Detection of Antibodies against the Four Subtypes of Ebola Virus in Sera from Any Species Using a Novel Antibody-Phage Indicator Assay

AbstractThe natural host for Ebola virus, presumed to be an animal, has not yet been identified despite an extensive search following several major outbreaks in Africa. A straightforward approach used to determine animal contact with Ebola virus is by assessing the presence of specific antibodies in serum. This approach however has been made very difficult by the absence of specific reagents required for the detection of antibodies from the majority of wild animal species. In this study, we isolated a human monoclonal antibody Fab fragment, KZ51, that reacts with an immunodominant epitope on Ebola virus nucleoprotein (NP) that is conserved on all four Ebola virus subtypes. The antibody KZ51 represents a major specificity as sera from all convalescent patients tested (10/10) and sera from guinea pigs infected with each of the four Ebola virus subtypes competed strongly with KZ51 for binding to radiation-inactivated Ebola virus. These features allowed us to develop a novel assay for the detection of seroconversion irrespective of Ebola virus subtype or animal species. In this assay, the binding of KZ51 Fab-phage particles is used as an indicator assay and the presence of specific antibodies against Ebola virus in sera is indicated by binding competition. A prominent feature of the assay is that the Fab-phage particles may be prestained with a dye so that detection of binding can be directly determined by visual inspection. The assay is designed to be both simple and economical to enable its use in the field

Hexamerization-enhanced CD20 antibody mediates complement-dependent cytotoxicity in serum genetically deficient in C9

We examined complement-dependent cytotoxicity (CDC) by hexamer formation-enhanced CD20 mAb Hx-7D8 of patient-derived chronic lymphocytic leukemia (CLL) cells that are relatively resistant to CDC. CDC was analyzed in normal human serum (NHS) and serum from an individual genetically deficient for C9. Hx-7D8 was able to kill up to 80% of CLL cells in complete absence of C9. We conclude that the narrow C5b-8 pores formed without C9 are sufficient for CDC due to efficient antibody-mediated hexamer formation. In the absence of C9, we observed transient intracellular increases of Ca2 + during CDC (as assessed with FLUO-4) that were extended in time. This suggests that small C5b-8 pores allow Ca2 + to enter the cell, while dissipation of the fluorescent signal accompanying cell disintegration is delayed. The Ca2 + signal is retained concomitantly with TOPRO-3 (viability dye) staining, thereby confirming that Ca2 + influx represents the most proximate mediator of cell death by CDC

The INNs and outs of antibody nonproprietary names

An important step in drug development is the assignment of an International Nonproprietary Name (INN) by the World Health Organization (WHO) that provides healthcare professionals with a unique and universally available designated name to identify each pharmaceutical substance. Monoclonal antibody INNs comprise a –mab suffix preceded by a substem indicating the antibody type, e.g., chimeric (-xi-), humanized (-zu-), or human (-u-). The WHO publishes INN definitions that specify how new monoclonal antibody therapeutics are categorized and adapts the definitions to new technologies. However, rapid progress in antibody technologies has blurred the boundaries between existing antibody categories and created a burgeoning array of new antibody formats. Thus, revising the INN system for antibodies is akin to aiming for a rapidly moving target. The WHO recently revised INN definitions for antibodies now to be based on amino acid sequence identity. These new definitions, however, are critically flawed as they are ambiguous and go against decades of scientific literature. A key concern is the imposition of an arbitrary threshold for identity against human germline antibody variable region sequences. This leads to inconsistent classification of somatically mutated human antibodies, humanized antibodies as well as antibodies derived from semi-synthetic/synthetic libraries and transgenic animals. Such sequence-based classification implies clear functional distinction between categories (e.g., immunogenicity). However, there is no scientific evidence to support this. Dialog between the WHO INN Expert Group and key stakeholders is needed to develop a new INN system for antibodies and to avoid confusion and miscommunication between researchers and clinicians prescribing antibodies

Towards effective immunotherapy of myeloma: enhanced elimination of myeloma cells by combination of lenalidomide with the human CD38 monoclonal antibody daratumumab

Background: In our efforts to develop novel effective treatment regimens for multiple myeloma we evaluated the potential benefits of combining the immunomodulatory drug lenalidomide with daratumumab. Daratumumab is a novel human CD38 monoclonal antibody which kills CD38+ multiple myeloma cells via antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity and apoptosis. Design and Methods To explore the effect of lenalidomide combined with daratumumab, we first carried out standard antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity assays in which the CD38+ multiple myeloma cell line UM-9 and primary multiple myeloma cells isolated from patients were used as target cells. We also tested the effect of lenalidomide on daratumumab-dependent cell-mediated-cytotoxicity and complement-dependent cytotoxicity of multiple myeloma cells directly in the bone marrow mononuclear cells of multiple myeloma patients. Finally, we determined the daratumumab-dependent cell-mediated cytotoxicity using peripheral blood mononuclear cells of multiple myeloma patients receiving lenalidomide treatment. Results Daratumumab-dependent cell-mediated cytotoxicity of purified primary multiple myeloma cells, as well as of the UM-9 cell line, was significantly augmented by lenalidomide pre-treatment of the effector cells derived from peripheral blood mononuclear cells from healthy individuals. More importantly, we demonstrated a clear synergy between lenalidomide and daratumumab- induced antibody-dependent cell-mediated cytotoxicity directly in the bone marrow mononuclear cells of multiple myeloma patients, indicating that lenalidomide can also potentiate the daratumumab-dependent lysis of myeloma cells by activating the autologous effector cells within the natural environment of malignant cells. Finally, daratumumab-dependent cellmediated cytotoxicity was significantly up-regulated in peripheral blood mononuclear cells derived from 3 multiple myeloma patients during lenalidomide treatment. Conclusions Our results indicate that powerful and complementary effects may be achieved by combining lenalidomide and daratumumab in the clinical management of multiple myelom