Anti-NKG2D single domain-based antibodies for the modulation of anti-tumor immune response

The natural killer group 2 member D (NKG2D) receptor is a C-type lectin-like activating receptor mainly expressed by cytotoxic immune cells including NK, CD8(+) T, gamma delta T and NKT cells and in some pathological conditions by a subset of CD4(+) T cells. It binds a variety of ligands (NKG2DL) whose expressions is finely regulated by stress-related conditions. The NKG2DL/NKG2D axis plays a central and complex role in the regulation of immune responses against diverse cellular threats such as oncogene-mediated transformations or infections. We generated a panel of seven highly specific anti-human NKG2D single-domain antibodies targeting various epitopes. These single-domain antibodies were integrated into bivalent and bispecific antibodies using a versatile plug-and-play Fab-like format. Depending on the context, these Fab-like antibodies exhibited activating or inhibitory effects on the immune response mediated by the NKG2DL/NKG2D axis. In solution, the bivalent anti-NKG2D antibodies that compete with NKG2DL potently blocked the activation of NK cells seeded on immobilized MICA, thus constituting antagonizing candidates. Bispecific anti-NKG2DxHER2 antibodies that concomitantly engage HER2 on tumor cells and NKG2D on NK cells elicited cytotoxicity of unstimulated NK in a tumor-specific manner, regardless of their apparent affinities and epitopes. Importantly, the bispecific antibodies that do not compete with ligands binding retained their full cytotoxic activity in the presence of ligands, a valuable property to circumvent immunosuppressive effects induced by soluble ligands in the microenvironment.

Automated summary

The NKG2D receptor, expressed by cytotoxic immune cells, plays a central role in immune responses against cellular threats. Specific antibodies targeting NKG2D can modulate immune responses and show both activating and inhibitory effects. Bispecific antibodies targeting NKG2D and other molecules such as HER2 on tumor cells can induce cytotoxicity in a tumor-specific manner. These antibodies can retain cytotoxic activity in the presence of ligands, overcoming immunosuppressive effects in the microenvironment.