CD38-specific nanobody-based bispecific antibody recruiters (BARs) redirect complement-dependent cytotoxicity toward multiple myeloma cells

Bispecific antibody recruiters (BARs) are an innovative class of immunotherapeutics that redirect endogenous antibodies to tumor cells. BARs comprise a tumor-binding and an antibody-binding module, enabling endogenous antibodies to opsonize tumor cells and induce FC-dependent effector functions such as complement-dependent cytotoxicity (CDC). CD38 is a validated target for myeloma therapy, as evidenced by the success of CD38-specific monoclonal antibodies daratumumab and isatuximab. Nanobodies are single variable immunoglobulin domains derived from camelid heavy chain antibodies. Here, we report the generation of nanobody-based BARs recognizing CD38 and assess their cytotoxicity against CD38-expressing myeloma cells in vitro and ex vivo. We constructed three CD38-specific BARs recognizing distinct, non-overlapping CD38 epitopes (E1-BAR, E2-BAR, and E3-BAR) by genetically fusing CD38-specific nanobodies to a human immunoglobulin κ light chain-specific nanobody. All BARs exhibited simultaneous binding to CD38 and IgGκ. Nonlinear regression revealed EC50 values of 0.73 nM (E1-BAR), 0.21 nM (E2-BAR), and 0.97 nM (E3-BAR). In patient-derived myeloma cells, E1-BAR reduced viability to 29 ± 18%, while E2-BAR and E3-BAR achieved 62 ± 49% and 57 ± 41%, respectively. In vivo, BAR half-life was markedly increased by IgGκ binding. Our results demonstrate the feasibility of CD38-specific nanobody-based BARs as therapeutics for multiple myeloma.