Transduction of rIL-2 Expanded CD4 + and CD8 + Ovarian TIL-Derived T Cell Lines with the G1Na (neo R ) Replication-Deficient Retroviral Vector

We have expanded ovarian tumor-infiltrating lymphocytes (TIL) in low concentrations of recombinant interleukin-2 (rIL-2) to conduct intraperitoneal adoptive immunotherapy trials in patients with ovarian cancer. We have previously demonstrated that certain T cell lines and clones derived from ovarian TIL exhibit in vitro autologous tumor-specific cytotoxicity and/or cytokine production (interferon-γ, tumor necrosis factor-α) preferentially in response to autologous tumor cells. Studies that utilize a marker gene introduced into the DNA of TIL can provide useful information on specific uptake or localization of TIL at tumor sites and on the survival of TIL in vivo. We have conducted a series of preclinical experiments in which we have successfully transfected TIL with G1Na, which encodes the gene for neomycin phosphotransferase (neo^R). Neo^R was detected in at least 10% of CD8⁺ cells (mean = 10.4%) and between 2.5 and 20% of CD4⁺ TIL (mean = 8.5%). Transduction of ovarian TIL with G1Na caused no substantial changes to the T cell phenotypes or in vitro cytotoxicities against ovarian and hematogenous tumor cell targets, or on the rIL-2 requirements of TIL for growth and proliferation. In addition, the intact G1Na provirus in transduced TIL cells was rescuable by replication-competent retrovirus and was transferred into the genome of NIH-3T3 fibroblasts, which were rendered resistant to G418. An enhanced polymerase chain reaction (PCR) procedure utilizing detection by ethidium bromide staining was developed. The enhanced PCR detected 1 in 100,000 neo^R-labeled cells. Furthermore, detection of the G1Na genome in transduced TIL by in situ hybridization with an RNA probe provided evidence for expression of the neo^R gene in transduced TIL. Results obtained from these studies suggest that ovarian TIL-derived T cell lines transduced with the neo^R gene post infection with the G1Na retroviral vector can be utilized to examine the in vivo trafficking pattern of ovarian TIL-derived T cell lines expanded in low concentrations of rIL-2 and their survival.

Overview summary

Ovarian tumor-infiltrating lymphocytes (TIL)-derived T cell lines (CD3⁺CD8⁺ and CD3⁺CD4⁺) obtained from patients with epithelial ovarian carcinoma have been successfully transduced with the neo^R gene using a retroviral vector. The transduced T cells were shown to be detected by an enhanced, ethidium bromide-based polymerase chain reaction (PCR) assay at levels of 1:100,000. The transduced T cells retained their phenotypic characteristics, such as dependence on interleukin-2 (IL-2) for growth and cytotoxic lysis of tumor cells. Linear integrity of the transduced gene was demonstrated by rescue with a replication-competent retrovirus, and the transduced cells were shown to be transcriptionally active by detection of transduced cells using in situ hybridization.