Effectiveness of Combined Interleukin 2 and B7.1 Vaccination Strategy Is Dependent on the Sequence and Order: A Liposome-mediated Gene Therapy Treatment for Bladder Cancer 1 William A. Larchian, 2 Yutaka Horiguchi, Smita K. Nair, William R. Fair, Warren D. W. Heston, and Eli Gilboa Department of Urology, The Cleveland Clinic Foundation, Cleveland, Ohio 44195 [W. A. L.]; Department of Urology, Memorial Sloan- Kettering Cancer Center, New York, New York 10021 [Y. H., W. R. F., W. D. W. H.]; and Center for Genetic and Cellular Therapies, Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710 [S. K. N., E. G.] ABSTRACT We have developed a novel liposome-mediated immuno- gene therapy using interleukin 2 (IL-2) and B7.1 in a murine bladder cancer model. A carcinogen-induced murine bladder cancer cell line, MBT-2, was transfected with cationic liposome 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide/dioleolylphosphatidylethanolamine and IL-2 plasmid. The optimized transfection condition generated IL-2 levels of 245–305 ng/10 6 cells/24 h, 100-fold higher than the levels seen with retrovirus transfection. Ninety percent of the peak level of IL-2 production was maintained for up to 11 days after trans- fection. Animal studies were conducted in C3H/HeJ female mice with 2  10 4 MBT-2 cells implanted orthotopically on day 0. Multiple vaccination schedules were performed with i.p. injection of 5  10 6 IL-2 and/or B7.1 gene-modified cell prep- arations. The greatest impact on survival was observed with the day 5, 10, and 15 regimen. Control animals receiving retrovirally gene-modified MBT-2/IL-2 cell preparations had a median survival of 29 days. Animals receiving the IL-2 liposo- mally gene-modified cell preparation alone had a median sur- vival of 46 days. Seventy-five percent of animals receiving IL-2 followed by B7.1 gene-modified tumor vaccines were the only group to show complete tumor-free survival at day 60. All of these surviving animals rejected the parental MBT-2 tumor rechallenge and survived at day 120 with a high CTL response. In conclusion, liposome-mediated transfection demonstrates a clear advantage as compared with the retroviral system in the MBT-2 model. Multi-agent as opposed to single-agent cytokine gene-modified tumor vaccines were beneficial. These “target- ed” sequential vaccinations using IL-2 followed by B7.1 gene- modified tumor cells significantly increased a systemic immune response that translated into increased survival. INTRODUCTION The majority of human neoplasms are treated by the tradi- tional modalities of surgery, radiation, or chemotherapy, either independently or in combination. The development of immuno- therapeutic models over the past 15 years has led to multiple human protocols for the treatment of cancer (1). These models are predicated on the basic assumption that tumor-specific an- tigens exist and that the patient’s immune system fails to either recognize or effectively respond to these antigens (2, 3). The goal of immunotherapy is therefore to increase tumor antigen recognition and enhance the antitumor response (4). In studies using the systemic administration of cytokines, such as IL-2, 3 profound inhibitory effects on tumor progression in animal models were seen, but only a limited therapeutic benefit was observed when IL-2 was administered to cancer patients (5–7). This limited efficacy was due in part to the toxicity resulting from the high doses of IL-2 required to stimulate an immune response in humans (8). Other studies using repeated injections of IL-2 directly into the tumor site have attempted to initiate the regression of established tumors or to induce immunological memory (9, 10). These alternative therapeutic options, although generating great interest, have met with sporadic clinical success to date (11). A new form of immunomodulation using gene transfer techniques is now being actively investigated for a variety of malignancies (12). This treatment requires the insertion of a plasmid DNA encoding a cytokine gene directly into tumor cells. These “gene-modified” tumor cells then produce this cytokine, resulting in enhanced tumor antigen recognition and a documented increase in an antigen-specific immune response (13–15). We have previously investigated this new technique in the MBT-2 murine bladder cancer model (16, 17). The MBT-2 tumor was induced in C3H mice by oral administration of N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide, a potent carcino- gen that resulted in bladder neoplasms in 80 –90% of animals Received 9/16/99; revised 4/6/00; accepted 4/7/00. 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. 1 Supported by an American Foundation for Urologic Disease Fel- lowship and the Peter T. Joseph Foundation (W. A. L.). 2 To whom requests for reprints should be addressed, at The Cleveland Clinic Foundation, Department of Urology, 9500 Euclid Avenue, A-100, Cleveland, OH 44195. Phone: (440) 329-7315; Fax: (440) 329- 7316; E-mail: LARCHIW@ccf.org. 3 The abbreviations used are: IL-2, interleukin 2; FBS, fetal bovine serum; DMRIE, 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl am- monium bromide; DOPE, dioleolylphosphatidylethanolamine; APC, an- tigen-presenting cell. 2913 Vol. 6, 2913–2920, July 2000 Clinical Cancer Research Research. on October 14, 2021. © 2000 American Association for Cancer clincancerres.aacrjournals.org Downloaded from