Abstract A68: An IL-12/Shh-C domain fusion protein based IL-12 autocrine loop for sustained natural killer cell activation
Authors: Zhu Lining , Wei Yanzhang , Yu Xianzhong 
Published: June 2012

Background: It has been known that resting natural killer (NK) cells do not possess high levels of cytotoxicity or inflammatory cytokines that are needed for efficient tumor cell killings. Ex vivo activated NK cells, when transferred back in vivo, need a large dose of IL-2 or IL-12 to maintain such an activated status, which is a main concern for systemic toxicity. The aim of the present study is to establish an NK cell self-activation system by employing the autoprocessing domain of mouse sonic hedgehog gene to form an IL-12/Shh-C fusion gene. This fusion protein can go through autocatalytic processing to form cholesterol-modified IL-12 molecules, which would in turn bind back to the NK cells in which it is produced in an autocrine fashion.

Methods: EGFP and IL-12 sequences were fused in frame with mouse sonic hedgehog C-terminal domain (Shh-C) to form eGFP/shh-C and IL-12/shh-C fusions. IL-12 without shh-C tail was used as control. Pseudo-lentiviral particles were produced and used to transduce purified murine NK cells. Subsequently, cell surface markers, cell proliferation rates, and IFN-Y secretion of NK cells transduced with different viral particles were analyzed with flow cytometry, alamar blue cell proliferation assay, and cytokine ELISA respectively. For in vivo studies, C57BL/6 mice received transduced NK cells intravenously without IL-2 support 3 days after receiving tail vein injection of B16 melanoma tumor cells and lung tissues were analyzed to determine the infiltration of modified NK cells into the tumor microenvironment.

Results: The transduction rates of the three (eGFP/shh-C, IL-12, IL-12/shh-C) lentivirus-transduced NKcell populations 5 days after infection are ranging from 20% to 32%, and were maintained within this range at least for up to 10 days after transduction. CD25 (IL-2 receptor) was found to be substantially higher in eGFP/shh-C infected (2.59-fold), IL-12 infected (2.67-fold), and IL-12/shh-C (2.04-fold) infected NK cells respectively, compared to untreated NK cells. EGFP/shh-C infected, IL-12 infected, and IL-12/shh-C infected NK cells were also observed to have significantly increased Mac-1 expressions (25%, 27%, and 18% respectively). Perforin production in IL-12/shh-C infected NK cells was doubled compared to that in untreated NK cells. In the presence of 60 lU/mL and 6000 lU/mL IL-2, IL-12/shh-C infected NK cells have 63.9% (p&lt;0.05) and 216.7% increase of proliferation rate respectively when compared to eGFP/shh-C infected NK cells, suggesting that the fusion protein decreased the requirement of NK cells for IL-2. The amounts of IFN-Y in the supernatant of IL-12/shh-C virus transduced NK cells 5 days and 7 days after transduction were 40% (p=0.008) and 48% (p=0.00045) higher respectively compared to the untreated group. Results from the cytotoxicity assay indicated that lentivirus transduction did not alter NK cell-associated cytotoxicity. Immunofluorescent staining in the lung tissue slides from B16-bearing mice suggested that donor NK cells successfully infiltrated into tumor nodules in lung tissues.

Conclusion: The IL-12/Shh-C domain fusion protein could function, once autoprocessed to form cholesterol anchored IL-12, to maintain the activated status of NK cells in vitro, as evidenced by the greatly reduced amount of IL-2 required as well as the greatly increased secretion of IFN-Y by NK cells in vitro. In addition, IL-12/shh-C virus transduced NK cells successfully infiltrated into B16 tumor nodules in lung tissues.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A68.