SAR 650984, a Therapeutic Anti-CD38 Monoclonal Antibody, Blocks CD38-CD31 Interaction in Multiple Myeloma

CD38, a type II transmembrane glycoprotein with both ecto-enzyme activity and receptor function, is highly expressed at the surface of many hematological cancer cells. Recently, SAR 650984 (SAR), a novel humanized anti-CD38 monoclonal antibody in early clinical development, has shown an impressive and durable single agent activity in patients with multiple myeloma (MM). To further characterize the biological function of CD38 in MM cells, we generated RPMI8226 MM cells overexpressing CD38 (R-CD38) by lentiviral infection followed by blascitidin selection. R-CD38, maintained in 10 mg/ml of blascitidin-containing media, expresses 6-fold greater CD38 mRNA and protein levels than control RPMI8226 cells, as confirmed by RNAseq and quantitative flow cytometry. To study whether CD38 levels affect cell signaling cascade in MM cells, the phosphorylated forms of various signaling proteins were analyzed using Kinexä Antibody Microarray. At least 20 and 8 phospho-signaling molecules were significantly increased and reduced, respectively, in R-38 vs control RPMI8226 cells. Upregulated kinases include HO1, CREB1, IRAK1, and ERK1/2, whereas calreticulin and RSK1/2 are downregulated. RNAseq profiling on Illumina HiSeq was next done to identify CD38-targeted genes in MM cells. A volcano plot shows that 123 genes are induced and 32 repressed with FC>= 4 (positive or negative) and p value <=0.01. Enrichment in functional categories in these 155 gene differentially regulated genes further revealed predominant changes in plasma membrane, cytoskeleton, cellular adhesion, synaptic plasticity, neuronal transmission, transport, and Ca2+ regulation. Gene set enrichment analysis on log2(R-CD38/control RPMI8226) profile also indicates downregulated genes involved in interferon response and MHC class I, whereas myogenic and astrocytic marks are upregulated. Ongoing studies include NextBio/Meta analysis in SAR-treated R-38 vs RPMI8226 cells. Importantly, we found that R-CD38 cells are more sensitive than control RPMI8226 to lysis induced by SAR-mediated antibody-dependent cellular cytotoxicity (ADCC) using at least 3 donor effector cells, confirming CD38 overexpression in R-CD38 vs control cells. In addition, SAR directly induces apoptosis of R-CD38 cells but not control cells, with neither effector cells nor FcgR cross-linking (Abstract #72699). Although overexpression of CD38 in R-CD38 cells did not alter cell proliferation, R-CD38 showed increased adherence to bone marrow stromal cells (BMSCs) derived from MM patients (n=3) relative to controls. Importantly, SAR (0-10 mg/ml) inhibited MM cell adherence in a dose-dependent manner. Similar SAR effect in MM cell adhesion was seen when HS-5 stromal cells were used. In order to investigate the role of CD31, a physiological ligand for CD38, in MM cell adhesion to bone marrow (BM) microenvironment, we next performed MM cell adhesion assays using CD14+ monocytes and HUVEC cells which express significantly higher CD31 levels than BMSCs and HS-5 cells. At the same concentrations of SAR, % of inhibition in adhesion of MM cell lines (R-CD38, MOLP8, MM1S, H929) to monocytes was approximately 10-fold higher than to BMSC. Furthermore, SAR blocked MM cell adhesion to HUVEC more potently than adhesion to BMSC. These results indicate that SAR decreases MM cell adhesion to BM accessory cells via blockage of CD31-CD38 interaction, thus suggesting a potential new mechanism of action of SAR to elicit its anti-MM activity.