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

Abstract

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 CD3ε 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 CD3ε 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 IFNγ 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 IFNγ, and culture of T cells and these BiTEs with plate-bound rMICA showed ligand density dependent production of IFNγ from both CD4+ and CD8+ T cells. There was two-fold more IFNγ produced per CD8+ T cell and five-fold greater percentage of CD8+ T cells producing IFNγ 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.

Remote control of CAR T cell therapies by thermal targeting

“…The genomic HSPA6 promoter was amplified from genomic DNA using PCR primers listed in a previous publication 81 . The NKG2DL BiTE sequence was described previously 64 and modified to include an Igκ leader sequence to facilitate secretion from T cells as well as a HisTag for construct detection. This combined sequence was synthesized (ATUM) and cloned downstream of synthetic thermal switches.…”

“…Last, we explored thermal control to expand CAR T recognition of tumor-associated antigens to include NKG2D ligands (NKG2DLs), which are upregulated on a wide range of cancers as well as suppressor cells [61][62][63][64] . Targeting NKG2DLs is limited by their expression in healthy cells such as intestinal epithelial cells and bone marrow stromal cells 65 .…”

“…Targeting NKG2DLs is limited by their expression in healthy cells such as intestinal epithelial cells and bone marrow stromal cells 65 . To enable thermal control of CAR T cells to target NKG2DL+ cells, we cloned a previously described NKG2DL-BiTE containing CD3-recognition domains from the OKT3 antibody linked to the extracellular domain of the human NKG2D receptor 64 . Our vector (TS-BiTE) included an Igκ leader sequence for BiTE secretion, a HisTag reporter, and a constitutive αCD19 CAR (Fig.…”

Summary of a workshop on preclinical and translational safety assessment of CD3 bispecifics

“…Dr. Charles Sentman (Dartmouth College) presented his work with CD3 bispecific molecules targeting MHC Class I polypeptiderelated sequence A or B7H6 tumor antigens using scFv or NKG2D (Godbersen et al 2017). The anti-CD3 binding domain was a scFv derived from OKT3.…”

Neural Stem Cells Secreting Bispecific T Cell Engager to Induce Selective Anti-Glioma Activity

“…BiTEs engage TILs and cancer cells in an MHCindependent manner, and are therefore unaffected by MHC downregulation that occurs in GBM cells [35][36][37][38]. The specificity of BiTE's tumor antigen-directed scFv is imperative to harness the full therapeutic potential of the recombinant molecule [39]. BiTE anti-cancer activity requires BiTE binding to malignant and immune cells simultaneously, single arm binding to tumor antigen or to CD3ε is therapeutically ineffective [40,41].…”