chTNT-3/hu IL-12 Fusion Protein for the Immunotherapy of Experimental Solid Tumors

Abstract

Fusion proteins are emerging as a promising approach for targeting cytokines to the tumor site in order to generate an effective anti-tumor response. In this study, a fusion protein, chTNT-3/huIL-12, consisting of the necrosis targeting antibody, chTNT-3, and human interleukin-12 (IL-12), was constructed and expressed using the glutamine synthetase gene amplification system in NS0 cells. For these studies, IL-12 was chosen since it has been shown to be a powerful anti-tumor cytokine. To generate the fusion protein, an expression vector was prepared by linking the huIL-12 p35 subunit cDNA to the 3′ end of the chTNT-3 heavy chain cDNA and the p40 subunit was added to a separate vector. The activity of the expressed chTNT-3/huIL-12 was confirmed by standard IL-12 bioactivity assays which demonstrated that the fusion protein induced similar levels of peripheral blood lymphocyte (PBL) proliferation as free recombinant IL-12. In addition, the lytic activity of the fusion protein was demonstrated in both naive and IL-2-activated lymphocytes using cytotoxicity assays against three human pancreatic and prostatic cancer cell lines (CAPAN, DU145, and PC3-MA). Human PBL incubated with this fusion protein showed an increase in IFN-γ production which was augmented dramatically by pre-incubation with IL-2. Finally, the immunotherapeutic potential of the fusion protein was demonstrated in the human PBL-SCID mouse model where a 44% reduction in DU145 prostatic tumor growth was obtained compared to control treated mice. These results demonstrate that tumor-targeted human IL-12 may be an effective immunotherapeutic reagent for the treatment of solid tumors in man.