Comparing recombinant human rabies monoclonal antibody (ormutivimab) with human rabies immunoglobulin (HRIG) for postexposure prophylaxis: A phase III, randomized, double-blind, non-inferiority trial.

OBJECTIVES: To evaluate the immunogenicity and safety of an anti-rabies monoclonal antibody (mAb), ormutivimab, compared with human rabies immunoglobulin (HRIG). METHODS: This phase III trial was designed as a randomized, double-blind, non-inferiority clinical trial in patients aged ≥18 years with suspected World Health Organization category Ⅲ rabies exposure. The participants were randomized 1:1 to ormutivimab and HRIG groups. After thorough wound washing and injection of ormutivimab/HRIG on day 0, the vaccination was administered on days 0, 3, 7, 14, and 28. The primary endpoint was the adjusted geometric mean concentration (GMC) of rabies virus-neutralizing activity (RVNA) on day 7. The endpoint of safety included the occurrence of adverse reactions and serious adverse events. RESULTS: A total of 720 participants were recruited. The adjusted-GMC of RVNA (0.41 IU/ml) on day 7 in ormutivimab group was not inferior to that in the HRIG group (0.41 IU/ml), with ratio of adjusted-GMC of 1.01 (95% confidence interval: 0.91, 1.14). The seroconversion rate of the ormutivimab group was higher than that of the HRIG group on days 7, 14, and 42. Most local injection sites and systemic adverse reactions reported from both groups were mild to moderate in severity. CONCLUSION: ormutivimab + vaccine can protect victims aged ≥18 years with category Ⅲ suspected rabies exposure as a component of postexposure prophylaxis. ormutivimab has a weaker influence on the immunity response of rabies vaccines. CLINICAL TRIALS REGISTRATION: ChiCTR1900021478 (the Chinese Clinical Trial Registry of World Health Organization).

Population Pharmacodynamic Analyses of Human Anti-Rabies Virus Monoclonal Antibody (Ormutivimab) in Healthy Adult Subjects.

Ormutivimab is the first recombinant human anti-rabies monoclonal antibody (rhRIG) approved for clinical application in China. In this study, a population pharmacodynamic (PPD) model was established to compare the neutralizing antibody activities of Ormutivimab and human rabies immunoglobulin (HRIG), alone or combined with human rabies vaccine (Vero), in a phase II clinical trial, and to recommend a target dose for the phase III trial. The model was verified to fit the PPD data well. The stability of the model was verified by the bootstrap method. The level of neutralizing antibodies in vivo increased rapidly after administration of Ormutivimab or HRIG. Neutralizing antibodies with a strong activity were produced at 7 days (Ormutivimab + vaccine) or 10 days (HRIG + vaccine) after induction by the vaccine in vivo. Compared to that induced by HRIG + vaccine, the level of the neutralizing antibodies induced by Ormutivimab + vaccine peaked higher and faster. The levels of neutralizing antibodies induced by Ormutivimab + vaccine and HRIG + vaccine were similar within 21 days after administration. According to these results and the safety data, 20 IU·kg-1 was recommended as the target dose in the confirmatory study of Ormutivimab. Registration: ClinicalTrials.gov #NCT02559921.

Efficacy of ormutivimab, a novel recombinant human anti-rabies monoclonal antibody, in post-exposure prophylaxis animal models.

Human rabies is a serious public health problem that can't be ignored. Rabies immune globulin (RIG) is an indispensable component of rabies post-exposure prophylaxis (PEP). However, current PEP relies on RIG purified from pooled human or equine plasma, which are either in chronic shortage or associated with safety concerns. Monoclonal antibodies have become widely accepted as safer and more cost-effective alternatives to RIG products in recent years. Here, we assessed the neutralization breadth of human monoclonal antibody ormutivimab and its protective efficacy in PEP models. Ormutivimab was able to neutralize a broad panel of Chinese prevalent street RABVs with neutralizing potency form 198-1487.6 IU/mL. Furthermore, ormutivimab offered comparable protection to that with HRIG both at standard doses (20 IU/kg) and higher doses (100 IU/kg and 200 IU/kg). The interference of ormutivimab on vaccine potency was also analyzed and found slightly reduced neutralizing antibody titers similar to HRIG. The broad-spectrum neutralization activities, highly protective potency, and rapid onset of action make ormutivimab an effective candidate for human rabies PEP.

