Abstract 488: MULT1E/mIL-12: A novel bi-functional protein for NK cell activation.

Abstract

Abstract Natural killer (NK) cells have the potential to be very effective killers of tumor cells. They are closely governed by inhibitory and activating receptors like NKG2D, whose ligands are normally upregulated in cells that are stressed, like cancer cells. Advanced cancer cells, however, have found ways to reduce these ligands, leaving them less detectable by NK cells. Along with these receptors, NK cells also require activating cytokines, like IL-12, to function. The infusion of high doses of IL-12 is effective in activating an immune response against tumor cells but is usually toxic. The goal of this study is to develop a novel bi-functional fusion protein for enhanced NK cell activation. The proposed fusion protein combines the extracellular domain of the NKG2D ligand MULT1 (MULT1E) and mouse IL-12 (mIL-12). It is hypothesized that when expressed by tumor cells, the protein will activate NK cells using the NKG2D receptor, and also deliver mIL-12 to the NK cells where it can interact with the IL-12R and enhance NK cells’ cytotoxicity. The fusion gene was constructed using the cDNA sequence encoding MULT1E and a short linker sequence at the 3’ end to replace the start codon and the signal sequence of mIL-12 and cloned into a mammalian expression vector. The vector was transfected into mouse TC-1 lung carcinoma cells. After characterizing the levels of protein expression using RT-PCR, qRT-PCR, fluorescent microscopy, ELISA, and FACS, three stable clones expressing high, moderate, or low levels of the protein were selected for study. To assess the biological activity of the protein in vitro, mouse NK cells were cultured with the stable clones and NK cell activation was measured by the production of interferon-γ (IFN-γ) using ELISA. NK cells cultured with clones expressing high or moderate levels of the protein expressed significantly higher levels of IFN-γ than the regular TC-1 cells. Interestingly, the clone expressing a moderate amount of the protein induced a significantly higher level of IFN-γ production than the clone expressing a high amount or a clone expressing mIL-12 alone. Furthermore, this clone showed a reduction in cell growth that was not observed in the other clones. Similar results were seen when the NK cells were cultured with regular TC-1 cells with the addition of supernatant from each clone. To examine the in vivo antitumor effects of the protein, C57BL/6 mice were injected intravenously with the clones. The results show that although there was no significant difference, clones expressing high and moderate levels of the protein grew slower than clones expressing either a low level or mIL-12 alone as assayed by lung tumor nodule count and total lung weight. It is also of note that all the clones grew significantly slower than the regular TC-1 cells in vivo. Although the study is preliminary and further studies are needed, the data suggest that the MULT1E/mIL-12 bi-functional fusion protein is an effective activator of NK cells for cancer treatment. Citation Format: Ashlee H. Tietje, Jinhua Li, Xianzhong Yu, Yanzhang Wei. MULT1E/mIL-12: A novel bi-functional protein for NK cell activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 488. doi:10.1158/1538-7445.AM2013-488