In vitro and in vivo delivery of novel anticancer fusion protein MULT1E/FasTI via adenoviral vectors.

We previously demonstrated that a novel fusion protein MULT1E/FasTI expressed by TC-1 tumor cells inhibited tumor growth by simultaneously activating NKG2D expressing cells, such as NK cells, through the MULT1E portion and sending a death signal into cells through the Fas portion (Kotturi et al., Gene Therapy, 2008). In this study, an adenoviral gene delivery system was used to deliver this fusion protein. Our data indicate that adenoviral vector can efficiently deliver the MULT1E/FasTI fusion protein into TC-1 cells both in vitro and in vivo as assayed by RT-PCR, FACS analysis, caspase-3 activity and decreased in vivo tumor growth. This study further confirms that MULTE/FasTI represents a powerful bi-functional, therapeutic protein for the treatment of cancers.

Tumor cells expressing a fusion protein of MULT1 and Fas are rejected in vivo by apoptosis and NK cell activation.

Tumor cells evade immunosurveillance by elements of the innate immune system, such as natural killer (NK) cells, by downregulating or 'shedding' certain cell-surface molecules like mouse UL16-binding protein-like transcript 1 (MULT1) that can activate NK cells through NK cell receptors such as NKG2D; they also avoid Fas-mediated apoptosis by downregulating its expression. In the present study we report the design and evaluation of the antitumor activity of a novel fusion protein, MULT1E/FasTI, consisting of the extracellular domain of MULT1 and the transmembrane and intracellular domains of Fas. The fusion construct (pMULT1E/FasTI) was transfected into the mouse pulmonary carcinoma cell line TC-1; and stable cell clones expressing the fusion protein were established. In-vitro cell culture studies demonstrated that the binding of the NKG2D/Fc, a recombinant protein of mouse NK cell receptor, to MULT1E/FasTI expressed on tumor cells was able to elicit apoptosis as assayed by Annexin V-fluorescein isothiocyanate staining and caspase-3 enzyme-linked immunosorbent assay and to activate NKG2D-expressing cells, such as NK cells. In-vivo subcutaneous tumor studies demonstrated that tumor cells expressing MULT1E/FasTI grew significantly slower than cells without the protein. Pulmonary metastasis studies showed that most of the mice completely rejected tumor cells expressing MULT1E/FasTI. This approach may generate a new therapeutic agent for tumor treatment when combined with tumor cell-specific gene delivery vehicles such as oncolytic adenovirus vectors.