A Bispecific Protein Targeting the NKG2D Receptor on Natural Killer Cells: In Vitro and In Vivo activity of ULBP2-CEA

Abstract 2095

Natural killer (NK) cells are effector lymphocytes of the innate immune system. NK cell activity is tightly regulated by inhibitory and activating surface receptors to ensure that NK cells recognize and eliminate target cells, whereas healthy cells escape from the NK cell-mediated immune surveillance. The inability of the immune system to recognize and kill malignant target cells has been partly attributed to the ineffective activation of NK cells. It is well accepted, that the activating NK receptor NKG2D (natural killer group 2 member D) plays an important role in tumor rejection. The Immunoligand ULBP2-CEA is a recombinant bispecific protein designed to enhance NK cell-mediated lysis of CEA-expressing target cells. Consisting of the NKG2D-ligand ULBP2 (UL16 binding protein 2) and an anti-CEA single chain antibody, this construct provides the ability of cross-linking NK cells and CEA-expressing cells for specific tumor targeting. CEA (carcinoembryonic antigen) is frequently overexpressed in colorectal and other carcinomas. The binding and specificity of ULBP2-CEA fusion protein was tested in FACS analysis. Transfected human CEA⁺ murine colon carcinoma cells (MC38-CEA) were incubated with ULBP2-CEA and recombinant NKG2D receptor (NKG2DR). The protein was able to bind both, tumor cells and recombinant NKG2DR simultaneously. No specific cell binding was detectable in control experiments using fusion constructs with another binding moiety (e.g. ULBP2-BB4) or soluble NKG2DR alone. In cytotoxicity assays ULBP2-CEA enhances the susceptibility to NK cell mediated lysis of CEA-expressing cells. CEA⁺ human colon carcinoma cells (LS174T) were incubated with NK cells isolated from healthy donors in the presence or absence of ULBP2-CEA. The increase of cell lysis was significant at all effector-target ratios compared to the controls demonstrating specific targeted in vitro efficacy of ULBP2-CEA. In order to investigate anti-tumor activity in vivo we use a syngeneic mouse model. MC38-CEA cells were implanted subcutaneously in CEA transgenic C57BL/6 mice and tumor growth was assessed using in vivo bioluminescence imaging (BLI). First results on the in vivo efficacy of ULBP-CEA and effects on the activation status of splenic and peripheral blood NK and T cells upon treatment will be discussed. This syngeneic mouse model is a useful tool to study efficacy and mode of action of ULBP2-CEA in vivo.