2007;98:1297C1302. agonist anti-CD137 monoclonal antibodies (mAbs), which was substantiated against poorly GNG12 immunogenic B16 melanomas (B16-OVA and B16.F10) and TC-1 lung carcinomas. Effector CD8+ T cells were sufficient to mediate complete tumor eradications. Accordingly, there was an intensely synergistic enhancement of cytotoxic T lymphocytes (CTL)-mediated immunity against the tumor antigens OVA and tyrosine-related protein-2 (TRP-2). This train of phenomena led to long-lasting tumor-specific immunity against rechallenge, attained transient control of the progression of concomitant tumor lesions that were not directly treated with SFV-IL-12 and caused autoimmune vitiligo. Importantly, we found that SFV-IL-12 intratumoral injection induces bright expression of CD137 on most tumor-infiltrating KD 5170 CD8+ T lymphocytes, thereby providing more abundant targets for the action of the agonist antibody. This efficacious combinatorial immunotherapy strategy offers feasibility for clinical translation since anti-CD137 mAbs are already undergoing clinical trials and development of clinical-grade SFV-IL-12 vectors is in progress. Introduction Interleukin-12 (IL-12) is a potent immunotherapeutic cytokine usually expressed by activated KD 5170 macrophages and dendritic cells. IL-12 has shown strong antitumoral activity mediated by activation of cytotoxic T lymphocytes (CTL), T-helper cell type 1 responses, NK and NKT cells, as well as by inhibition of angiogenesis.1,2 Most of these effects are mediated by the induction of interferon (IFN).3 Several viral vectors, such as adenovirus, retrovirus, or alphavirus, have been used to deliver IL-12 to animal tumor models, resulting in localized expression of the cytokine and antitumor efficacy.4,5,6 However, in spite of successful preclinical studies, phase I clinical trials performed with adenovirus or canarypox vectors-expressing IL-12 only showed minor therapeutic effect.7,8 These results indicated a need for more potent vectors and for the development of combinatorial strategies.9 Alphavirus vectors based on Semliki Forest virus (SFV) have shown some advantages over adenoviral vectors in preclinical studies of cancer treatment, such as higher expression levels, broad tropism, and induction of immunogenic apoptosis in tumor cells.10,11 This last property can lead to the release of tumor antigens that can be uptaken by antigen-presenting cells, favoring an ensuing antitumor immune response.12 The SFV vector is based on a viral RNA genome in which the region coding for the structural proteins has been replaced by an heterologous gene.13 SFV vectors-expressing IL-12 have shown to be very efficient in inducing therapeutic antitumor responses in tumor models of colon adenocarcinoma, sarcoma, and glioma in mice,10,14,15 orthotopic hepatocellular carcinoma in rats,11 or spontaneous hepatocellular carcinoma in woodchucks.16 treatment with agonist agents acting on CD137 (4-1BB) expressed on primed T cells results in enhancement of tumor-eradicating cytotoxic T-cell responses.17 These therapeutic effects have been observed with conventional monoclonal antibodies (mAbs) and single chain Fv antibodies attached to tumor cells.18 Although originally described as an inducible molecule on activated T-cells,19 CD137 is not only expressed on antigen-activated T-cells but also on other cell types such as activated NK cells,20 dendritic cells,21 and endothelial cells in tumor vessels.22 Agonist mAbs given as monotherapy to tumor-bearing mice rely mainly on CTL antitumor responses, although an involvement for NK cells KD 5170 has been reported in a number of cases.23 Particularly, therapeutic CD137 stimulation greatly enhanced the NK-mediated ADCC activity of mAbs recognizing tumor-associated surface molecules.24 Moreover, anti-CD137 mAbs enhanced lymphocyte infiltration into tumors as a result of stimulating tumor endothelial cells to behave as those in inflamed cells.22 Agonist mAbs directed to human being CD137 (BMS663516 and PF-05082566) are undergoing clinical KD 5170 tests for malignancy treatment, the results of which KD 5170 are eagerly awaited.25 Synergistic treatments that involved adenoviral gene transfer of IL-12 and.