Method validation of Q-PCR detection of host residual DNA in antibody drug based on protein A magnetic beads.

The residual DNA derived from host cells in antibody drugs have potential safety risks. In this paper, the antibody in the test sample was removed by magnetic bead separation method, and the residual DNA were quantitatively determined by Q-PCR method. The residual DNA in the sample was analyzed according to the standard curve. We validated the species specificity, accuracy, precision, quantitative restrictions, reproducibility of this method. The results showed the linearrange was of 1 × 10-1～1 × 102 pg/µL and the curve linear was good, this method can specifically detect CHO cell DNA. Compared with the method of extracting residual DNA by magnetic beads, the method has the advantages of simplicity, rapidity and low cost, and can be used for quantitative determination of the residual host cell DNA in antibody drugs producted by CHO cells.

Rabies virus glycoprotein serology ELISA for measurement of neutralizing antibodies in sera of vaccinated human subjects.

BACKGROUND: Vaccination provides protection against infection by inducing VNAs mainly against RABV surface GP. The measurement of VNAs to RABV is commonly used to assess the level of immunity in humans and animals after vaccination. A VNA titer of  ≥ 0.5 IU/mL of sera indicates adequate response to vaccination. Here, we report the development and validation of a RABV GP serology ELISA kit for semi-quantitative measurement of VNA titers in sera of vaccinated human subjects. METHODS: Using a recombinant RABV GP expressed in mammalian cells as the capture antigen, the ELISA method was established using HuMAb NM57 reference initially and HRIG reference subsequently. The limit of detection (LOD), linear range, reproducibility, and precision of the method were examined. Specificity and sensitivity were established to assess the diagnostic accuracy. RESULTS: RABV GP for ELISA plate coating and optimal dilution of human serum sample was 1 µg/mL and 1:20, respectively. Multiple assays were carried out by different technicians at different laboratories for assay standardization. Using the HRIG reference, the LOD was found to be 0.02-0.06 IU/mL and the linear range was 0.2-10.0 IU/ mL. The inter-assay CVs were in the range of 6.60-10.79%, indicating the reproducibility. None of the 12 known negative human sera, tested positive by ELISA, highlighting the specificity. A total of 415 unknown positive human sera were double-blind tested by the RFFIT and ELISA. The VNA titer cut-off value of ELISA was set at 1.5 IU/mL to ensure no false-positive. The diagnostic specificity and sensitivity were 100% and 91.1%, respectively. CONCLUSIONS: The validation data characterize this ELISA as a suitable method for semi-quantitative measurement of VNA titers in human serum samples to assess vaccination status. The ELISA kit can offer simplicity, speed, low cost and high throughput, making it a practical tool for monitoring the immune response following vaccination.

[Research progress of IgG4 in isotype selection of antibody drugs].

Many specific therapeutic antibody drugs have been developed for different indications. In drug development, it has been found that the antibody isotype framework can not only affect the physical and chemical properties of therapeutic antibodies, but also influence the activity and therapeutic effect. As a result, IgG isotype selection should be considered carefully in antibody drug development strategies. Because of the unique biological characteristics, IgG4 isotype has been used in some therapeutic antibodies for which effector functions are not desired. In order to provide new ideas for the development of antibody drugs, the research and application progress of IgG4 isotype in therapeutic antibody drug development has been reviewed.

[Testing immunogenicity of recombinant antibody by surface plasmon resonance].

