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Redirected Perforin-Dependent Lysis of Colon Carcinoma by Ex Vivo Genetically Engineered CTL¹

Phillip K. Darcy^2,*, Nicole M. Haynes^*, Marie B. Snook^*, Joseph A. Trapani^*, Loretta Cerruti, Stephen M. Jane and Mark J. Smyth^*

^* Cellular Cytotoxicity Laboratory, The Austin Research Institute, Heidelberg, Victoria, Australia; and Rotary Bone Marrow Research Laboratory, Royal Melbourne Hospital, Parkville, Victoria, Australia

The redirection of autologous lymphocytes to predefined tumor target Ags has considerable potential for the immunotherapeutic treatment of cancer; however, robust experimental systems for comparing various approaches have not been developed. Herein, we have generated a single chain variable domain anti-carcinoembryonic Ag (CEA) Fc receptor I -chain fusion (scFv anti-CEA) receptor and demonstrated high-level expression of this chimeric receptor in naive mouse T lymphocytes by retroviral gene transduction. These gene-modified CTL were able to lyse CEA⁺ targets and secrete high levels of IFN- following Ag stimulation. Depletion studies demonstrated that specific tumor cell cytotoxicity was mediated by gene-modified CD8⁺ T cells. Importantly, in increasingly stringent tests of efficacy in vivo, transduced CTL were sequentially shown to reject CEA⁺ colon carcinoma cells in a Winn assay and then reject established s.c. colon carcinoma in scid or syngeneic mice. Furthermore, using gene-targeted and scFv anti-CEA receptor-transduced donor CTL, perforin and IFN- were demonstrated to be absolutely critical for the eradication of colon carcinoma in mice. In summary, we have developed a highly efficient gene transfer system for evaluating chimeric receptor expression in cytotoxic lymphocytes. This series of experiments has revealed the utility of scFv anti-CEA chimeras in providing mouse T cells the capacity to reject colon carcinoma in an Ag- and perforin-specific manner.