization through phylogenetic analyses and Mantel's test. RESULTS: Compartmentalization was frequent in the LT setting. HCV quasispecies were more homogeneous after LT in both the plasma and PBMC compartments, with a significant decrease in quasispecies complexity (P = .003) and genetic distances (P = .004) after transplantation. Our analysis identified 8 PBMC-related amino acid residues in HVR1. CONCLUSIONS: HCV compartmentalization between plasma and PBMCs and the emergence of leukotropic variants could be potentiated by immunosuppression in liver transplant recipients. The identification of defined leukotropic variants may contribute to the understanding of virus-host interactions after transplantation.

Early evolution of hepatitis C virus (HCV) quasispecies after liver transplant for HCV-related disease.

BACKGROUND: End-stage liver disease as a result of chronic hepatitis C virus (HCV) infection is the main indication for liver transplant (LT), but allografts are systematically infected with HCV soon after transplant. Viral quasispecies are poorly described during the early posttransplant period. METHODS: For 17 patients who received an LT for HCV disease, plasma viral quasispecies evolution was determined by sequence analysis of hypervariable region 1 of the E2 envelope gene before transplant (BT), after 7 days (D7), and after 1 month (M1). T helper (Th)1/Th2 cytokine levels were determined concomitantly. RESULTS: HCV quasispecies showed a significant decrease in amino acid diversity at D7 and M1, compared with BT (P<.05). A correlation was observed between low plasma tumor necrosis factor-alpha levels at D7 and decreased quasispecies amino acid complexity at the same date. Nucleic acid diversity was lower for genotype 1 than for genotype 3 infection (P<.05). The complexity and diversity of amino acids were lower in patients with hepatocellular carcinoma (HCC) BT than in those without HCC (P<.05). Conserved amino acid residues within quasispecies were shared by the whole cohort before and after LT. CONCLUSION: Viral structural and/or host immunological features could favor the emergence of fitter HCV strains after LT.

Early decrease in circulating dendritic cells number after liver transplantation could favor hepatitis C virus recurrence.

Cirrhosis and hepatocarcinoma related to hepatitis C virus (HCV) lead to more than 30% of liver transplantations. Host- and virus-related mechanisms, involved in the recurrence of HCV infection of the liver graft, are not yet well known. A weak CD4+ T-cell response was shown to be involved in the outcome of re-infection but whether dendritic cell numbers are modified in patients transplanted for HCV-related disease has never been evaluated. Eight transplanted patients for HCV-related disease and eight non-HCV-infected transplanted controls were included. Blood plasmacytoid dendritic cells and myeloid dendritic cells were quantified before transplantation, at day 7 and 1 month after transplantation. Plasma interferon (IFN)-alpha and interleukin (IL)-12 were concomitantly measured. The results showed a significant decrease in the relative (P < 0.0001) and absolute (P = 0.0002) values of blood plasmacytoid dendritic cells at day 7 after transplantation when compared to the values obtained before transplantation, increasing again 1 month later, in both HCV-infected patients and controls. The same tendency was observed for myeloid dendritic cell relative values (P = 0.0004) and plasma IL-12 (P < 0.05). IFN-alpha appeared to be less often detectable for HCV-infected patients. These results obtained on dendritic cell numbers could explain partially the early and systematic recurrence of HCV infection on the liver graft and contribute to better adapted therapeutic strategies.