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The dependency of activated natural killer (NK) cells on the continuoussupport of exogenous interleukin (IL)-2 for their in vivo survival, tumor localizationand consequently, their antitumor effect, is a major obstacle for NK cell-mediatedtumor therapy. In the present study, a fusion gene between IL-12 and mouse sonichedgehog C-terminal domain (Shh-C) was constructed. The fusion protein was autocatalyticallyprocessed to form cholesterol-modified IL-12 molecules and an autocrine loop ofIL-12 was established for the sustained activation of NK cells. The transducedNK cells matured more rapidly in vitro with the enhanced expression of granule-relatedproteins. NKIL-12/Shh-C cells reached the same proliferation rate as NK cellstransduced with enhanced green fluorescent protein (EGFP)/Shh-C (NKEGFP/Shh-C)with &lt;10-fold IL-2 supplementation, suggesting that the fusion protein reducedthe dependency of NK cells on IL-2. The amount of interferon‑γ (IFN-γ) in thesupernatants of NKIL-12/Shh-C cells 5 and 7 days after transduction was significantlyhigher than that in the supernatants of NKIL-12 cells. Immunofluorescent stainingof lung tissues from B16-bearing mice which had received an intravenous injectionof lentivirus-transduced NK cells without exogenous IL-2 confirmed that donorNK cells successfully infiltrated into the lung tissues. The survival time ofthe mice which had received NKIL-12/Shh-C cells was significantly prolonged comparedto the mice which had received NKEGFP/Shh-C cells.

An IL-12/Shh-C domain fusion protein-based IL-12 autocrine loop for sustained natural killer cell activation