With the development of bio-technological drugs, drug immunogenicity evaluation has become key factor of clarifying safety and efficacy of these drugs. It has become the focus to establish a stable and reliable evaluation system. Due to the advantages such as continuous real-time monitoring, surface plasmon resonance (SPR) technology has been widely used in bio-technological drugs immunogenicity assessments. Our study applied this technology to detect anti-drug antibody (ADA) of a recombinant human anti-rabies monoclonal antibody NM57 in the sera of 48 volunteers admitted in phase I clinical trials. This method could satisfy the basic requirements of detection of ADA.

A Na+/Ca2+ exchanger-like protein (AtNCL) involved in salt stress in Arabidopsis.

Calcium ions (Ca(2+)) play a crucial role in many key physiological processes; thus, the maintenance of Ca(2+) homeostasis is of primary importance. Na(+)/Ca(2+) exchangers (NCXs) play an important role in Ca(2+) homeostasis in animal excitable cells. Bioinformatic analysis of the Arabidopsis genome suggested the existence of a putative NCX gene, Arabidopsis NCX-like (AtNCL), encoding a protein with an NCX-like structure and different from Ca(2+)/H(+) exchangers and Na(+)/H(+) exchangers previously identified in plant. AtNCL was identified to localize in the Arabidopsis cell membrane fraction, have the ability of binding Ca(2+), and possess NCX-like activity in a heterologous expression system of cultured mammalian CHO-K1 cells. AtNCL is broadly expressed in Arabidopsis, and abiotic stresses stimulated its transcript expression. Loss-of-function atncl mutants were less sensitive to salt stress than wild-type or AtNCL transgenic overexpression lines. In addition, the total calcium content in whole atncl mutant seedlings was higher than that in wild type by atomic absorption spectroscopy. The level of free Ca(2+) in the cytosol and Ca(2+) flux at the root tips of atncl mutant plants, as detected using transgenic aequorin and a scanning ion-selective electrode, required a longer recovery time following NaCl stress compared with that in wild type. All of these data suggest that AtNCL encodes a Na(+)/Ca(2+) exchanger-like protein that participates in the maintenance of Ca(2+) homeostasis in Arabidopsis. AtNCL may represent a new type of Ca(2+) transporter in higher plants.

Generation and characterization of neutralizing human recombinant antibodies against antigenic site II of rabies virus glycoprotein.

The currently recommended treatment for individuals exposed to rabies virus (RV) is post-exposure prophylaxis (PEP) through the combined administration of rabies vaccine and rabies immune globulin (RIG). Human monoclonal antibodies (mAbs) that neutralize RV offer an opportunity to replace RIG for rabies PEP. Here, a combinatorial human Fab library was constructed using antibody genes derived from the blood of RV-vaccinated donors. Selections of this library against purified RV virions resulted in the identification of 11 unique Fab antibodies specific for RV glycoprotein. Of the Fab antibodies, five were converted to full human IgG1 format. The human IgG antibodies revealed high binding affinity and neutralizing activities against RV fixed strains through a rapid fluorescent focus inhibition test in vitro as well as the early stage protective function after exposure to RV infection in vivo. Furthermore, epitope mapping and binding competition analysis showed that all of obtained human neutralizing and protective antibodies were directed to the antigenic site II of RV glycoprotein. Our results provide not only important insight into the protective immune response to RV in humans, but also more candidates eligible for use in a mAb cocktail aimed at replacing RIG for rabies post-exposure prophylaxis.

A "GC-rich" method for mammalian gene expression: a dominant role of non-coding DNA GC content in regulation of mammalian gene expression.

High mammalian gene expression was obtained for more than twenty different proteins in different cell types by just a few laboratory scale stable gene transfections for each protein. The stable expression vectors were constructed by inserting a naturally-occurring 1.006 kb or a synthetic 0.733 kb DNA fragment (including intron) of extremely GC-rich at the 5' or/and 3' flanking regions of these protein genes or their gene promoters. This experiment is the first experimental evidence showing that a non-coding extremely GC-rich DNA fragment is a super "chromatin opening element" and plays an important role in mammalian gene expression. This experiment has further indicated that chromatin-based regulation of mammalian gene expression is at least partially embedded in DNA primary structure, namely DNA GC-content.

