Mimicking An Induced Self Phenotype by Coating Lymphomas with the Nkp30-Ligand B7-H6 Promotes Antitumoral Natural Killer Cell Cytotoxicity

Abstract 103

Induced self-expression of ligands for stimulatory receptors facilitates natural killer (NK) cell-mediated elimination of stressed cells. Stimulatory receptors include Natural killer group 2 member D (NKG2D) and Nkp30, which control cytotoxic activities of NK cells and are important in immune surveillance against tumors. Specific modulation of NK cell cytotoxicity by selectively increasing the surface density of activating ligands on tumor cells may therefore represent an innovative approach to develop novel treatment strategies. A novel fusion protein was designed to enhance NK cell-based immune responses against B-lineage lymphomas by increasing the cell surface density of the recently identified Nkp30 ligand B7-H6 on tumor cells. The recombinant protein consisted of the ectodomain of B7-H6 and a CD20-directed human single chain fragment variable (scFv) as targeting device. The resulting fully-human protein designated B7-H6:CD20-scFv was eukaryotically expressed and purified by affinity chromatography. B7-H6:CD20-scFv indeed had bifunctional properties as reflected by its ability to simultaneously bind to the CD20 antigen and to the Nkp30 receptor. CD20-positive lymphoma cells opsonized with B7-H6:CD20-scFv alerted human NK cells as indicated by upregulated surface expression levels of the early inducible activation marker CD69. Activation was accompanied by induced CD107a cell surface exposure indicating enhanced NK cell degranulation. In cytotoxicity assays using human NK cells from healthy donors as effector cells, B7-H6:CD20-scFv triggered killing of lymphoma-derived B-cell lines. B7-H6:CD20-scFv was active in a strictly antigen-specific manner as demonstrated by blocking experiments and was not able to mediate killing of cell lines not expressing the CD20 target antigen. B7-H6:CD20-scFv mediated killing of lymphoma cells in a dose-dependent manner starting at nanomolar concentrations. Target cell death induced by B7-H6:CD20-scFv occurred by apoptosis and involved caspase cleavage. Moreover, B7-H6:CD20-scFv induced NK cell-mediated lysis of fresh tumor cells from 8/8 CLL and 5/5 MCL patients with variable CD20 expression levels. In comparison to ULBP2:CD20-scFv, a similarly constructed fusion protein of the NKG2D ligand ULBP2 and a CD20-directed scFv, the B7-H6:CD20-scFv had a lower potency (EC50 values for B7-H6:CD20-scFv and ULBP2:CD20-scFv were 100 and 4 nM, respectively) but nevertheless achieved similar maximum extents of lysis. Interestingly, when B7-H6:CD20-scFv was added together with ULBP2:CD20-scFv to a mixture of NK cells and target cells, synergistic cytotoxic effects were induced. The combined treatment resulted in a higher percentage of NK cells that responded and exposed the degranulation marker CD107a on the cell surface in comparison to samples containing only one of the two agents. As a consequence a significantly higher extent of lysis was achieved. These results strongly indicate a co-operation between Nkp30 and NKG2D signalling which use different downstream signalling pathways. Thus, mimicking an induced self phenotype of tumors by coating lymphomas with B7-H6:CD20-scFv either alone or in combination with molecules triggering NKG2D may provide an innovative strategy to enhance specific anti-tumoral NK cell cytotoxicity.