FUNCTIONAL AND MOLECULAR CHARACTERIZATION OF TUMOR-INFILTRATING LYMPHOCYTES TRANSDUCED WITH TUMOR-NECROSIS-FACTOR-ALPHA CDNA FOR THE GENE-THERAPY OF CANCER IN HUMANS

TNF is effective in causing the regression of selected murine tumors w hen administered at high concentrations. Therapeutic levels in humans cannot be obtained systemically, however, because of dose-limiting tox icity. The development of immunotherapy with IL-2 and tumor-infiltrati ng lymphocytes (TIL), which can accumulate at tumor sites in some pati ents, and of efficient retroviral techniques for gene transfer into eu karyotic cells has allowed new therapeutic approaches using TNF. We ha ve retrovirally transduced human TIL with the gene for TNF in an attem pt to deliver high concentrations of TNF to the tumor site without dos e-limiting systemic toxicity. Successful gene insertion was confirmed by Southern hybridization in 16 of 16 transduced and selected TIL cult ures from 15 different patients, with an estimated 28 to 93% transduce d cells within each culture. Transduced selected TIL cultures produced greater amounts of TNF, compared with nontransduced controls, in 11 o f 16 cultures evaluated. However, overall production of TNF was >30-fo ld lower, compared with a transduced and highly selected tumor cell li ne control (MEL-TNF). In addition, steady state levels of vector-deriv ed transcript in nine of 10 transduced selected TIL cultures were <14% of the amount seen in the MEL-TNF control line. In an attempt to incr ease TNF production, TIL were tran5duced with a mutated form of TNF co ntaining the IFN-gamma signal peptide in place of the transmembranous region, to enhance secretion into the endoplasmic reticulum. By using this vector, TNF production increased by an average of fivefold. These studies demonstrate that TIL can be genetically modified to express a nd secrete a protein for use in targeted cancer therapy but that parti al expression blockades exist that prevent maximal cytokine production by introduced genes in TIL.