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Genetic immunotherapy of lung cancer using conditionally replicating adenovirus and adenovirus-interferon-β

Abstract

Genetic immunotherapy is considered an ideal treatment modality for cancer because of its systemic nature. This study was designed to develop a potent novel genetic immunotherapy by combining conditionally replicating adenovirus (CRAd) and replication-defective adenovirus expressing interferon-β (ad-IFN-β). We investigated the efficacy of this therapy in an immunocompetent mouse tumor model. Transduction with CRAd (Δ24RGD) induced cytolysis in a mouse lung cancer cell line (Lewis lung carcinoma (LLC)). Combined transduction of ad-IFN-β and Δ24RGD in the LLC cells induced a greater and more prolonged production of IFN-β. Media transfer from the LLC-Δ24RGD-ad-IFN-β to untransduced LLC cells induced the production of IFN-β; these results confirmed the replication and release of ad-IFN-β. LLC cells transduced with ad-IFN-β and Δ24RGD had decreased tumorigenicity in syngeneic mice. Tumor vaccination with irradiated LLC-ad-IFN-β-Δ24RGD showed a significant increase in the survival of tumor-bearing syngeneic mice compared with mice with a single transduced LLC vaccination; this was mediated by an enhanced cytotoxic T-lymphocyte response against the LLC cells. The results of this study showed that cotransduced Δ24RGD to ad-IFN-β aided the replication of ad-IFN-β in the LLC cells. A high local concentration of IFN-β and local release of tumor antigen by CRAd induced strong antitumor immunity. This combination strategy might provide a powerful means by which ad-cytokines and CRAd can be combined and other adenoviruses expressing different cytokines might also be used.

Introduction

Cancer remains a life-threatening illness in most cases. The current treatment modalities, such as surgery, chemotherapy and radiation, have limitations with regard to their effects on the improvement of cancer survival, including lung cancer. The need for new treatment modalities for patients with cancer has led to research in gene therapy, immunotherapy and new targeted therapies. Genetic immunotherapy has a great theoretical advantage over the current regimens; genetic immunotherapy is not destructive and cancer selective and uses the host's immune surveillance. However, many obstacles exist for the use of immunotherapy in the clinical setting, such as the absence of potent tumor-specific immune stimulants, the identification of ideal administration routes and the presence of defective host immune systems.1 Recombinant adenoviruses expressing cytokines have been studied for a long time; however, to date, clinical trials with adenovirus (ad)-cytokine have shown only limited responses.2, 3, 4, 5

The concept of an oncolytic adenovirus (conditionally replicating adenovirus (CRAd)) was introduced in 1996. A mutant adenovirus with a deletion of the E1B gene (ONYX-015) can replicate in and cause lysis of human cancer cells with defective p53, but not cells with intact p53.6, 7 The Δ24 is another version of the CRAd with a 24-bp deletion in E1A; this adenovirus can replicate in cancer cells defective in the pRB/p16 pathway. Mutant E1 protein produced from Δ24 loses the potential of binding and inactivating pRb in normal cells that permits adenovirus to replicate; however, it can act as a normal E1 protein in cancer cells defective in the pRB/p16 pathway.8

With the aim of improving antitumor effects, another modification of CRAd referred to as an armed therapeutic adenovirus has been introduced.9 Several therapeutic genes, such as drug-sensitizing genes10 and apoptosis-inducing genes,11 have been inserted into the E1 or E3 region of the CRAd. These viruses have dual antitumor effects (direct oncolysis due to tumor-specific replication and tumor-killing effects of the co-inserted therapeutic genes). An armed therapeutic adenovirus with therapeutic genes has been shown to improve the antitumor effects compared with the original CRAd; however, the antitumor effects previously reported are locoregional.10, 11

Another version of an armed therapeutic adenovirus is the combination of oncolytic gene therapy and cytokine gene therapy. Cytokine genes have been inserted into CRAds. These adenoviruses were effectively replicated in and caused lysis of cancer cells, and also produced cytokines in the tumor microenvironment. Release of tumor antigens and the attraction of immune effector cells by high levels of cytokines have been shown to induce strong antitumor immunity.12, 13, 14, 15, 16