[Experimental choroidal neovascularization is inhibited by subretinal administration of Endostatin].

OBJECTIVE: Endostatin is an endogeneous angiogenesis inhibitor. The purpose of this study was to investigated the effect of Endostatin on the eyes of rats with experimental choroidal neovascularization (CNV). METHODS: Experimental CNV was induced by laser photocoagulation. Animals were given subretinal injections of recombinant human Endostatin 20 microl (5 g/L) or 0.9% chlorine sodium. The intensity of fluorescein leakage from the photocoagulated lesions was studied 13 days after photocoagulation. The area of CNV at each rupture site was measured using high molecular weight FITC-dextran (MW 2 x 10(6)) for high resolution angiography in RPE-choroid-sclera flat mounts. In addition, 8 eyes in each group were removed and fixed 14 days after photocoagulation, cut into thin sections. The sections were examined by light microscopy. Immunolocalization of Endoglin (CD105) and factor VIII on sections of CNV lesions was studied by immunohistochemical evaluation. RESULTS: After Endostatin injection, fluorescein leakage from the CNV lesions decreased significantly compared with the control eyes. The average area of CNV at sites of the Bruch's membrane rupture showed significant difference in eyes injected with Endostatin compared with control eyes. Endothelial cells demonstrated strong immunoreactivity of CD105 and factor VIII in CNV lesions of control eyes. CD105-positive cell were not detected in normal chorioretinal tissues. CONCLUSIONS: The development of CNV can be inhibited by injection of Endostatin, which suggest that Endostatin may be beneficial in treating CNV and that further studies can be considered to evaluate this possibility.

Purification and characterization of recombinant human anti-HAV monoclonal antibody.

In order to obviate the drawbacks of plasma immunoglobulins, the whole molecular recombinant human anti-HAV (hepatitis A virus) monoclonal antibody (anti-HAV IgG) produced and secreted by rCHO cells was purified and its physicochemical properties were extensively characterized. The rCHO cells were cultured in serum-free medium and the supernatants were collected. The recombinant human IgG molecules were sequentially purified by ultrafiltration, rProtein A Sepharose Fast Flow affinity chromatography, ion exchange chromatography and diafiltration. In affinity chromatography, prior to the target protein elution, an intermediate high salt wash step was inserted, different pH and salt concentrations were evaluated for the capacity of removing host cell DNA. The yield of the downstream purification process was approximately 40%. The purity of anti-HAV IgG thus generated was assayed with SEC-HPLC method, integration result showed that the monomeric IgG content was more than 99%. Western-blot was carried out with AP-antiHuman IgG (Fab specific) and AP-antiHuman IgG (Fc specific) respectively, the blot result demonstrated that the anti-HAV IgG is human antibody with Fab and Fc structure. The specific anti-HAV activity determined by ELISA was 100 IU/mg, with anti-HAV immunoglobulin as the working standard reference. Ligand leakage in the eluate of the affinity column was approximately 32 ng/mg IgG, while after further purification steps, it was decreased to less than 2 ng/mg IgG. Residual host cell DNA was monitored with solid dot blot assay, DNA can be removed effectively with intermediate high salt wash step in the affinity chromatography. Free sulfhydryl content of anti-HAV IgG was assayed with fluorescent spectrophotometer, the low molecular weight bands appeared in non-reducing SDS-PAGE may be caused by the presence of free sulfhydryl. The endotoxin content was less than 1EU/ mg examined by standard LAL test procedures. Anti-HAV IgG prepared with this process is able to fulfill the regulatory requirements of State Food and Drug Administration for recombinant products.