Gene Therapy (1997) 4, 361–366 1997 Stockton Press All rights reserved 0969-7128/97 $12.00 Combination gene transfer to potentiate tumor regression T Ohno1,4, Z Yang1,4, X Ling2, M Jaffe1,4, EG Nabel2, D Normolle4 and GJ Nabel1,4 1Howard Hughes Medical Institute, University of Michigan Medical Center, Departments of 2Internal Medicine and 3Biological Chemistry, Ann Arbor, MI; and 4Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, MI, USA Recent efforts to treat malignancy using gene transfer have shown, a significant reduction of tumor size was observed met with varying degrees of success. In this paper, we with each vector. Combination treatment, in which both report the results of studies using two recombinant adeno- vectors were administered, resulted in a trend toward a viral vectors to examine the efficacy of combination gene reduced tumor growth greater than with either vector alone. transfer to cause tumor regression in vivo. One of these In order to characterize the mechanism of tumor vectors encodes the murine MHC class I gene, H-2K b regression, cytolytic T lymphocyte (CTL) assays against (ADV-Kb), which induces an immune response that stimu- the allogeneic molecule, H-2K b, were performed. Mice lates tumor regression. The second vector encodes the treated with ADV-K b showed specific CTL activity against human p21 cyclin dependent kinase inhibitor (ADV-p21). the H-2Kb molecule, demonstrating that the immune This gene product arrests cell cycle progression and pre- response against the H-2Kb gene product involved in vents proliferation of tumor cells. Both vectors were tested tumor regression was potentiated by expression of the p21 in a murine model in vivo for antitumor effect. As previously gene which affects cell cycle progression. Keywords: gene therapy; cancer; p21; MHC Introduction through the administration of either a retroviral vector or DNA–liposome complexes in vivo. This strategy is It has been suggested that most malignancies have designed to induce an immune response to syngeneic evolved mechanisms to evade immune detection, but the tumor cells, initially directed to the allogeneic MHC class ability to induce an immune response against tumors has I gene (HLA-B7 for humans or H-2Ks for mice), conse- been demonstrated after immunologic activation, and the quently generating tumor-specific immune responses.20 stimulation of specific antitumor immune recognition Alternative genetic targets may also be candidates to offers the potential to provide therapy for malignancy. A limit tumor growth after gene transfer in vivo. Among variety of approaches for inducing immune recognition these approaches, genes which affect cell cycle pro- of tumors or increasing the strength of the immune gression, specific cyclin/cyclin-dependent kinase inhibi- response have been studied. Among the methods tors (CKIs), have shown antitumor effects in animal mod- developed to elicit immune rejection are the stimulation els.21 Expression of the p21 CKI using gene transfer of major histocompatibility-restricted lysis through a techniques suppressed the growth of malignant cells in 1 cytotoxic T cell (CTL) response, tumor infiltrating lym- vitro and in vivo.21 In the previous study, the arrest of cell 2–4 5 phocytes, immunization with tumor cells, adminis- cycle progression was due to growth arrest associated 6 tration of cytokines or tumor-associated antigen sensi- with terminal differentiation. In this report, we have 6–8 9,10 tized lymphoid cells, natural killer cells, and tested the efficacy of gene transfer of the murine MHC macrophages. Others have attempted to introduce new gene, H-2Kb, in combination with the gene for p21, using antigens into tumor cells by treatment with drugs or adenoviral vectors in vivo. We sought to determine 11–13 virus infection. Furthermore, the expression of cyto- whether combination gene transfer with an immunomod- kine genes by genetic modification of tumor cells has ulatory and antiproliferative gene could potentiate the been shown to induce an antitumor response, mediated antitumor immune response. by lymphocytes or other inflammatory cells.14–18 We have previously developed a gene transfer protocol which utilizes the human MHC class I molecule, HLA- Results B7, for human trials,19 or a murine gene product, H-2Ks, in mouse studies,20 for the modification of tumor cells Construction of adenoviral vectors Recombinant adenoviral vectors which encode the H-2Kb gene (ADV-Kb) and the human p21 gene (ADV-p21) were Correspondence: GJ Nabel, Howard Hughes Medical Institute, University constructed using homologous recombination techniques of Michigan Medical Center, Departments of Internal Medicine and Bio- logical Chemistry, 1150 W. Medical