Previously, we introduced a new strategy with combination gene therapy using the oncolytic adenovirus (Δ24) with E1-deleted, replication-defective adenovirus containing therapeutic genes instead of the oncolytic adenovirus containing the therapeutic genes. When both adenoviruses infected the same cancer cells, the mutant E1 protein from the Δ24 aided the E1-deleted adenovirus replicate in the cells (transcomplementation). As a result, both the Δ24 and E1-deleted adenoviruses replicated in and caused lysis of the cells, and spread laterally to the neighboring cancer cells. Furthermore, the tumor-restricted replication of the E1-deleted adenovirus induced a greater and more prolonged production of the therapeutic gene product with strong antitumor effects.17, 18 In contrast to previous experiments, we used the murine lung cancer cell line for this experiment to evaluate antitumor immunity in syngeneic, immunocompetent mice. Can Δ24 with or without RGD engineered into the fiber knob protein efficiently infect murine cancer cells and induce the replication of cotransduced replication-defective adenovirus? This is an important issue to be answered in the experiments presented herein.

Interferon-β is known to stimulate antitumor immunity by regulating antibody production and activating natural killer, cytotoxic T lymphocytes (CTLs) and macrophage activity.19, 20 Intrapulmonary instillation of adenovirus-interferon-β (ad-IFN-β) has been very effective in treating murine bronchogenic adenocarcinoma by direct killing due to natural killer cells and induction of CTL.19, 21

This study was designed to evaluate a potent novel genetic immunotherapy with CRAd (Δ24RGD) and ad-IFN-β combined to induce tumor antigen release caused by CRAd and a greater and more prolonged production of IFN-β from tumor-restricted replication of ad-IFN-β.

Materials and methods

Recombinant adenoviruses

An adenovirus expressing mouse IFN-β was constructed using the BD Adeno-X expression system (BD Biosciences Clontech, Palo Alto, CA). Briefly, mouse IFN-β cDNA in pORF (InVivogen, San Diego, CA) was inserted into multiple cloning sites of the pShuttle 2. The mouse IFN-β was retrieved from the pShutte 2-IFN-β by digestion with I-CeuI and Pl-SceI, and then cloned into the BD adeno-X viral DNA by in vitro ligation. After digestion with Swa I and transformation into Escherichia coli, a large amount of adeno-X-IFN-β was obtained. Adeno-X-IFN-β was then transfected into 293 cells, and the generation of recombinant ad-IFN-β was confirmed by the appearance of the cytopathic effects. The ad-IFN-β was confirmed again by DNA sequencing of the cloning site. In addition, production of the mouse IFN-β was confirmed by enzyme-linked immunosorbent assay (ELISA) (mouse interferon-β ELISA kit; PBL Biomedical Laboratories, Piscataway, NJ).

The conditionally replicating adenovirus (CRAd), Δ24RGD, was provided by David T Curiel (University of Alabama at Birmingham). Δ24 and Δ24-luc (Δ24 containing the luciferase gene in the E3 region) were provided by Victor van Beusechem (VU University, the Netherlands). The Δ24RGD is a Δ24 with an Arg-Gly-Asp (RGD) sequence in the adenoviral fibers, known to interact with αv-integrin to enhance tumor infection.22 Adenovirus-luciferase (ad-luc) is a recombinant E1-deleted, replication-defective adenovirus with a CMVie promoter and luciferase gene in the E1 region of the adenoviral genome.

All the adenoviruses were propagated in the 293 cells and were concentrated and purified with the BD adeno-X purification kit (BD Biosciences Clontech). The titers of the adenoviruses were determined by the TCID 50 method.

Cells and animals

The mouse lung carcinoma cell line (Lewis lung carcinoma (LLC)) and A549 (human lung adenocarcinoma) were purchased from the American Type Culture Collection (Manassas, VA). Male C57BL/6 mice (6 weeks) were purchased from the Orient Corporation (Seongnam, Korea) and were originally from Charles River Laboratories International (Wilmington, MA).

Oncolysis of LLC by Δ24RGD

To confirm the replication and cytolysis of Δ24RGD in the mouse cancer cell line, LLC cells were transduced with Δ24RGD at various concentrations (0.1 multiplicity of infection (m.o.i.) to 500 m.o.i.) in 96-well plates (3 × 103 cells per well) for 1 h and then incubated in complete media for 96 h. Cell growth was measured by absorbance at 490 nm by a 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide assay kit (CellTiter 96 AQueous One Solution Cell Proliferation Assay; Promega Corporation, Madison, WI) in 12-well plates (1 × 105 cells per well) and read by an ELISA reader (Spectra Max Plus 384; Molecular Devices, Sunnyvale, CA). The same experiment was carried out in a human lung cancer cell line (A549) with lower concentrations of Δ24RGD.

