Reconstructing the human hematopoietic niche in immunodeficient mice: opportunities for studying primary multiple myeloma.

Interactions within the hematopoietic niche in the BM microenvironment are essential for maintenance of the stem cell pool. In addition, this niche is thought to serve as a sanctuary site for malignant progenitors during chemotherapy. Therapy resistance induced by interactions with the BM microenvironment is a major drawback in the treatment of hematologic malignancies and bone-metastasizing solid tumors.

Penetration of antibody-opsonized cells by the membrane attack complex of complement promotes Ca(2+) influx and induces streamers.

We have reported that during complement-mediated cytolysis of B cells promoted by the CD20 mAbs rituximab or ofatumumab (OFA), long, thin structures that we call streamers (≥ 3 cell diameters) are rapidly generated and grow out from the cell surface. Streamers appear before cells are killed and contain opsonizing mAbs and membrane lipids. By exploiting the differential Ca(2+) requirements of discrete steps in the complement cascade, we determined that mAb-opsonized cells first tagged with C3b using C5-depleted serum are killed on addition of serum and EDTA, but the cells do not produce streamers.

Daratumumab, a novel therapeutic human CD38 monoclonal antibody, induces killing of multiple myeloma and other hematological tumors.

CD38, a type II transmembrane glycoprotein highly expressed in hematological malignancies including multiple myeloma (MM), represents a promising target for mAb-based immunotherapy. In this study, we describe the cytotoxic mechanisms of action of daratumumab, a novel, high-affinity, therapeutic human mAb against a unique CD38 epitope. Daratumumab induced potent Ab-dependent cellular cytotoxicity in CD38-expressing lymphoma- and MM-derived cell lines as well as in patient MM cells, both with autologous and allogeneic effector cells.

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.

High throughput screening for antibody induced complement-dependent cytotoxicity in early antibody discovery using homogeneous macroconfocal fluorescence imaging.

Complement-dependent cytotoxicity (CDC) represents an important Fc-mediated effector function of antibodies and is a quality often sought in candidates for therapeutic antibody development in cancer. Antibodies inducing potent CDC are relatively rare as the ability to induce CDC is strongly dependent on the antigen and epitope recognized as well as antibody isotype. To allow the identification of antibodies with optimal CDC characteristics in early stages of antibody discovery, we developed a homogeneous high throughput CDC assay, compatible with 384 and 1536 well formats and which therefore allows direct functional screening of very large panels of antibodies.

Colony lift assay using cell-coated filters: a fast and efficient method to screen phage libraries for cell-binding clones.

For efficient screening of phage antibody libraries obtained by selection on whole cells, we have developed a modified colony lift assay using cell-coated filters. Both cells growing in suspension as well as adherent cells can be coated onto nitrocellulose filters and used to detect bacterial colonies responsible for the production of cell-binding (specific) single chain variable fragment (scFv) antibodies. We demonstrate, using a selected library developed in our laboratory (named "AB" library) as a model system, that the frequency of specific clones as detected by colony lift assay using cell-coated filter is comparable to the frequency of positive clones as detected by the "classical" method (i.