Establishment of a Monoclonal Antibody against Human NTCP That Blocks Hepatitis B Virus Infection

Abstract

Hepatitis B virus (HBV) infects 240 million people worldwide. Current therapy profoundly suppresses HBV replication but requires long-term maintenance therapy. Therefore there is still a medical need for an efficient HBV cure. HBV enters host cells by binding via the preS1-domain of the viral L protein to the Na + /Taurocholate Cotransporting Polypeptide (NTCP). Thus, NTCP should be a key target for the development of anti-HBV therapeutics. Indeed, Myrcludex B, a synthetic form of the myristoylated preS1 peptide, effectively reduces HBV/HDV infection and has been approved as Hepcludex® in Europe for the treatment of patients with chronic hepatitis D virus (HDV) infection. We established a monoclonal antibody (mAb) N6HB426-20 that recognizes the extracellular domain of human NTCP and blocks HBV entry in vitro into human liver cells but has much less of an inhibitory effect on bile acid uptake. In vivo , administration of the N6HB426-20 mAb prevented HBV viremia for an extended period of time after HBV inoculation in a mouse model system without strongly inhibiting bile acid absorption. Among the extracellular loops (ECLs) of NTCP, regions of amino acids (aa) 84-87 in ECL1 and aa 157-165 near ECL2 of transmembrane domain 5 are critically important for HBV/HDV infection. Epitope-mapping and the 3D model of the NTCP structure suggested that the N6HB426-20 mAb may recognize aa 276/277 at the tip of ECL4 and interfere with an binding of HBV to the aa 84-87 region. In summary, we identified an in vivo neutralizing NTCP-targeting antibody capable of preventing HBV infection. Further improvements in efficacy of this drug will pave the way for its clinical applications. IMPORTANCE A number of entry inhibitors are being developed to enhance the treatment of HBV patients with oral nucleos(t)ide analogues (NA). To amplify the effectiveness of NA therapy, several efforts have been made to develop therapeutic mAbs with neutralizing activity against HBs antigens. However, the neutralizing effect of these mAbs may be muted by a large excess of HBsAg-positive noninfectious particles in the blood of infected patients. The advantage of NTCP-targeted HBV entry inhibitors is that they remain effective regardless of viral genotype, viral mutations and the presence of subviral particles. Although N6HB426-20 requires a higher dose than Myrcludex to obtain equivalent suppression of HBV in a model mouse system, it maintained the inhibitory effect for a long time post administration in proportion to the half-life of an IgG mAb. We believe that further improvements will make this antibody a promising treatment option for patients with chronic hepatitis B.

Inhibiting Sodium Taurocholate Cotransporting Polypeptide in HBV-Related Diseases: From Biological Function to Therapeutic Potential

“…The inhibition of NTCP by N6HB426-20 depends on interacting with the 276th or 277th residue of ECL4 to sterically block the binding of HBV to ECL1 of NTCP. 89 In an experiment using PHHs as an infection model, N6HB426-20 effectively inhibited HBV infection with an IC 50 of 8 nM for the HBsAg concentration. Treatment with N6HB426-20 prevented HBV viremia in a mouse model for a longer time period.…”

“…Very recently, another mAb N6HB426-20 was established. The inhibition of NTCP by N6HB426-20 depends on interacting with the 276th or 277th residue of ECL4 to sterically block the binding of HBV to ECL1 of NTCP . In an experiment using PHHs as an infection model, N6HB426-20 effectively inhibited HBV infection with an IC 50 of 8 nM for the HBsAg concentration.…”

Methods for Engineering Binders to Multi-Pass Membrane Proteins

“…To ensure the selection of binders against a native epitope, target proteins in multiple formulations/formats can be used alternately. For example, to engineer antibodies against Na + /taurocholate co-transporting polypeptide (NTCP), a key receptor for the hepatitis B virus, Takemori et al immunized NTCP-knockout mice with NTCP solubilized in detergent (1% DMM), reconstituted in liposomes, and expressed in transfected cells [38]. After 4-6 rounds of immunization, they screened the supernatants of hybridomas using a flow cytometric assay and identified mAb N6HB426, which efficiently inhibited HBV infection by binding to the extracellular domain of NTCP.…”

Regulation of the HBV Entry Receptor NTCP and its Potential in Hepatitis B Treatment

“…The concentration that blocks HBV and HDV entry is 100-fold lower than that required to inhibit bile acid transport by NTCP ( Volz et al, 2013 ; Bogomolov et al, 2016 ; Cheng et al, 2021 ). At present, Myrcludex B has been approved in Europe for the treatment of patients with chronic HDV infection under the trade name Hepcludex ® ( Takemori et al, 2022 ). It has been reported that cyclosporin A (CsA), another peptide inhibitor that is usually used as an immunosuppressant in organ transplantation, as well as its analogs, can inhibit HBV infection by targeting NTCP and interfering with the interaction between preS1 and NTCP, thereby blocking HBV entry into hepatocytes ( Watashi et al, 2014b ; Nkongolo et al, 2014 ).…”

“…For example, MA18/7 and 2H5-A14 target the preS1 region and neutralize HBV infection ( Glebe et al, 2003 ; Li et al, 2017 ). In addition, a monoclonal antibody against NTCP—N6HB426-20—has been shown to recognize the extracellular domain of human NTCP (residues 276/277 at the tip of extracellular loop-4 of NTCP) and block HBV entry into human liver cells in vitro , while exerting substantially less of an inhibitory effect on bile acid uptake ( Takemori et al, 2022 ). With further improvements, this antibody may be a promising treatment option for patients with chronic hepatitis B.…”