Daratumumab and Nanobody-Based Heavy Chain Antibodies Inhibit the ADPR Cyclase but not the NAD(+) Hydrolase Activity of CD38-Expressing Multiple Myeloma Cells

SIMPLE SUMMARY: Multiple myeloma is a hematological malignancy of antibody-producing plasma cells in the bone marrow. Nucleotides released from cells in the tumor microenvironment act as inflammatory danger signals. CD38 and other enzymes on the surface of cancer cells hydrolyze these nucleotides to immunosuppressive mediators, thereby hampering anti-tumor immune responses. Daratumumab and other CD38-specific antibodies mediate killing of tumor cells by natural killer cells, macrophages, and the complement system. Here, we investigated whether CD38-specific antibodies also inhibit the enzyme activity of CD38-expressing tumor cells, thereby providing a potential second mode of action. Our results showed that daratumumab and nanobody-based heavy chain antibodies inhibit the ADPR cyclase but not the NAD(+) hydrolase activity of CD38. Thus, there remains a need for better CD38-inhibitory antibodies. ABSTRACT: The nucleotides ATP and NAD(+) are released from stressed cells as endogenous danger signals. Ecto-enzymes in the tumor microenvironment hydrolyze these inflammatory nucleotides to immunosuppressive adenosine, thereby, hampering anti-tumor immune responses. The NAD(+) hydrolase CD38 is expressed at high levels on the cell surface of multiple myeloma (MM) cells. Daratumumab, a CD38-specific monoclonal antibody promotes cytotoxicity against MM cells. With long CDR3 loops, nanobodies and nanobody-based heavy chain antibodies (hcAbs) might bind to cavities on CD38 and thereby inhibit its enzyme activity more potently than conventional antibodies. The goal of our study was to establish assays for monitoring the enzymatic activities of CD38 on the cell surface of tumor cells and to assess the effects of CD38-specific antibodies on these activities. We monitored the enzymatic activity of CD38-expressing MM and other tumor cell lines, using fluorometric and HPLC assays. Our results showed that daratumumab and hcAb MU1067 inhibit the ADPR cyclase but not the NAD(+) hydrolase activity of CD38-expressing MM cells. We conclude that neither clinically approved daratumumab nor recently developed nanobody-derived hcAbs provide a second mode of action against MM cells. Thus, there remains a quest for “double action” CD38-inhibitory antibodies.