Induction of NK Cell Reactivity in Breast Cancer by Fc-Engineered NKG2D-Ig Fusion Proteins

Abstract 253

NK cells are cytotoxic lymphocytes that play an important role in anti-tumor immunity. Their capability to mediate Fc-receptor dependent effector functions like antibody dependent cellular cytotoxicity (ADCC) largely contributes to the clinical success of anti-tumor antibodies like Herceptin (Trastuzumab®), which is approved for treatment of breast cancer displaying HER2/neu-overexpression. Notably, only about 20% of breast cancer patients show overexpression of HER2/neu. Moreover, this antigen is also expressed on healthy cells, and application of Herceptin is associated with side effects. In contrast, ligands of the activating immunoreceptor NKG2D (NKG2DL) are widely expressed on malignant cells, but generally absent on healthy tissues. We aimed to take advantage of the tumor-restricted expression of NKG2DL by using them as tumor-antigens for Fc-optimized NKG2D-Ig fusion proteins targeting breast cancer cells for NK cell ADCC and IFN-γ production. NKG2D-Ig fusion proteins with distinct modifications in their Fc portion were generated by amino acid exchange as previously described (Lazar 2006; Armour 1999). Compared to wildtype NKG2D-Fc (NKG2D-Fc-WT) or Herceptin, our mutants (S239D/I332E and E233P/L234V/L235A/ΔG236/A327G/A330S) displayed highly enhanced (NKG2D-Fc-ADCC) and abrogated (NKG2D-Fc-KO) affinity to the NK cell FcγRIIIa receptor (CD16), respectively. This resulted in lacking (NKG2D-Fc-KO) or highly enhanced (NKG2D-Fc-ADCC) NK cell activation. In cultures of NK cells and breast cancer cells, NKG2D-Fc-KO significantly reduced NK cell reactivity due to blockade of NKG2DL-mediated activating signals, while NKG2D-Fc-WT substantially enhanced NK reactivity by induction of ADCC and cytokine production. Notably, the effect of our NKG2D-Fc-ADCC by far exceeded that of NKG2D-Fc-WT and, in case of HER2/neu low targets, also that of Herceptin, resulting in potently enhanced NK anti-tumor reactivity. Together, our results demonstrate that Fc-engineered NKG2D-Fc-ADCC fusion proteins can effectively target NKG2DL-expressing cancer cells for NK anti-tumor reactivity. In line with the hierarchically organized potential of the various activating receptors governing NK reactivity and due to its highly increased affinity to CD16 NKG2D-Fc-ADCC potently enhances NK cell reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL. Due to the tumor-restricted expression of NKG2DL, NKG2D-Fc-ADCC may thus constitute an attractive means for immunotherapy, especially of HER2/neu-low or -negative breast cancer.