HUMAN-GENE TRANSFER - CHARACTERIZATION OF HUMAN TUMOR-INFILTRATING LYMPHOCYTES AS VEHICLES FOR RETROVIRAL-MEDIATED GENE-TRANSFER IN MAN

Tumor-infiltrating lymphocytes (TILs) are cells generated from tumor suspensions cultured in interleukin 2 that can mediate cancer regression when adoptively transferred into mice or humans. Since TILs proliferate rapidly in vitro, recirculate, and preferentially localize at the tumor site in vivo, they provide an attractive model for delivery of exogenous genetic material into man. To determine whether efficient gene transfer into TILs is feasible, we transduced human TILs with the bacterial gene for neomycin-resistance (Neo(R)) using the retroviral vector N2. The transduced TIL populations were stable and polyclonal with respect to the intact Neo(R) gene integration and expressed high levels of neomycin phosphotransferase activity. The Neo(R) gene insertion did not alter the in vitro growth pattern and interleukin 2 dependence of the transduced TILs. Analyses of T-cell receptor gene rearrangement for β- and γ-chain genes revealed the oligoclonal nature of the TIL populations with no major change in the DNA rearrangement patterns or the levels of mRNA expression of the β and γ chains following transduction and selection of TILs in the neomycin analog G418. Human TILs expressed mRNA for tumor necrosis factors (α and β) and interleukin 2 receptor P55 but not for interleukin 1β, granulocyte/macrophage colony-stimulating factor, interleukin 6, and interferon γ when grown with high-dose interleukin 2 without subsequent activation with mitogen or specific antigen. This pattern of cytokine-mRNA expression was not significantly altered following the transduction of TILs. The Neo(R) gene-transduced TILs could thus be used to follow the trafficking and survival of TILs in vivo, and clinical protocols using these transduced TILs in cancer patients have begun. The studies demonstrate the feasibility of TILs as suitable cellular vehicles for the introduction of therapeutic genes into patients receiving autologous TILs.