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 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 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 to B2-OKT3. No T cell pre-treatment was required for IFN production upon co-culture of B2-OKT3 or huNKG2D-OKT3 with T cells and target cells. The effector memory T cell compartment was the primary source of IFN, and culture of T cells and these BiTEs with plate-bound rMICA showed ligand density dependent production of IFN from both CD4⁺ and CD8⁺ T cells. There was two-fold more IFN produced per CD8⁺ T cell and five-fold greater percentage of CD8⁺ T cells producing IFN compared to CD4⁺ T cells. In addition, both BiTEs elicited significant anti-tumor responses against human metastatic melanoma tumor samples using autologous or healthy donor T cells. These data demonstrate the robust anti-tumor activity of these NKG2D ligand binding bispecific proteins and support their further development for clinical use.

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