Fusion Proteins of Ligands for NKG2D and CD20-Directed ScFvs Sensitize Lymphomas for NK Cell-Mediated Lysis

Abstract 2843

Natural killer group 2 member D (NKG2D) is an important activating receptor controlling cytotoxicity of natural killer (NK) cells and T cells and plays an important role in immune surveillance against tumors. For redirecting NK cells to B-lymphoid tumor cells two recombinant bifunctional antibody-based fusion proteins were designed in order to coat malignant cells with ligands for NKG2D and attract NK cells. Therefore, a human CD20-directed single-chain fragment variable (scFv) was fused to NKG2D-specific ligands, either MHC class I chain-related protein A (MICA) or unique long 16-binding protein 2 (ULBP2). These two fully human fusion proteins, designated MICA:CD20 and ULBP2:CD20, respectively, were expressed in eukaryotic cells and purified to homogeneity. Size exclusion chromatography revealed that both purified proteins predominantly formed monomers. MICA:CD20 and ULBP2:CD20 specifically and simultaneously bound to CD20 and NKG2D and efficiently mediated lysis of lymphoma cell lines with mononuclear cells from healthy donors as effector cells. Analysis of the activation status of NKG2D-positive T cells and NK cells revealed that MICA:CD20 and ULBP2:CD20 activated resting NK cells, but not T cells, indicating that NK cells were the relevant effector cell population for the two molecules. In cytotoxicity assays using human NK cells from healthy donors, both agents sensitized lymphoma cell lines as well as fresh tumor cells for NK cell-mediated lysis. MICA:CD20 and ULBP2:CD20 induced lysis at low nanomolar concentrations with half maximum effective concentrations between 1 and 4 nM depending on target cells. Interestingly, ULBP2:CD20 exhibited a higher cytolytic potential than MICA:CD20 in terms of maximum lysis. Importantly, MICA:CD20 and ULBP2:CD20 induced lysis of 13/13 tested primary tumor cell samples from patients with different B cell malignancies including chronic lymphocytic leukemia, mantle cell lymphoma and marginal zone lymphoma. Interestingly, cell surface expression of endogenous MICA and ULBP2 was low or not detectable on fresh tumor cells. In addition, ULBP2:CD20 was also capable of inducing lysis of tumor cells in cytotoxicity experiments using autologous patient-derived NK cells as effector cells, indicating that the triggering signal was sufficient to overcome inhibition by interactions between killer cell immunoglobulin-like receptors and MHC class I molecules. Moreover, both MICA:CD20 and ULBP2:CD20 synergistically enhanced antibody-dependent cellular cytotoxicity (ADCC) by the monoclonal antibody daratumumab directed against CD38 which is co-expressed together with CD20 on certain B cell lymphomas. This approach of simultaneously triggering ADCC and natural cytotoxicity by these bifunctional fusion proteins may represent a promising strategy to achieve stronger NK cell-mediated antitumor responses.