Enhancing Natural Killer Cell-Mediated Lysis of Lymphoma Cells By Combining Therapeutic Antibodies with CD20-Specific Immunoligands Engaging NKG2D or NKp30

Antibody-dependent cell-mediated cytotoxicity (ADCC) represents a major effector function of many therapeutic antibodies. Thus, enhancing ADCC is a promising approach to further improve antibody therapy. Here, the CD20-specific immunoligands ULBP2:7D8 and B7-H6:7D8, which engage the stimulatory NK cell receptors natural killer group 2 member D (NKG2D) and NKp30, respectively, were compared for their abilities to boost ADCC in an attempt to design an effective antibody combination strategy.

The immunoligands are designed as single chain molecules, with a single chain fragment variable (scFv) of the CD20 antibody 7D8 fused to UL16-binding protein (ULBP) 2 or B7 homologue 6 (B7-H6), which are ligands of the activating NK cell receptors NKG2D and NKp30, respectively. By binding to lymphoma cells the immunoligands designated as ULBP2:7D8 and B7-H6:7D8 mimicked an induced self phenotype and thereby triggered NK cells to kill lymphoma and leukemia cells. Both immunoligands augmented ADCC by NK cells synergistically when combined with the lymphoma-directed antibodies rituximab or daratumumab recognizing CD20 and CD38, respectively. Antibody combinations with ULBP2:7D8 resulted in higher cytotoxicity (up to 10-fold lower EC50-values) in comparison to combinations with B7-H6:7D8, which in individual experiments failed to boost ADCC. Thus, NK cells were triggered more efficiently when NKG2D rather than NKp30 was co-ligated together with FcγRIIIA. Although a combination of ULBP2:7D8 and B7-H6:7D8 produced synergistic effects, no significant improvements were obtained by combining the three agents rituximab, B7-H6:7D8 and ULBP2:7D8. Enhancement of ADCC by the immunoligands was also achieved when NK cells from lymphoma or leukemia patients were analyzed as effector cells. ULBP2:7D8 in particular increased lysis not only of allogeneic but also of autologous tumor cells.

In summary, co-targeting of NKG2D was more effective in promoting NK cell-mediated ADCC than co-ligation of NKp30 and may represent a promising approach to further enhance the efficacy of therapeutic antibodies. Based on these results we propose a ‘dual-dual-targeting’ concept by co-targeting of two surface antigens on tumor cells and concomitant engagement of two different activating NK cell receptors.