@article {M{\"a}rklin143, author = {Melanie M{\"a}rklin and Ilona Hagelstein and Samuel P. Koerner and Kathrin Rothfelder and Martin S. Pfluegler and Andreas Schumacher and Ludger Grosse-Hovest and Gundram Jung and Helmut R. Salih}, title = {Bispecific NKG2D-CD3 and NKG2D-CD16 fusion proteins for induction of NK and T cell reactivity against acute myeloid leukemia}, volume = {7}, number = {1}, elocation-id = {143}, year = {2019}, doi = {10.1186/s40425-019-0606-0}, publisher = {BMJ Specialist Journals}, abstract = {Background Monoclonal antibodies (mAbs) mediate their effects in great part by inducing ADCC of NK cells, and multiple efforts aim to increase this function by engineering mAbs optimized Fc-parts. Even more potent antitumor immunity can be induced by strategies to stimulate T cells with their profoundly higher effector potential. However, upon increased immunostimulatory potential, the necessity to target highly tumor-specific antigens becomes critically important to reduce side effects.Methods We here report on bispecific fusion proteins (BFP) that target ligands of the immunoreceptor NKG2D (NKG2DL), which are widely expressed on malignant cells but generally absent on healthy tissue. They consist of the extracellular domain of NKG2D as targeting moiety fused to Fab-fragments of CD3 (NKG2D-CD3) or CD16 (NKG2D-CD16) antibodies.Results NKG2D-CD16 displayed increased affinity to the FcγRIII on NK cells compared to engineered Fc-parts, which are contained in optimized mAbs that presently undergo clinical evaluation. In line, NKG2D-CD16 induced superior activation, degranulation, IFN-γ production and lysis of acute myeloid leukemia (AML) cell lines and patient AML cells. NKG2D-CD3 in turn potently stimulated T cells, and comparison of efficacy over time revealed that NKG2D-CD16 was superior upon short term application, while NKG2D-CD3 mediated overall more potent effects which manifested after longer times. This can be attributed to treatment-induced proliferation of T cells but not NK cells.Conclusions Taken together, we here introduce novel {\textquotedblleft}antibody-like{\textquotedblright} BFP that take advantage of the highly tumor-restricted expression of NKG2DL and potently activate the reactivity of NK cells or T cells for immunotherapy of AML.Abbreviations:7-AAD7-aminoactinomycin DAbAntibodyADCCAntibody dependant cellular cytotoxicityALLAcute lymphoblastic leukemiaAMLAcute myeloid leukemiaBFPBispecific fusion proteinBiTEBispecific T cell engagerbsAbBispecific antibodyCARChimeric antigen receptorE:TEffector to targetELISAEnzyme-linked immunosorbent assayEUEndotoxinFACSFluorescence-activated cell sortingFcγRFc-gamma-receptorIFN-γInterferon-γmAbmonoclonal antibodyMFIMean fluorescence intensityMICMHC class I chain-relatedNKG2DNatural killer group 2DNKG2DLNKG2D ligandPBMCPeripheral blood mononuclear cellPEPhycoerythrinPIPropidium iodideSDIEAmino acid modifications S239D and I332ESDS-PAGESodium dodecyl sulfate polyacrylamide gel electrophoresisSEMStandard error of the meanSFISpecific fluorescence intensityTCRT cell receptorULBPUL16 binding protein}, URL = {https://jitc.bmj.com/content/7/1/143}, eprint = {https://jitc.bmj.com/content/7/1/143.full.pdf}, journal = {Journal for ImmunoTherapy of Cancer} }