title: Increased susceptibility to immune destruction of B16BL6 tumor cells engineered to express a novel pro-Smac fusion protein.
abstract: Recent developments in immunology have provided new strategies to induce and augment the immune response to cancer. Nonetheless, objective clinical responses after vaccination are rare and even when high frequencies of tumor-specific T cells are achieved after adoptive immunotherapy, tumor cells continue to evade the immune response. We hypothesize that 1 mechanism of resistance of tumor cells to destruction by T cells is an elevated threshold for the induction of apoptosis. Inhibitor of apoptosis proteins (IAPs) are overexpressed in various tumors and have been associated with treatment failure and poor prognosis. As the mitochondrial peptide second mitochondria-derived activator of caspase (Smac) can antagonize IAPs, we designed a GFP-Smac fusion protein with a granzyme B (GrB) cleavage site. This fusion protein should be cleaved when tumor-specific cytolytic T cells recognize the tumor and, using the pore-forming protein perforin, insert GrB into the target. Here we report that transfer of a construct encoding a novel eGFP-Smac fusion protein (pro-Smac) containing a specific cleavage site for GrB, into the poorly immunogenic mouse melanoma cell line, B16BL6-D5 (D5), sensitizes tumor cells for killing by tumor-specific wild type, but not perforin-deficient (perforin-knockout), effector T cells in vitro and in vivo. These results describe the first example of a tumor-specific, T-cell-mediated approach to amplify the GrB-mediated cytotoxicity pathway with a pro-Smac fusion protein and provide an innovative approach to overcome IAPs and improve the efficacy of immunotherapy.
authors.affiliation: Laboratory of Molecular and Tumor Immunology, Robert W. Franz Cancer Research Center, Earle A. Chiles Research Institute, Providence Portland Medical Center, OR 97213, USA.
authors.forename: Dominik
authors.initials: D
authors.lastname: Rüttinger
authors.forename: Rui
authors.initials: R
authors.lastname: Li
authors.forename: Christian H
authors.initials: CH
authors.lastname: Poehlein
authors.forename: Dan
authors.initials: D
authors.lastname: Haley
authors.forename: Edwin B
authors.initials: EB
authors.lastname: Walker
authors.forename: Hong-Ming
authors.initials: HM
authors.lastname: Hu
authors.forename: Bernard A
authors.initials: BA
authors.lastname: Fox
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D003601:Cytotoxicity Tests, Immunologic; D005260:Female; D005434:Flow Cytometry; D053804:Granzymes / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016219:Immunotherapy, Adoptive / Q000379:methods*; D008175:Lung Neoplasms / Q000276:immunology / Q000556:secondary / Q000628:therapy; D008546:Melanoma, Experimental / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D052899:Pore Forming Cytotoxic Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000378:metabolism / Q000637:transplantation; D014162:Transfection
chemical_list: D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; C499605:Diablo protein, mouse; D024101:Mitochondrial Proteins; D052899:Pore Forming Cytotoxic Proteins; D011993:Recombinant Fusion Proteins; C483886:perforin, mouse; D049452:Green Fluorescent Proteins; D053804:Granzymes; C509214:Gzmb protein, mouse; D053148:Caspase 3