Manami Iida

Monoclonal Antibodies against Extracellular Domains of Claudin-1 Block Hepatitis C Virus Infection in a Mouse Model

ABSTRACT Hepatitis C virus (HCV) entry into host cells is a complex process requiring multiple host factors, including claudin-1 (CLDN1). Safe and effective therapeutic entry inhibitors need to be developed. We isolated a human hepatic Huh7.5.1-derived cell mutant that is nonpermissive to HCV, and comparative microarray analysis showed that the mutant was CLDN1 defective. Four hybridomas were obtained, which produced monoclonal antibodies (MAbs) that interacted with the parental Huh7.5.1 cell but not with the CLDN1-defective mutant. All MAbs produced by these hybridomas specifically bound to human CLDN1 with a very high affinity and prevented HCV infection of Huh7.5.1 cells in a dose-dependent manner, without apparent cytotoxicity. Two selected MAbs also inhibited HCV infection of human liver-chimeric mice without significant adverse effects. CLDN1 may be a potential target to prevent HCV infection in vivo. Anti-CLDN1 MAbs may hence be promising candidates as novel anti-HCV agents. IMPORTANCE Safe and effective therapeutic entry inhibitors against hepatitis C virus (HCV) are very useful for combination therapies with other anti-HCV drugs, such as direct-acting antivirals. In this study, we first showed an effective strategy for developing functional monoclonal antibodies (MAbs) against extracellular domains of a multimembrane-spanning target protein, claudin-1 (CLDN1), by using parental cells expressing the intact target membrane protein and target-defective cells. The established MAbs against CLDN1, which had a very high affinity for intact CLDN1, efficiently inhibited in vitro and in vivo HCV infections. These anti-CLDN1 MAbs are promising leads for novel entry inhibitors against HCV.

Development of an Anti–Claudin-3 and -4 Bispecific Monoclonal Antibody for Cancer Diagnosis and Therapy

Most malignant tumors are derived from epithelium, and claudin (CLDN)-3 and CLDN-4 are frequently overexpressed in such tumors. Although antibodies have potential in cancer diagnostics and therapy, development of antibodies against CLDNs has been difficult because the extracellular domains of CLDNs are too small and there is high homology among human, rat, and mouse sequences. Here, we created a monoclonal antibody that recognizes human CLDN-3 and CLDN-4 by immunizing rats with a plasmid vector encoding human CLDN-4. A hybridoma clone that produced a rat monoclonal antibody recognizing both CLDN-3 and -4 (clone 5A5) was obtained from a hybridoma screen by using CLDN-3– and -4–expressing cells; 5A5 did not bind to CLDN-1–, -2–, -5–, -6–, -7–, or -9–expressing cells. Fluorescence-conjugated 5A5 injected into xenograft mice bearing human cancer MKN74 or LoVo cells could visualize the tumor cells. The human-rat chimeric IgG1 monoclonal antibody (xi5A5) activated FcγRIIIa in the presence of CLDN-3– or -4–expressing cells, indicating that xi5A5 may exert antibody-dependent cellular cytotoxicity. Administration of xi5A5 attenuated tumor growth in xenograft mice bearing MKN74 or LoVo cells. These results suggest that 5A5 shows promise in the development of a diagnostic and therapeutic antibody for cancers.

Discovery of anti-claudin-1 antibodies as candidate therapeutics against hepatitis C virus

Claudin-1 (CLDN1), a known host factor for hepatitis C virus (HCV) entry and cell-to-cell transmission, is a target molecule for inhibiting HCV infection. We previously developed four clones of mouse anti-CLDN1 monoclonal antibody (mAb) that prevented HCV infection in vitro. Two of these mAbs showed the highest antiviral activity. Here, we optimized the anti-CLDN1 mAbs as candidates for therapeutics by protein engineering. Although Fab fragments of the mAbs prevented in vitro HCV infection, their inhibitory effects were much weaker than those of the whole mAbs. In contrast, human chimeric IgG1 mAbs generated by grafting the variable domains of the mouse mAb light and heavy chains inhibited in vitro HCV infection as efficiently as the parental mouse mAbs. However, the chimeric IgG1 mAbs activated Fcγ receptor, suggesting that cytotoxicity against mAb-bound CLDN1-expressing cells occurred through the induction of antibody-dependent cellular cytotoxicity (ADCC). To avoid ADCC-induced side effects, we prepared human chimeric IgG4 mAbs. The chimeric IgG4 mAbs did not activate Fcγ receptor or induce ADCC, but they prevented in vitro HCV infection as efficiently as did the parental mouse mAbs. These findings indicate that the IgG4 form of human chimeric anti-CLDN1 mAb may be a candidate molecule for clinically applicable HCV therapy.