Measurement of luciferase expression from Δ24-luc or ad-luc and Δ24RGD-cotransduced cells

We measured luciferase expression using the Luciferase Assay System (Promega Corporation) to confirm the replication of ad-luc with the aid of mutant E1 protein from the Δ24 in the mouse cell line. The A549 (human lung cancer cell line) and LLC (mouse lung cancer cell line) cells in 12-well plates (1 × 105 cells per well) were transduced with ad-luc (10 m.o.i. or 100 m.o.i.)±Δ24 (100 m.o.i.) or Δ24-luc (100 m.o.i.) for 1 h. Luciferase expression was measured after 48 h by measuring relative light units using the LMax II384 (Molecular Devices).

Measurement of IFN-β production from ad-IFN-β or ad-IFN-β+Δ24RGD in the mouse lung cancer cell line (LLC)

Interferon-β production was measured from the supernatant of cells transduced with ad-IFN-β±Δ24RGD to determine the effects of the cotransduced Δ24RGD in the ad-IFN-β-transduced LLC cells. The LLC cells were transduced with ad-IFN-β (10 and 100 m.o.i.)±Δ24RGD (10 and 100 m.o.i.) for 1 h in 12-well plates (1 × 105 cells per well) and then incubated for 72 h. The IFN-β in the supernatant of each group was measured using a mouse IFN-β ELISA kit, according to the manufacture's protocol (PBL Biomedical Laboratories).

IFN-β production from irradiated LLC transduced with ad-IFN-β or ad-IFN-β+Δ24RGD

To determine the duration and amount of IFN-β production, we measured the amount of IFN-β in the supernatant daily for 5 days from the LLC-ad-IFN-β (100 m.o.i.) or LLC-ad-IFN-β (100 m.o.i.)–Δ24RGD (10 m.o.i.). We transduced LLC cells in 12-well plates (1 × 105 cells per well) with ad-IFN-β (100 m.o.i.) or ad-IFN-β (100 m.o.i.)+Δ24RGD (10 m.o.i.). We treated the adenovirus-transduced LLC cells with radiation (1 Gy) after transduction because the LLC cells, used as a tumor vaccine, were also treated with radiation to reduce tumorigenicity while maintaining viability and cytokine production. Daily production of IFN-β in the supernatant were measured by a mouse IFN-β ELISA kit.

Media transferable bystander effect by release of ad-IFN-β due to Δ24RGD

To determine the replication and release of ad-IFN-β from the cells after cotransduction with Δ24RGD, we transduced LLC cells with ad-IFN-β (100 m.o.i.)±Δ24RGD (10 m.o.i.) for 48 h in 12-well plates (1 × 105 cells per well), transferred the media (1 ml) to the untransduced LLC cells in another 12-well plate (1 × 105 cells per well) and measured the IFN-β in the supernatant of the second plate after 48 h.

In vivo tumorigenicity assay

The LLC cells were transduced with ad-IFN-β (100 m.o.i.) alone, Δ24RGD (100 m.o.i.) alone, or ad-IFN-β (100 m.o.i.)+Δ24RGD (100 m.o.i.) for 1 h in 10 cm plates, and then incubated for 48 h. Then, the LLC cells (1.0 × 106 per mouse) were diluted in 200 μl of phosphate-buffered saline and injected subcutaneously into the flank of the C45BL/6 mice. Subcutaneous tumor formation was observed and measured by the following formula: tumor volume = 0.5 × (length) × (width).2 The analysis of variance test was used for statistical analysis.

Treatment of established tumor

To simulate a clinical situation, we applied this strategy to established tumors. Briefly, the LLC (1.0 × 105 per mouse) cells were injected into the subcutaneous tissue of the right flank of the mice (C57BL/6) and tumor formation was observed over 7 days. Then, the LLC (5 × 105 per mouse) cells were transduced with ad-IFN-β (100 m.o.i.) alone, Δ24RGD (100 m.o.i.) alone, or ad-IFN-β (100 m.o.i.)+Δ24RGD (100 m.o.i.), and then injected into the left flank of the mice as tumor vaccines. All LLC cells injected as tumor vaccines were irradiated (1 Gy) to reduce tumorigenicity, but maintain the production of cytokines just before the injection. The survival of the tumor-bearing animals was evaluated.

