An Fc-optimized NKG2D-Ig fusion protein for induction of NK cell reactivity against breast cancer.

Abstract

3054 Background: The anti-tumor activity and clinical success of the monoclonal antibody trastuzumab, approved for treatment of HER2/neu-overexpressing breast cancer, is at least partially mediated by induction of antibody dependent cellular cytotoxicity (ADCC). However, only about 20% of patients show HER2/neu overexpression, and trastuzumab treatment is associated with side effects. The ligands of the activating immunoreceptor NKG2D (NKG2DL) are widely expressed on malignant cells, but generally absent on healthy tissue. We aimed to take advantage of this tumor-restricted expression by using NKG2DL as target-antigens on breast cancer cells. To this end we generated NKG2D-Ig fusion proteins with modified Fc moieties and studied their ability to induce NK cell anti-tumor reactivity. Methods: The Fc parts within the constructs were modified by amino acid exchange as previously described (Lazar 2006; Armour 1999). Direct effects on tumor cell viability as well as induction of NK cell activation, degranulation, cytotoxicity and IFN-γ release in cultures with breast cancer cell lines expressing different HER2/neu levels were determined. Results: Compared to NKG2D-Fc containing a wildtype Fc part (NKG2D-Fc-WT) or trastuzumab, 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 Fc receptor, respectively. In contrast to trastuzumab, no direct effect of the constructs on tumor cell viability was observed. In cultures of NK cells and breast cancer cells, NKG2D-Fc-KO significantly reduced NK reactivity due to blocking immunostimulatory NKG2D-NKG2DL interaction. NKG2D-Fc-WT substantially enhanced NK reactivity by induction of ADCC, while the effects of NKG2D-Fc-ADCC by far exceeded that of NKG2D-Fc-WT and, in case of HER2/neu-low targets also that of Herceptin. Conclusions: Fc-engineered NKG2D-Ig fusion protein effectively target breast cancer cells for NK anti-tumor reactivity. Due to the tumor-restricted expression of NKG2DL, NKG2D-Fc-ADCC may constitute an attractive means for immunotherapy especially of HER2/neu-low or -negative breast cancer.