Abstract
Induced self expression of the NKp30 ligand B7-H6 facilitates NK cell-mediated elimination of stressed cells. A fusion protein consisting of the ectodomain of B7-H6 and the CD20 single-chain fragment variable 7D8 was generated to mimic an induced self phenotype required for NK cell-mediated target cell elimination. B7-H6:7D8 had bifunctional properties as reflected by its ability to simultaneously bind to the CD20 Ag and to the NKp30 receptor. B7-H6:7D8 bound by CD20(+) lymphoma cells activated human NK cells and triggered degranulation. Consequently, the immunoligand B7-H6:7D8 induced killing of lymphoma-derived cell lines as well as fresh tumor cells from chronic lymphocytic leukemia or lymphoma patients. B7-H6:7D8 was active at nanomolar concentrations in a strictly Ag-specific manner and required interaction with both CD20 and NKp30. Remarkably, NK cell cytotoxicity was further augmented by concomitant activation of Fcγ receptor IIIa or NK group 2 member D. Thus, B7-H6:7D8 acted synergistically with the CD20 Ab rituximab and the immunoligand ULBP2:7D8, which was similarly designed as B7-H6:7D8 but engaging the NK group 2 member D receptor. In conclusion, to our knowledge, B7-H6:7D8 represents the first Ab-based molecule stimulating NKp30-mediated NK cell cytotoxicity for therapeutic purposes and provides proof of concept that Ag-specific NKp30 engagement may represent an innovative strategy to enhance antitumoral NK cell cytotoxicity.
Highlights
Ramos cells were preincubated with B7-H6:7D8 (ULBP2:7D8 as negative control) at 50 mg/ml followed by the fusion protein NKp30-Fcko at 100 mg/ml
The extracellular domain (ECD) including the secretion leader of B7-H6 was genetically fused to the human CD20-specific single-chain fragment variable (scFv) derived from the mAb 7D8 (Fig. 1A, 1B) [34]
In good agreement to results obtained with Ramos cells, synergistic effects of ULBP2:7D8 and B7-H6:7D8 were observed when freshly isolated lymphoma or leukemia cells were used as targets (Fig. 8D), with some samples expressing low amounts of cell surface ULBP2 (Supplemental Fig. 1D)
Summary
Ramos and Raji cells (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were cultured in RPMI 1640 GlutaMAX-I medium (Invitrogen, Karlsruhe, Germany) supplemented with 10% FCS (Invitrogen), 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen). For generation of NKp30-Fcko, a fusion protein between NKp30 and a human IgG1 Fc mutant with highly reduced Fc receptor and C1q binding (amino acid substitutions, L234F/L235E/P331S; Ref. 29), the coding sequence for the ECD of NKp30 was amplified from cDNA of mononuclear cells by standard procedures using primers 59-ACGTGCTAGCCACCATGGCCTGGATGCTGTTGCTCATC-39 and 59-ACGTAC-. To analyze binding of B7-H6:7D8 and B7-H6:4D5, Ramos or MDA-MB-361 cells were incubated with either protein on ice for 45 min, washed with 2 ml PBA buffer and subsequently stained with a secondary Alexa Fluor 488-coupled anti-penta His Ab (Qiagen) on ice for 30 min. Ramos cells were preincubated with B7-H6:7D8 (ULBP2:7D8 as negative control) at 50 mg/ml followed by the fusion protein NKp30-Fcko at 100 mg/ml. Ag expression levels were analyzed by determination of NK cell-specific Ag binding capacities of NKp30 and NKG2D-specific Abs (both from R&D Systems) using Qifikit (Dako, Hamburg, Germany) according to the manufacturers’ protocols. Synergy and strong synergy were indicated by CI values of 0.3–0.7 and 0.1–0.3, respectively
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