CTL assay with an IFN-γ ELISPOT assay

To investigate the immune mechanisms, at the cellular level, of combining Δ24RGD and ad-IFN-β, we performed an interferon-γ enzyme-linked immunospot (ELISPOT) assay for the measurement of IFN-γ produced from CTL in spleen cells. The LLC cells transduced with Δ24RGD (100 m.o.i.) alone, ad-IFN-β (100 m.o.i.) alone, or Δ24RGD (100 m.o.i.)+ad-IFN-β (100 m.o.i.) and irradiated with 1 Gy were injected into the subcutaneous tissues of the C56BL/6 mice. The mice were killed 2 weeks later and the spleens were extracted and minced. The splenocytes were isolated and stimulated with irradiated LLC (50 Gy) cells at a ratio of 10:1 for 24 h. The splenocytes (1 × 105 per well) were incubated in a 96-well plate coated with mouse anti-IFN-γ antibodies for 24 h, and then stained according to the manufacturer's protocol (mouse IFN-γ ELISPOT assay kit; R & D Systems, Minneapolis, MN); the resulting spots were quantified using an image analyzer.23

Results

Cytolysis of LLC after transduction with Δ24RGD

We investigated whether Δ24RGD could infect, replicate and induce cytolysis in the mouse cancer cell line (LLC). Transduction with Δ24RGD induced cytolysis of LLC; however, it required a high dose of Δ24RGD. Cytolysis became evident at a Δ24RGD of 100 m.o.i. or more in the LLC. However, a Δ24RGD of 5 m.o.i. induced almost complete cytolysis in the human lung cancer cell line (A549) (Figure 1). We confirmed that Δ24RGD could infect and replicate in the mouse cancer cell line, even though it was more resistant to Δ24RGD than the human cancer cell line.

Figure 1
figure1

Cytolysis of Lewis lung carcinoma (LLC) (mouse lung cancer cell line) and A549 (human lung cancer cell line) after transduction with Δ24RGD. LLC cells (3 × 103 cells per well) were transduced with Δ24RGD (0.1–500 multiplicity of infection (m.o.i.)) and cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide assay (absorbance at 490 nm) at 96 h. A549 cells were also transduced with Δ24RGD (0.1–10 m.o.i.). Transduction with Δ24RGD, over 100 m.o.i., induced significant cytolysis in LLC cells. A549 cells were much more susceptible to Δ24RGD-induced cytolysis. Δ24RGD, of 5 m.o.i., induced complete cytolysis of A549 at 48 h. Data are represented as the mean±s.d. in triplicate wells.

Mouse lung cancer (LLC) cells transduced with ad-luc and Δ24RGD expressed higher luciferase than cells transduced with ad-luc

We confirmed that cotransduction with Δ24RGD and ad-luc induced a greater expression of luciferase in the mouse cancer cell line. Mouse lung cancer (LLC) cells and human lung cancer cells (A549) were transduced with ad-luc and Δ24RGD. In the LLC cells, the combined transduction of Δ24RGD (100 m.o.i.) and ad-luc (100 m.o.i.) resulted in a more than 50-fold increase in luciferase expression compared with ad-luc (100 m.o.i.) alone. In addition, combined transduction of Δ24RGD and ad-luc induced a slightly higher luciferase expression than the equivalent Δ24-luc. We confirmed the same outcome with the human lung cancer cell line (A549) at a lower adenovirus dose (10 m.o.i. each) (Figure 2).

Figure 2
figure2

Enhanced luciferase expression from adenovirus-luciferase (ad-luc) and Δ24 cotransduced Lewis lung carcinoma (LLC) and A549 cells. LLC cells and A549 cells (1.0 × 105 per well in 12-well plates) were transduced with an indicated multiplicity of infection (m.o.i.) of ad-luc±Δ24 for 1 h. Relative light units (RLUs) of luciferase expression were measured at 48 h after transduction. In LLC, combined transduction of Δ24 (100 m.o.i.)) and ad-luc (100 m.o.i.) induced more than a 50-fold increase in luciferase expression compared with ad-luc (100 m.o.i.) alone. The same phenomenon was found in the human lung cancer cell line (A549) at lower adenovirus doses (10 m.o.i. each). Data are represented as the mean±s.d. in triplicate wells.

