NKG2D Ligand-Targeted Bispecific T-Cell Engagers Lead to Robust Antitumor Activity against Diverse Human Tumors.

Two new bispecific T-cell engaging (BiTE) molecules with specificity for NKG2D ligands were developed and functionally characterized. One, huNKG2D-OKT3, was derived from the extracellular portion of the human NKG2D receptor fused to a CD3 epsilon binding single-chain variable fragment (scFv), known as OKT3. NKG2D has multiple ligands, including MICA, which are expressed by a variety of malignant cells. A second molecule, B2-OKT3, was created in the tandem scFv BiTE format that targets MICA on tumor cells and CD3 epsilon on human T cells. Both BiTEs specifically activated T cells to kill human tumor cell lines. Cytotoxicity by B2-OKT3, but not huNKG2D-OKT3, is blocked by soluble rMICA. The huNKG2D-OKT3 induced greater T-cell cytokine production in comparison with B2-OKT3. No T-cell pretreatment was required for IFN gamma production upon coculture of B2-OKT3 or huNKG2D-OKT3 with T cells and target cells. The effector memory T-cell compartment was the primary source of IFNg, and culture of T cells and these BiTEs with plate-bound rMICA showed ligand density-dependent production of IFNg from both CD4(+) and CD8(+) T cells. There was 2-fold more IFN gamma produced per CD8(+) T cell and 5-fold greater percentage of CD8(+) T cells producing IFN gamma compared with CD4(+) T cells. In addition, both BiTEs elicited significant antitumor responses against human metastatic melanoma tumor samples using autologous or healthy donor T cells. These data demonstrate the robust antitumor activity of these NKG2D ligand-binding bispecific proteins and support their further development for clinical use. (C) 2017 AACR.