Claudin-1 Binder Enhances Epidermal Permeability in a Human Keratinocyte Model

Tight junctions (TJs) are complex biochemical structures that seal the intercellular space and prevent the free movement of solutes across epithelial cell sheets. Modulating the TJ seal is a promising option for increasing the transdermal absorption of drugs. Within TJs, the binding of the claudin (CLDN) family of tetratransmembrane proteins through cis- and trans-interactions is an integral part of seal formation. Because epidermal TJs contain CLDN-1 and CLDN-4, a binder for these CLDNs may be a useful modulator of the permeability of the epidermal barrier. Here, we investigated whether m19, which can bind to CLDN-1/-4 (also CLDN-2/-5), modulates the integrity of epidermal TJs and the permeability of cell sheets to solutes. Treatment of normal human epidermal keratinocytes (NHEKs) with the CLDN binder reduced the integrity of TJs. A CLDN-1–specific binder (a monoclonal antibody, clone 7A5) also weakened the TJ seal in NHEKs. Although m19 attenuated the TJ barrier in human intestinal epithelial cells (Caco-2), 7A5 did not. Treatment of NHEKs with 7A5 enhanced permeation of a paracellular permeation marker. These findings indicate that CLDN-1 is a potential target for modulating the permeability of the epidermis, and that our CLDN-1 binder is a promising candidate molecule for development as a dermal absorption enhancer.

Creation of a Claudin-2 Binder and Its Tight Junction–Modulating Activity in a Human Intestinal Model

Disruption of the gastrointestinal epithelial barrier is a hallmark of chronic inflammatory bowel diseases (IBDs). The transmembrane protein claudin 2 (CLDN2) is a component of epithelial tight junctions (TJs). In the intestines of patients with IBDs, the expression of the pore-forming TJ protein CLDN2 is upregulated. Although CLDN2 is involved in these leaky barriers, whether it can be a target to enhance TJ integrity is unknown because a CLDN2-specific inhibitor has not been developed. Here, we used DNA immunization to generate a monoclonal antibody (mAb) that recognized an extracellular loop of CLDN2. Treatment of epithelial cell monolayers with the mAb increased barrier integrity. In addition, the anti-CLDN2 mAb attenuated the decrease in TJ integrity induced by the proinflammatory cytokine tumor necrosis factor-α (TNF-α), and cotreatment of cells with anti–TNF-α mAb and anti-CLDN2 mAb showed additive attenuating effects. These findings indicate that CLDN2 may be a target for enhancing TJ integrity, and CLDN2 binder may be an enhancer of mucosal barrier integrity and a potential therapeutic option for IBDs.

Development of a Quenchbody for the Detection and Imaging of the Cancer-Related Tight-Junction-Associated Membrane Protein Claudin

Claudins (CLs) are membrane proteins found in tight junctions and play a major role in establishing the intercellular barrier. However, some CLs are abnormally overexpressed on tumor cells and are valid clinical biomarkers for cancer diagnosis. Here, we constructed antibody Fab fragment-based Quenchbodies (Q-bodies) as effective and reliable fluorescent sensors for detecting and visualizing CLs on live tumor cells. The variable region genes for anti-CL1 and anti-CL4 antibodies were used to express recombinant Fab fragments, and clones recognizing CL4 with high affinity were selected for making Q-bodies. When two fluorescent dyes were conjugated to the N-terminal tags attached to the Fab, the fluorescent signal was significantly increased after adding nanomolar-levels of purified CL4. Moreover, addition of the Q-body to CL4-expressing cells including CL4-positive cancer cells led to a clear fluorescence signal with low background, even without washing steps. Our findings suggested that such Q-bodies would serve as a potent tool for specifically illuminating membrane targets expressed on cancer cells, both in vitro and in vivo.