Center Drive, 4520 MSRB I, Ann (Figure 1a and Refs 21 and 22). These vectors were exam- Arbor, MI 48109-0650, USA ined for their ability to express the allogeneic MHC class Received 27 August 1996; accepted 23 December 1996 I molecule, H2Kb (ADV-Kb) or to inhibit the growth of Combination immune and antiproliferative gene transfer T Ohno et al 362 Figure 1 Construction of recombinant adenoviruses. (a) Strategy for the construction of adenoviral vectors. The recombinant adenoviruses were con- structed by homologous recombination between sub360 genomic DNA, an Ad5 derivative with a deletion in the E3 region, and H-2Kb expression plasmid, pAd-RSV-Kb or p21 expression plasmid described previously.22 This Figure shows the construct of ADV-Kb. These recombinant adenoviral vectors have the deleted sequences in the E1A and E1B region, impairing the ability of these viruses to replicate and transform nonpermissive cells. (b) Flow cytometric analysis of the expression of H-2Kb molecules on Renca cells infected with ADV-Kb. Cells were infected with either ADV-Kb or control vector, ADV-DE1 at an MOI of 200. Twenty-four hours after infection, cells were stained with a murine anti-H-2Kb antibody (solid line) or an isotype control antibody (dashed line) as a first antibody and FITC-conjugated anti-mouse IgG antibody as a second antibody. Cells infected with ADV-Kb (left panel), ADV-DE1 (middle panel) and Renca-Kb which were stable transfectants of Renca cells with the H-2Kb gene (right panel) were analyzed by flow cytometry (FACScan). Renca cells (p21).15 When Renca cells were infected with Treatment of established tumors with adenoviral vectors ADV-Kb at a multiplicity of infection (MOI) of 200, cells in vivo were able to express the H-2Kb protein on the cell surface, To examine the efficacy of ADV-Kb and ADV-p21 for the in contrast to cells transduced with the control adeno- treatment of tumors in vivo, BALB/c mice were pre- virus, ADV-DE1 (Figure 1b). Similar to previous studies, immunized twice with 1 × 106 irradiated splenic lympho- Renca cells transduced with ADV-p21 reduced prolifer- cytes of C57BL6 mice, then injected subcutaneously on ation, in contrast to the control vector ADV-DE1 (Figure the right flank with Renca cells (106) on day 0. Six days 2). No expression of H-2Kb was observed in these cells after inoculation, tumors of approximately 2 × 2 mm were transduced with ADV-p21 (data not shown). Thus, each established in each mouse. Adenoviral infection was vector was able specifically to modify the phenotype of initiated by direct injection into tumor tissues once each Renca cells with respect to cell surface antigen expression day for 5 days during the first week and twice at 2 day (ADV-Kb) or cell cycle progression (ADV-p21) in vitro. intervals during the second week (a total of seven Combination immune and antiproliferative gene transfer T Ohno et al 363 The growth rates in groups treated with ADV-Kb, ADV-p21 or both were significantly less than the growth rates in the PBS-treated group (Figure 3, P , 0.01). The groups treated with ADV-DE1/ADV-Kb or ADV- DE1/ADV-p21 showed a lower rate of tumor growth when compared to the group treated with ADV-DE1 alone (Figure 3, P = 0.23 and P = 0.07, respectively); how- ever, only the mice treated with ADV-p21/ADV-Kb showed a trend toward lower growth rates than those treated with ADV-DE1 alone (Figure 3, P = 0.02). Tumor growth was almost completely arrested by day 15 in seven of nine mice (tumors were less than 3 × 3 mm) in the group treated with the combination of ADV-Kb and ADV-p21. Figure 2 Effect of ADV-p21 on proliferation of Renca cells in vitro. 104 We also examined the rate of generation of tumor-free D Renca cells were infected with ADV-p21 and ADV- E1 at an MOI of mice in each group (Table 1). The group treated with PBS 200. Cell numbers were counted using trypan blue exclusion on days 2, had no tumor-free mice. The group treated with ADV- 4, 6 and 7 to examine the effect of p21 on cell growth. DE1 or ADV-Kb alone showed one tumor-free mouse out of eight treated mice evaluated (12.5%). In the group injections). Mice received 2 × 108 p.f.u. (50 ml per mouse) treated with ADV-p21 alone, three out of seven (42.9%) of adenovirus in each injection. Treatment with either p21 mice were tumor-free, and in the combination group of b or Kb was conducted by injecting 1 × 108 p.f.u. each of ADV-K and p21, five out of eight (62.5%) mice were ADV-DE1 mixed with an equal amount of either ADV- tumor-free on day 44. Thus, the frequency of tumor Kb or ADV-p21 to adjust the total viral dose among the regression correlated with the ability to inhibit local groups. In the combination experiment, 1 × 108 p.f.u. each tumor growth in this model.