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Background: The efficacy assessment of human anti-IgE monoclonal antibodies (mAbs) in animal models before clinical trials is hampered due to the lack of cross-reactivity of anti-IgE mAbs between species. Objective: We developed CRE-DR (an anti-dog IgE monoclonal antibody), an anti-IgE mouse mAb that recognizes canine and human IgE, and then examined its IgE specificity and cross-reactivity between three animal and human species. Methods: After mouse immunization with a synthetic peptide derived from canine IgE (282NTNDWIEGETYYC294), we generated a hybridoma producing CRE-DR. The CRE-DR purified from the ascites of hybridoma-inoculated mice was used for ELISA and Western blot analysis to examine reactivity to dog, human, and rodent IgEs as well as recombinant bovine serum albumin (BSA)- conjugated to canine, human, and rodent IgE amino acid peptides corresponding to the immunizing sequence. We then performed enzyme-linked immunosorbent assays (ELISAs) for dog IgE using sera from dogs with atopic dermatitis (AD) after inhibition with canine IgE and IgG. The amino acid sequence recognized by CRE-DR was identified by ELISA using synthetic peptides. Results: CRE-DR is a monoclonal mouse IgG1κ specific for dog IgE, and the ELISA values in atopic dog sera were inhibited by dog IgE, but not dog IgG. The binding of CRE-DR to human IgE was relatively maintained, but not to rodent IgEs, which results were confirmed with the BSA-conjugated IgE peptides of the various species. The CRE-DR reactivity was supported by the comparison of amino acid sequence of CRE-DR epitope, DWIEGETYYC, in dog IgE; one, two, and three amino acids were substituted in the human, rat, and mouse IgE epitopes, respectively. Conclusions and Clinical Relevance: CRE-DR is a mAb cross-reactive to dog and human IgEs, which can allow the use of a dog model of allergy to test the efficacy of a CRE-DR-derived anti-IgE therapeutic mAb before human clinical trials. [ABSTRACT FROM AUTHOR]

Due to their lower production cost compared with monoclonal antibodies, single-chain variable fragments (scFvs) have potential for use in several applications, such as for diagnosis and treatment of a range of diseases, and as sensor elements. However, the usefulness of scFvs is limited by inhomogeneity through the formation of dimers, trimers, and larger oligomers. The scFv protein is assumed to be in equilibrium between the closed and open states formed by assembly or disassembly of VH and VL domains. Therefore, the production of an scFv with equilibrium biased to o the closed state would be critical to overcome the problem in inhomogeneity of scFv for industrial or therapeutic applications. In this study, we obtained scFv clones stable against GA-pyridine, an advanced glycation end-product (AGE), by using a combination of a phage display system and random mutagenesis. Executing the bio-panning at 37 °C markedly improved the stability of scFvs. We further evaluated the radius of gyration by small-angle X-ray scattering (SAXS), obtained compact clones, and also visualized open-close dynamics of these scFvs by high-speed atomic force microscopy (HS-AFM), revealing that one of the compact clones was biased to the closed state. Finally, nuclear magnetic resonance (NMR) analysis revealed that peak intensity and line width became homogeneous, supporting that dynamic features and/or formation of oligomers was improved in the thus-obtained clone. These findings should contribute to the future industrial and therapeutic use of scFvs. [ABSTRACT FROM AUTHOR]

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 Na1/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/hepatitis D virus (HDV) infection and has been approved as Hepcludex in Europe for the treatment of patients with chronic 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 to 87 in ECL1 and aa 157 to 165 near ECL2 of transmembrane domain 5 are critically important for HBV/HDV infection. Epitope mapping and the three-dimensional (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 binding of HBV to the region from aa 84 to 87. 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 nucleoside/nucleotide 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 postadministration 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. [ABSTRACT FROM AUTHOR]