Affibody-Based BCMA x CD16 Dual Engagers for Activation of NK Cells Towards Multiple Myeloma

The re-direction of natural killer (NK) cells is an emerging strategy for cancer immunotherapy. We describe the development and characterization of the so far smallest NK cell re-directing hBCMA x CD16 dual engagers for potential multiple myeloma treatment based on combinations of 7 kDa non-immunoglobulin affibody affinity proteins (Fig. 1). Affibodies to human CD16a protein (low affinity Fc-gamma receptor IIIa) were selected by phage display which resulted in the identification of binders with individual affinities (KD) for CD16a in the range of 100 nM to 3 µM, depending on the CD16 allotype (158V/F) and clone used in the analysis. Pairwise epitope binding experiments showed that binders with non-overlapping epitopes could be identified, allowing for the construction of bi-paratopic heterodimers with subnanomolar affinity for CD16a. In addition, a bivalent construct based on the variant with the highest monovalent affinity, denoted A10, showed subnanomolar apparent affinity (avidity) for both CD16a allotypes. The binding epitope of A10 was shown not to overlap with the Fc (hinge) binding activity of CD16a. For the construction of hBCMA x CD16 dual engagers, different CD16a binding affibody constructs containing one or two CD16a binding affibodies were genetically fused to a high-affinity (KD of ca. 7 nM) hBCMA-specific affibody, or a null-variant thereof. These 15-23 kDa dual engager constructs showed simultaneous hBCMA and CD16a binding ability and could efficiently induce synapse formation, activate resting primary NK cells and trigger specific lysis of a panel of hBCMA-positive multiple myeloma cell lines (Fig. 2), without evidence of NK cell fratricide. Hence, we report a novel class of versatile and uniquely small NK cell engagers with potent and specific binding properties and functional profiles. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal