Murine dendritic cells infected with adenovirus vectors show signs of activation.

Dendritic cells (DC) are highly efficient antigen presenting cells being actively evaluated as vaccine components. A number of studies have shown adenovirus-mediated gene transfer to cultured DCs is feasible and that Ad-modified DCs are effective at inducing T cell immunity in vitro and establishing antitumor immunity in experimental tumor models in vivo. The current study evaluates the biologic effects of Ad infection on murine bone marrow-derived DCs (BMDC) in primary culture. Ad infection (MOI 200) of BMDC induced significant increases in IL 12 p40 protein in culture supernatants (6 x that of uninfected BMDC and similar to that observed with addition of LPS and CD40 crosslinking antibody). Supernatants from Ad infected BMDCs induced appreciable increases in IFNgamma from naive splenocytes in culture. Consistent with DC activation, FACs analysis showed BMDC infected with Ad vectors up-regulated the surface expression of B7-2, ICAM-1 and MHC II. Additional experiments evaluated the role of virus attachment, internalization and gene expression using IL-12 p40 production as a marker of DC activation. Neither heat-inactivated Ad nor peptides containing the RGD sequence (the primary component of Ad penton base which interacts with cell surface integrins) induced significant amounts of IL12 p40. In contrast, psoralen/UV-inactivated Ad showed similar levels of IL12 p40 production compared with intact Ad. These data suggest this phenomenon is dependent on viral entry into the cell and/or translocation to the nucleus, and is independent of either viral gene or transgene expression.

Regional treatment of hepatic micrometastasis by adenovirus vector-mediated delivery of interleukin-2 and interleukin-12 cDNAs to the hepatic parenchyma.

We hypothesize that adenovirus (Ad) vector-mediated delivery of the human interleukin-2 (IL-2) cDNA (AdIL2) or the murine IL-12 cDNA heterodimer (AdIL12) would produce high concentrations of cytokines in the local hepatic milieu to induce host responses sufficient to inhibit the growth of experimental colon carcinoma-derived hepatic metastases. Ad vectors administered intravenously, which is a route known to deliver >90% of the vector to the hepatic parenchyma, achieved significant levels of each cytokine locally, with minimal levels in the sera. To examine the therapeutic effect, the AdIL2 and AdIL12 vectors were evaluated in a hepatic metastasis model that was established by injecting 3 x 10(4) cells from the poorly immunogenic syngeneic C26 colon carcinoma cell line into the right lobe of the livers of BALB/c mice. Animals received AdIL2, AdIL12, or control virus (10(8) plaque-forming units each) intravenously for 2 days after tumor implantation, and tumor growth was compared with naive controls. The AdNull control tumors measured 116 +/- 25 mm2 at 2 weeks. The control virus showed no significant antitumor effect. In marked contrast, both AdIL2 and AdIL12 vectors that were delivered regionally had significant antitumor effects, with AdIL2-treated animals having an average tumor size of 16 +/- 8 mm2; AdIL12-treated tumors measured 6 +/- 6 mm2 (P < .01, both compared with control). Both the AdIL2 and AdIL12 vectors provided a significant survival advantage by log-rank analysis (P < .01), but only AdIL12 translated into an increase in mean survival from 27 (naive control) to 37 days. To evaluate whether these antitumor effects were T-cell-mediated, splenocytes from AdIL2-treated, AdIL12-treated, and naive control groups were stimulated in vitro with gamma-irradiated C26 tumor cells for 5 days and tested for C26 tumor cell cytolysis by an in vitro cytotoxicity assay. Splenocytes from both AdIL2- and AdIL12-treated animals showed a dose-dependent, T-cell-mediated, specific cytolysis of CT26 cells. AdIL12 and to a lesser extent AdIL2 induced natural killer cell activity, as determined by a dose-dependent increase in lysis of the natural killer-specific target cell YAC-1. Overall, these data suggest that regional Ad-mediated delivery of IL-2 and IL-12 cDNAs may be useful for local tumor control and may warrant further investigation as a potentially useful adjuvant for the treatment of hepatic micrometastasis.

Variability of human systemic humoral immune responses to adenovirus gene transfer vectors administered to different organs.

Administration of adenovirus (Ad) vectors to immunologically naive experimental animals almost invariably results in the induction of systemic anti-Ad neutralizing antibodies. To determine if the human systemic humoral host responses to Ad vectors follow a similar pattern, we evaluated the systemic (serum) anti-Ad serotype 5 (Ad5) neutralizing antibodies in humans after administration of first generation (E1(-) E3(-)) Ad5-based gene transfer vectors to different hosts. AdGVCFTR.10 (carrying the normal human cystic fibrosis [CF] transmembrane regulator cDNA) was sprayed (8 x 10(7) to 2 x 10(10) particle units [PU]) repetitively (every 3 months or every 2 weeks) to the airway epithelium of 15 individuals with CF. AdGVCD.10 (carrying the Escherichia coli cytosine deaminase gene) was administered (8 x 10(8) to 8 x 10(9) PU; once a week, twice) directly to liver metastasis of five individuals with colon cancer and by the intradermal route (8 x 10(7) to 8 x 10(9) PU, single administration) to six healthy individuals. AdGVVEGF121.10 (carrying the human vascular endothelial growth factor 121 cDNA) was administered (4 x 10(8) to 4 x 10(9.5) PU, single administration) directly to the myocardium of 11 individuals with ischemic heart disease. Ad vector administration to the airways of individuals with CF evoked no or minimal serum neutralizing antibodies, even with repetitive administration. In contrast, intratumor administration of an Ad vector to individuals with metastatic colon cancer resulted in a robust antibody response, with anti-Ad neutralizing antibody titers of 10(2) to >10(4). Healthy individuals responded to single intradermal Ad vector variably, from induction of no neutralizing anti-Ad antibodies to titers of 5 x 10(3). Likewise, individuals with ischemic heart disease had a variable response to single intramyocardial vector administration, ranging from minimal neutralizing antibody levels to titers of 10(4). Evaluation of the data from all trials showed no correlation between the peak serum neutralizing anti-Ad response and the dose of Ad vector administered (P > 0.1, all comparisons). In contrast, there was a striking correlation between the peak anti-Ad5 neutralizing antibody levels evoked by vector administration and the level of preexisting anti-Ad5 antibodies (P = 0.0001). Thus, unlike the case for experimental animals, administration of Ad vectors to humans does not invariably evoke a systemic anti-Ad neutralizing antibody response. In humans, the extent of the response is dictated by preexisting antibody titers and modified by route of administration but is not dose dependent. Since the extent of anti-Ad neutralizing antibodies will likely modify the efficacy of administration of Ad vectors, these observations are of fundamental importance in designing human gene therapy trials and in interpreting the efficacy of Ad vector-mediated gene transfer.

Adenovirus-mediated expression of interleukin-12 induces natural killer cell activity and complements adenovirus-directed gp75 treatment of melanoma lung metastases.

Based on the knowledge that adenovirus (Ad)-mediated expression of the murine gp75 melanoma antigen (Adgp75) will effectively immunize mice against H2-matched B16 melanoma cells, probably via cell- mediated immune mechanisms, we hypothesized that Ad-mediated delivery of the murine interleukin-12 (IL-12) complementary DNA heterodimer would have independent therapeutic effects on tumor growth, and that the combination of the two vectors would work synergistically to augment the antitumor response. We evaluated the therapeutic effect of each vector alone and in combination for efficacy in C57BL/6 mice with preestablished (2 d) B16 melanoma-derived pulmonary metastases, using the number of lung metastases as the efficacy parameter. Intraperitoneal administration of Adgp75 (10(8) PFU) reduced tumor burden to 45 +/- 7% of controls (P < 0.01), and AdIL12 administration (10(8) PFU, intraperitoneally) reduced the number of metastases to 43 +/- 7% of controls (P < 0.01). The combination of Adgp75 (10(8) PFU, intraperitoneally) and AdIL12 (10(8) PFU, intraperitoneally) provided further protection (15 +/- 3%; P < 0.01 as compared with naive control; P < 0.01 compared with Adgp75 or AdIL12 alone). Mice receiving AdIL12 showed increased natural killer cell (NK cell) function in an in vitro cytotoxicity assay, with a dose- dependent lysis of YAC-1 cells and, to a lesser extent, lysis of B16 cells. To assess the relative contribution of major histocompatibility complex I (MHC I) Dependent and Independent activity in combination therapy with Adgp75 plus AdIL12, we performed adoptive transfer experiments, using splenocytes from mice receiving Adgp75, AdIL12, or Adgp75 + AdIL12, from among which NK cells had been selectively depleted in vitro prior to adoptive transfer. Each group showed significant decreases in tumor burden resembling those with primary treatment. Interestingly, NK-cell depletion from among cells derived from the Adgp75- and AdIL12-treated mice significantly altered the therapeutic response (P < 0.01 compared with the Adgp75 + AdIL12 group), suggesting a significant role of NK-cell-mediated cytolysis in vivo, although there was still a significantly reduced tumor burden (P < 0.01 compared with that of naive controls). Collectively, these data support the concept that the combination of AdIL12 and Adgp75 provides additive effects against pulmonary metastases of B16 melanoma by MHC-independent (NK cell) means as well as MHC-dependent cytotoxic lymphocyte means, suggesting that this therapy may be a useful adjuvant in the treatment of metastatic melanoma.

Adenovirus-mediated expression of melanoma antigen gp75 as immunotherapy for metastatic melanoma.

Melanocyte differentiation antigens, such as the brown locus protein gp75, are potential biological targets for immunotherapy. We investigated whether expression of the murine gp75 cDNA mediated by an adenovirus (Ad) vector could induce melanoma rejection using this model self antigen that usually induces tolerance, and whether Ad vector-directed production of interleukin-2 (IL2) might augment this response. To evaluate this approach, Ad vectors were constructed containing the murine gp75 cDNA (Ad.gp75) and the human IL2 cDNA (Ad.IL2). Efficacy was evaluated in C57BI/6 mice challenged i.v. with 10(5) B16 cells, using the number of lung metastases as the efficacy parameter. Naive control mice developed 175 +/- 12 metastases by day 14. Controls receiving intranasal Ad.IL2 1 day after B16 cell injection, intraperitoneal (i.p.) mitomycin-C-treated B16 cells +/- i.p. Ad.IL2 before B16 cell challenge and Ad.beta gal-treated mice had similar numbers of metastases as controls (P > 0.1). In marked contrast, preimmunization with intradermal Ad.gp75 provided dramatic reduction in the number of lung metastases (52 +/- 7, 29% of control). Addition of regional (intranasal delivery to the lung) Ad.IL2 to intradermal Ad.gp75 preimmunization 1 day following tumor challenge provided further protection (18 +/- 6, 10% of control). Depletion of CD4+ and CD8+ T-cell subsets effectively blocked the protective effect seen following immunization. Adoptive transfer of macrophage-depleted splenocytes from Ad.gp75-immunized mice similarly afforded significant protection against B16 tumor cell challenge. Further, serum obtained 21 days following Ad.gp75 immunization showed no detectable anti-gp75 antibody by immunoprecipitation. These results suggest that immunization with Ad.gp75 induces cellular immune responses that are capable of rejecting B16 melanoma in a host that is usually tolerant to gp75 antigen.

Inhibition of vascular smooth muscle cell proliferation and neointimal accumulation by adenovirus-mediated gene transfer of cytosine deaminase.

BACKGROUND: Restenosis remains a significant problem after balloon angioplasty. Previous studies have demonstrated that recombinant adenoviruses are efficient vectors for gene transfer to the arterial wall and can be used to inhibit the proliferative aspect of restenosis. We sought to extend these observations using AdCMV.CD, an adenovirus that encodes cytosine deaminase (CD) and is capable of metabolizing 5-fluorocytosine (5-FC) to 5-fluorouracil. METHODS AND RESULTS: Infection of vascular smooth muscle cells (VSMC) with AdCMV.CD increases by two to three orders of magnitude the growth-inhibitory effects of 5-FC. The degree of VSMC inhibition in vitro was a function of 5-FC concentration and the level of CD expression. Cells infected with AdCMV.CD exhibited a profound bystander effect on the growth of neighboring cells, which did not require direct cell-to-cell contact. The predominant effect of AdCMV.CD on growth of VSMC appeared to be cytostatic, not cytotoxic. Assessment of this strategy in a rabbit femoral artery model of balloon-induced injury demonstrated that compared with animals in either of two control groups, animals treated with the active combination of infection with AdCMV.CD and 1-week treatment with parenteral 5-FC had a significant reduction at 30 days in the neointimal-to-medial ratio. CONCLUSIONS: Our results suggest that adenovirus-mediated gene transfer of CD along with 5-FC administration may be a useful strategy to treat the proliferative aspects of restenosis.

Adenoviral thymidine kinase prodrug gene therapy inhibits sarcoma growth in vivo.

Local recurrence of sarcoma is due to residual tumor cells remaining after surgical resection and is associated with decreased survival. We implemented adenoviral-mediated transfer of the herpes simplex thymidine kinase (HSTK) gene with subsequent ganciclovir (GCV) administration to treat a model of residual sarcoma, [3H]Thymidine uptake in MCA sarcoma cells was determined after infection with replication incompetent adenovirus of the AdMLP.HSTK construct in the presence of GCV. In vivo efficacy was evaluated in a model of residual sarcoma when 9 mg of MCA tumor was implanted into the latissimus muscle of Fischer 344 rats. Three days after implantation, animals were randomized to receive AdMLP.HSTK, AdCMV. Null, or viral suspension buffer intratumorally. From Day 4, animals were administered b.i.d. GCV (50 mg/kg) or saline ip. Tumors were excised on Day 14 and weighed. Statistical analysis was by Mann-Whitney U test. In vitro: [3H]thymidine incorporation was significantly decreased in MCA sarcoma cells infected with AdMLP.HSTK in the presence of GCV (P < 0.05). In vivo: Growth of MCA sarcoma treated with AdMLP.HSTK and GCV was significantly inhibited. Final tumor weights in the AdMLP.HSTK/GCV group were lower than all control groups (P < 0.05). A significant antitumor growth effect on MCA sarcoma was seen with adenoviral-mediated transfer of the HSTK gene and GCV administration, both in vitro and in an in vivo model of residual disease. This prodrug gene therapy strategy warrants investigation as an adjuvant modality in the management of sarcoma.

In vivo adenoviral-mediated gene transfer in the treatment of pancreatic cancer.

Gene therapy may allow targeted delivery of tumoricidal drugs to treat pancreatic cancer. Cytosine deaminase (CD) is a bacterial enzyme that converts the nontoxic agent 5-fluorocytosine (5FC) to the active chemotherapeutic agent 5-fluorouracil (5FU). Neoplastic cells induced to express the CD gene treated with 5FC may generate locally high concentrations of 5FU while minimising systemic toxicity. Replication deficient adenovirus vector carrying the CD gene (AdCMV.CD) was tested for therapeutic efficacy against the murine pancreatic carcinoma cell line Pan02. Pan02 cells were infected in vitro with AdCMV.CD or null vector (Ad.-Null) and were examined for expression of CD messenger RNA (mRNA) (Northern blot) and CD enzymatic function (spectrophotometry). mRNA transcripts of the CD gene increased in a dose-dependent manner after infection with AdCMV.CD. Conversion of 5FC to 5FU at a multiplicity of infection (MOI) of 20 was measured to be 51% after a 48-hr incubation. Growth inhibition was measured by MTT assay and thymidine uptake. Pan02 growth in vitro treated with AdCMV.CD and 5FC was inhibited by 80% as compared to cells treated with Ad.Null and 5FC. An in vivo model of pancreatic cancer was established by injecting 2.5 x 10(5) PAN02 cells subcutaneously into the flanks of C57BL/ 6 mice. Seven days later AdCMV.CD was injected into each tumor and 5FC was administered for 10 days. Treatment of mice with AdCMV.CD and 5FC inhibited tumor growth compared to mice who received AdCMV.CD only or 5FC only. These data demonstrate the therapeutic efficacy of an enzyme prodrug strategy in experimental pancreatic cancer.

Delayed-type hypersensitivity response to high doses of adenoviral vectors.

The present study evaluates the hypothesis that delayed-type hypersensitivity (DTH) contributes to the inflammatory reaction observed when high-dose adenoviral (Ad) vectors are administered to a previously immunized animal. Immunocompetent C57BL/6 mice immunized intraperitoneally with 10(9) pfu AdCMV.Null [an E1-, E3- Ad vector with a cytomegalovirus (CMV) promoter but no transgene] and challenged intradermally to the footpad with the same vector demonstrated significant footpad swelling 24 hr after challenge with 10(9) pfu, but not with a lower dose. Footpad histology revealed a mononuclear-granulocytic cellular infiltrate typical of that seen in DTH. Evaluation of the same doses of vector in immunodeficient mice nu/nu and RAG-2- on the C57BL/6 background, and nu/nu and severe combined immunodeficiency (SCID) on the BALB/c background demonstrated suppression of footpad swelling. However, the footpad response remained intact in beta 2-microglobulin deficient (beta 2-m-) mice, suggesting minimal or no role of major histocompatibility complex (MHC) class I-mediated mechanisms for the region of localized inflammation. Challenge with an Ad expressing the interleukin-2 cDNA to immunized C57BL/6 mice demonstrated augmented footpad swelling response. Finally, pretreatment with cyclosporin resulted in a 69% inhibition of the response compared to controls, whereas other immunosuppressants (cyclophosphamide, methotrexate, and hydrocortisone) had no inhibitory effect. These findings provide further insight into the dynamic interplay of immune processes ultimately leading to inflammation when high-dose Ad vectors are administered to a target organ.

Regional delivery of an adenovirus vector containing the Escherichia coli cytosine deaminase gene to provide local activation of 5-fluorocytosine to suppress the growth of colon carcinoma metastatic to liver.

To evaluate the hypothesis that regional delivery of an adenovirus vector containing the Escherichia coli cytosine deaminase gene (AdCMV.CD) together with systemic 5-FC could suppress the growth of metastatic colon cancer in the liver, the AdCMV.CD vector was injected 0.8-1 cm from the site of a human colon cancer tumor in the livers of nude mice. The growth of the human colon cancer cells was quantified by dot blot analysis of genomic DNA extracted from tumor-bearing liver, hybridized with a human-specific Alu probe. The combination of regional AdCMV.CD plus systemic 5-fluorocytosine (5-FC) suppressed the growth of the metastatic tumors over the 21 days of evaluation following vector administration. Histologic evaluation showed necrosis at the site of the tumor in the livers of mice treated with AdCMV.CD/5-FC, but not in control groups. Evaluation of the potential toxicity of AdCMV.CD plus 5-FC on the normal liver showed only mild, self-limited dose-related inflammation, with no deaths. These data suggest that the regional administration of AdCMV.CD together with systemic 5-FC may be a safe and effective strategy to suppress the growth of metastases of colorectal carcinoma in the liver.

In vivo replication-deficient adenovirus vector-mediated transduction of the cytosine deaminase gene sensitizes glioma cells to 5-fluorocytosine.

Viral vector-mediated transfer of chemosensitization genes represents a promising new approach to the treatment of cancer. Previous reports have demonstrated that transfection of the bacterial cytosine deaminase (cd) gene into mammalian cells can sensitize them to the otherwise nontoxic nucleoside, 5-fluorocytosine (5-FC). We now report that a replication-deficient adenovirus vector that transduces the cd gene (Ad.CMV-cd) highly sensitizes 9L gliosarcoma cells to 5-FC, and that gene transduction is associated with a potent bystander effect that is not dependent on direct cell-to-cell contact. Stereotactic injection of Ad.CMV-cd into established rat gliomas, followed by systemic administration of 5-FC in vivo, results in prolongation of survival.

In vivo adenovirus-mediated gene transfer of the Escherichia coli cytosine deaminase gene to human colon carcinoma-derived tumors induces chemosensitivity to 5-fluorocytosine.

To evaluate the concept that in vivo transfer of the Escherichia coli cytosine deaminase gene will confer sensitivity of a solid tumor to the prodrug 5-fluorocytosine (5FC), we constructed an adenovirus vector (AdCMV.CD) carrying the cytosine deaminase gene driven by the cytomegalovirus (CMV) promoter, infected HT29 colon carcinoma cells in vitro and in vivo, and evaluated cell growth over time. AdCMV.CD produced a functional cytosine deaminase protein in HT29 cells in vitro as evidenced by the ability of lysates from the infected cells to convert [3H]5FC to its active metabolite 5-fluorouracil (5FU). The AdCMV.CD vector effectively suppressed HT29 cell growth in vitro in the presence of 5FC in a dose-dependent manner. Infection with AdCMV.CD, when as few as 10% of cells expressed the cytosine deaminase gene, was associated with a bystander effect when combined with 5FC in cell mixing studies. Further, this bystander effect was not dependent on cell-to-cell contact as demonstrated by suppression of [3H]thymidine incorporation in HT29 cells when supernatant from AdCMV.CD-infected cells treated with 5FC was transferred cells. Consistent with these in vitro observations, when AdCMV.CD was directly injected into established subcutaneous HT29 tumors in nude mice receiving 5FC, there was a four-fold reduction in tumor size at day 15 compared to controls, and a five-fold reduction at day 28. These observations suggest that adenovirus-mediated gene transfer of the E. coli cytosine deaminase gene and concomitant administration of 5FC may have potential as a strategy for local control of the growth of tumor cells susceptible to 5FU.

In vivo suppression of injury-induced vascular smooth muscle cell accumulation using adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene.

Restenosis, a process characterized in part by excessive smooth muscle cell (SMC) proliferation in areas of vascular injury, occurs in up to 50% of patients undergoing balloon angioplasty. In an effort to develop a treatment strategy for restenosis, we constructed a replication-deficient recombinant adenovirus (AdMLP.HSTK) containing the herpes simplex virus thymidine kinase gene (HSV tk). This viral gene product phosphorylates the prodrug ganciclovir to form a nucleoside analog that inhibits DNA synthesis. Cultured primary rat SMCs infected with AdMLP.HSTK were completely growth-inhibited by incubation in ganciclovir-containing medium. In addition, when only a portion of the SMC population received the HSV tk transgene, an inhibitory effect on neighboring SMCs was evident. Evaluation of this strategy in vivo using a rat carotid balloon injury model demonstrated that local infection of injured arteries with AdMLP.-HSTK followed by 2 weeks of systemic ganciclovir treatment significantly (P < 0.01) reduced injury-induced SMC accumulation. In contrast, there was no suppression of injury-induced SMC accumulation in animals infected with AdMLP.HSTK but not receiving ganciclovir or in those animals infected with a control adenovirus and either treated or not treated with ganciclovir. These results demonstrate the potential utility of adenovirus-mediated gene transfer for treatment of restenosis after balloon injury.