Human reverse transcriptase-transduced human cytotoxic T cells suppress the growth of human melanoma in immunodeficient

Immunotherapy of melanoma by adoptive transfer of tumor-reactive T lymphocytesaimsatincreasingthenumberofactivatedeffectorsatthetumorsitethatcanmediatetumorregression.ThelimitedlifespanofhumanTlymphocytes,however,hampersobtainingsufficientcellsforadoptivetrans-fertherapy.WehaveshownpreviouslythatthelifespanofhumanTcellscanbegreatlyextendedbytransductionwiththehumantelomerasereversetranscriptase(hTERT)gene,withoutalteringantigenspecificityoreffectorfunction.WedevelopedamurinemodeltoevaluatetheefficacyofhTERT-transducedhumanCTLswithantitumorreactivitytoeradicateautologoustumorcells in vivo . We transplanted the human melanoma cell line melAKR or melAKR-Flu, transduced with a retrovirus encoding the influenza virus/ HLA-A2 epitope, in RAG-2 (cid:2) / (cid:2) IL-2R (cid:3) (cid:2) / (cid:2) double knockout mice. Adoptive transfer of the hTERT-transduced influenza virus-specific CTL clone INFA24orcloneINFA13inhibitedthegrowthofmelAKR-Flutumors invivo and not of the parental melAKR melanoma cells. Furthermore, the hTERT- transduced CTL clone INFA13 inhibited tumor growth to the same extent in vivo as the untransduced CTL clone, as determined by in vivo imaging of luciferase gene-transduced melAKR-Flu tumors, indicating that hTERT did not affect the in vivo function of CTL. These results demonstrate that hTERT-transduced human CTLs are capable of mediating antitumor activity in vivo in an antigen-specific manner. hTERT-transduced MART-1-specificCTLclonesAKR4D8andAKR103inhibitedthegrowthofsyngeneicmelAKRtumors in vivo . Strikingly, melAKR-Flu cells were equally killed by the MART-1-specific CTL clones and influenza virus-specific CTL clones in vitro ,butonlyinfluenza-specificCTLswereabletomediatetumorregression in vivo . The influenza-specific CTL clones were found to produce higher levels of IFN (cid:3) on tumor cell recognition than the MART-1-specific CTL clones, which may result from the higher functional avidity of the influenza virus-specific CTL clones. Also, melAKR-Flu tumors were growing faster than melAKR tumors, which may have surpassed the relatively modest antitumor effect of the MART-1-specific CTL, as compared with the influenza virus-specific CTL. Taken together, the adoptive transfer model de- scribedhereshowsthathTERT-transducedTcellsarefunctional invivo ,and allows us to evaluate the balance between functional activity of the CTL and tumorgrowthrate invivo ,whichdeterminestheefficacyofCTLstoeradicate tumors in adoptive transfer therapy. efficacy of human hTERT-transduced CTL clones to eradicate autol- ogous melanoma cells in an in vivo environment. In the present report, we describe adoptive transfer of hTERT-transduced CTL clones in RAG-2 (cid:3) / (cid:3) IL-2R (cid:2) (cid:3) / (cid:3) (RAG/ (cid:2) cKO) mice bearing human melanoma lung tumors. The effect of single or multiple doses of two influenza- reactive CTL clones or two MART-1-reactive CTL clones on the growth of a human melanoma in vivo was studied in relation to their functional activity in vitro . In this model, the criteria for CTL clones to be effective in mediating tumor regression in vivo upon adoptive transfer can be defined. the Functional Activity of Human CTL Clones with Extended Life Span in Vitro . To compare different human CTL clones for their individual efficacy to eradicate autolo- gous human melanoma cells in vivo , we generated two influenza virus-specific CTL clones, INFA13 and INFA24, and two melanoma- specific CTL clones, AKR4D8 and AKR103. All four of the CTL clones were transduced with the hTERT-IRES-GFP encoding retro- virus. During subsequent culture, we observed an accumulation of hTERT-IRES-GFP-expressing cells in all of the transduced T-cell cultures, which contained only transduced T cells after 1 or 2 months. The mean fluorescence intensities of GFP expression level of the tumor-bearing mice (Fig. 5). Viable CTLs were detect- able in the peripheral blood at a concentration of 25 cells/10,000 murine PBMC (0.25%), and (cid:8) 9,500 injected T cells (0.18%) are present in the lungs during the first 3 days after injection, followed by rapid clearance between day 3 and day 7 after injection. The tumor cells in the lungs started to increase in number after day 3 when most of the injected CTLs were cleared. These results show that the injected INFA24 CTLs mediated the antitumor effect at very low E:T cell ratios and were able to effect up to 40% reduction in tumor growth (Fig. 5). Moreover, these results suggest that the CTLs mediated their antitumor effect during the first 3 days after injection. CTL clone INFA13 was significantly more effective in vivo than clone INFA24. This difference was not apparent from the above-mentioned in vitro assays and may be the result of other yet-to-be- defined functional differences of the CTL that may affect the in vivo activity. These findings illustrate the limitations of in vitro assays to predict in vivo efficacy of CTL and the importance of testing CTLs for their functionality in vivo . Furthermore, although melAKR-Flu tumor cells were equally well killed in vitro by both the influenza virus-specific CTL clones and the MART-1-specific CTL clones, the influ- enza virus-specific CTL clones produced more IFN (cid:2) upon recognition of melAKR-Flu cells. These results may indicate a rate-limiting effect in the cytotoxicity assays caused by the level of sensitivity of target cells to lysis by CTL, which, therefore, do not reveal quantitative differences between highly lytic CTL clones. The importance of the CTL avidity for the in vivo efficacy upon adoptive transfer was demonstrated for tyrosinase-related protein 2-specific murine CTL in syngeneic mouse model (7) and for gp100-specific human CTL clones in a nude mouse model (6). These findings are consistent with our data. The influenza virus-specific CTL displayed a higher tetramer binding intensity and functional avidity, and produced more IFN (cid:2) upon activation with specific antigen, as compared with the MART- 1-specific CTL clones. This correlated well with the higher efficacy of the influenza virus-specific CTL in mediating tumor growth inhibition in vivo . The elevated level of IFN (cid:2) production by the influenza virus-specific CTL upon recognition of the melAKR-Flu tumor cells may have additionally enhanced the in vivo efficacy of these clones. Local IFN (cid:2) production in the tumor leads to an increased HLA class I expression on tumor cells, allowing better tumor cell recognition by the CTL. Adoptive transfer of murine CTL transduced with the IFN (cid:2) gene was found to be more effective in mediating tumor regression in a syngeneic setting than the parental CTL (35), suggesting a positive effect of IFN (cid:2) production by the CTL