[CANCER RESEARCH 64, 2153–2161, March 15, 2004] Human Telomerase Reverse Transcriptase-Transduced Human Cytotoxic T Cells Suppress the Growth of Human Melanoma in Immunodeficient Mice Natascha C. V. Verra, Annelies Jorritsma, Kees Weijer, Janneke J. Ruizendaal, Arie Voordouw, Pauline Weder, Erik Hooijberg, Ton N. M. Schumacher, John B. A. G. Haanen, Hergen Spits, and Rosalie M. Luiten Division of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands ABSTRACT Immunotherapy of melanoma by adoptive transfer of tumor-reactive T lymphocytes aims at increasing the number of activated effectors at the tumor site that can mediate tumor regression. The limited life span of human T lymphocytes, however, hampers obtaining sufficient cells for adoptive trans- fer therapy. We have shown previously that the life span of human T cells can be greatly extended by transduction with the human telomerase reverse transcriptase (hTERT) gene, without altering antigen specificity or effector function. We developed a murine model to evaluate the efficacy of hTERT- transduced human CTLs with antitumor reactivity to eradicate autologous tumor cells 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 / IL-2R / double knockout mice. Adoptive transfer of the hTERT-transduced influenza virus-specific CTL clone INFA24 or clone INFA13 inhibited the growth of melAKR-Flu tumors in vivo 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 activ- ity in vivo in an antigen-specific manner. hTERT-transduced MART-1- specific CTL clones AKR4D8 and AKR103 inhibited the growth of syngeneic melAKR tumors in vivo. Strikingly, melAKR-Flu cells were equally killed by the MART-1-specific CTL clones and influenza virus-specific CTL clones in vitro, but only influenza-specific CTLs were able to mediate tumor regression in vivo. The influenza-specific CTL clones were found to produce higher levels of IFN 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 influ- enza virus-specific CTL. Taken together, the adoptive transfer model de- scribed here shows that hTERT-transduced T cells are functional in vivo, and allows us to evaluate the balance between functional activity of the CTL and tumor growth rate in vivo, which determines the efficacy of CTLs to eradicate tumors in adoptive transfer therapy. INTRODUCTION The goal of immunotherapy is to bolster the immune system of the patient in such a way that it eradicates an established tumor. One way to achieve this goal is to increase the number of activated effector cells at the tumor site by adoptive transfer of in vitro generated and expanded CTLs. Indeed, infusion of human T cells has been shown to cause a delayed tumor growth in human xenograft mouse models (1–7). More importantly, adoptive transfer therapy has been success- ful in clinical settings as well. Transfer of virus-specific T cells has been shown to be effective in preventing reactivation of latent cyto- megalovirus infections in patients after organ transplantation (8, 9) and in the treatment of lymphoproliferative disorders caused by EBV infection (10, 11). Adoptive transfer therapy of cancer requires the isolation of T cells with tumor reactivity. Tumor-infiltrating lympho- cytes may be enriched for such T cells and were shown to be effective in mediating tumor regression in metastatic renal cell carcinoma patients after nephrectomy (12). Recently, the infusion of polyclonal T lymphocytes that were expanded from the tumor-infiltrating lym- phocytes, combined with high doses of IL-2, has been shown to induce substantial tumor regression in metastatic melanoma patients (13). In addition, Yee et al. (14) reported regression of individual tumor metastases by adoptive transfer of CD8+ T-cell clones that recognize the melanoma antigens MART-1 or gp100 in combination with low doses of IL-2 in metastatic melanoma patients. These clinical studies show that adoptive T-cell therapy is feasible to treat cancer patients, has low toxicity, and can be effective in mediating tumor regression. Adoptive transfer of tumor-specific CTLs has required large doses of at least 10 9 -10 10 T cells. This means that, after isolation from peripheral blood or tumor-infiltrating lymphocytes, CTLs need to be expanded in vitro. Whereas it is in general not a problem to expand freshly isolated polyclonal T cells to very large numbers, this is not the case with well-defined tumor antigen-specific T-cell clones. The application of adoptive transfer therapy of well-defined antigen- specific T-cell clones is, therefore, limited by the relatively low success rate of isolating sufficient numbers of specific T cells from individual patients. CTLs with tumor reactivity that are found in cancer patients are derived frequently from the memory T-cell pool. Human CD8+CD28- memory T cells have a replicative life span of maximally 40 population doublings (PDs) in vitro but most often much less (15), which limits large-scale expansion of these cells. For initial screening approximately 10 5 -10 6 cells are needed, which amounts to 17–20 PD when starting from one cell. Therefore, the isolation and cloning of tumor-reactive T cells selects for the rela- tively young T cells, or rare T cells with an exceptionally long life span, which may not be found in all cancer patients. Moreover, the limited life span of human T cells may also have contributed to the fact that the most prominent antitumor responses seen to date were obtained with relatively young cultures of tumor-infiltrating lympho- cytes (16). We have described previously that ectopic expression of the en- zyme complex, telomerase reverse transcriptase (hTERT) greatly ex- tends the life span of both human CD8+ and CD4+ T cells (17–19). Ectopic hTERT expression prevents telomere shortening in the cells, which occurs at each cell division or by oxidative DNA damage. Telomeres are DNA repeats at the distal ends of the chromosomes, which protect against chromosome end-to-end fusions (20). Critically short telomeres have an impaired function and may lead to cell cycle Received 5/13/03; revised 1/19/04; accepted 1/20/04. Grant support: Dutch Cancer Society NKI 1999-2048, NKI 2002-2587 (H. Spits) and NKI 2001-2419 (T. N. M. Schumacher). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: N. Verra and A. Jorritsma contributed equally to this work. K. Weijer, A. Voordouw, and H. Spits are currently at the Department of Cell Biology and Histology, AMC, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Nether- lands. J. Ruizendaal and E. Hooijberg are currently at the Department of Pathology, VUMC, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. R. Luiten is currently at the Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, E3-Q, Albinusdreef 2, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Requests for reprints: Hergen Spits, Department of Cell Biology and Histology, AMC, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Nether- lands. Phone: 31-20-5564966; Fax: 31-20-6974156; E-mail: hergen.spits@amc.uva.nl. 2153 Research. on February 16, 2016. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from