Enhancement of IFN-β production by combined transduction of ad-IFN-β and Δ24RGD

The expression of IFN-β in ad-IFN-β-transduced LLC was significantly enhanced by the combined transduction with Δ24RGD. The addition of Δ24RGD (10 m.o.i.) to ad-IFN-β enhanced IFN-β production by 3- (ad-IFN-β: 10 m.o.i.) to 13-fold (ad-IFN-β: 100 m.o.i.). Furthermore, addition of higher Δ24RGD (100 m.o.i.) enhanced IFN-β production by 83- (ad-IFN-β: 10 m.o.i.) to 130-fold (ad-IFN-β: 100 m.o.i.) (Figure 3).

Figure 3
figure3

Cotransduction of adenovirus expressing interferon-β (ad-IFN-β) and Δ24RGD increased IFN-β production in the Lewis lung carcinoma (LLC) cells. LLC cells (1.0 × 105 per well in 12-well plates) were transduced with ad-IFN-β (10, 100 multiplicity of infection (m.o.i.))±Δ24RGD (10, 100 m.o.i.) for 1 h and kept in complete media for 72 h. IFN-β from the supernatant was measured by a mouse IFN-β enzyme-linked immunosorbent assay (ELISA) kit at 72 h after transduction. Addition of Δ24RGD enhanced the production of adenovirus expressing IFN-β (ad-IFN-β)-transduced LLC. Data are represented as the mean±s.d. in triplicate wells.

Cotransduction of LLC with ad-IFN-β and Δ24RGD induced higher and more prolonged IFN-β production

Irradiated LLC cells transduced with adenovirus survived and produced IFN-β for 5 days. The LLC cells transduced with ad-IFN-β (100 m.o.i.) and Δ24RGD (10 m.o.i.) produced a much greater amount of IFN-β daily for the 5 days than did the cells transduced with ad-IFN-β (100 m.o.i.) alone (Figure 4). This finding confirmed the viability and cytokine production of the LLC treated with adenovirus and radiation used as a tumor vaccine in the treatment experiments.

Figure 4
figure4

Higher and prolonged production rate of interferon-β (IFN-β) after addition of Δ24RGD to adenovirus expressing IFN-β (ad-IFN-β) in the transduced and irradiated the Lewis lung carcinoma (LLC). LLC cells were transduced with ad-IFN-β (100 multiplicity of infection (m.o.i.))±Δ24RGD (10 m.o.i.) for 1 h and irradiated with 1 Gy and incubated in complete media. Daily production of IFN-β from the supernatant was measured. Ad-IFN-β (100 m.o.i.) and Δ24RGD- (10 m.o.i.) cotransduced LLC resulted in significantly higher IFN-β production daily, for up to 5 days, compared with the ad-IFN-β- (100 m.o.i.) transduced cells. Data are represented as the mean±s.d. in triplicate wells.

Release of intact ad-IFN-β from LLC transduced with ad-IFN-β and Δ24RGD

Media transfer of the LLC cells transduced with ad-IFN-β (100 m.o.i.) and Δ24RGD (10 m.o.i.) to the untransduced LLC cells resulted in an increased production of IFN-β (2152 pg per 5 × 105 cells per 48 h). By contrast, the media from the LLC transduced with ad-IFN-β induced only negligible amounts (15.6 pg per 5 × 105 cells per 48 h) of IFN-β (Figure 5). This confirmed our hypothesis that cotransduction of ad-IFN-β and Δ24RGD in the LLC cells would induce replication of ad-IFN-β and release it into the surrounding tumor microenvironment.

Figure 5
figure5

Supernatant from adenovirus expressing interferon-β (ad-IFN-β) and Δ24RGD-cotransduced the Lewis lung carcinoma (LLC) cells transferred intact ad-IFN-β to untransduced LLC cells. The supernatant from LLC cells in 12-well plates (1 × 105 cells per well) transduced with ad-IFN-β (100 multiplicity of infection (m.o.i.))±Δ24RGD (100 m.o.i.) for 48 h was transferred to untransduced LLC cells in 12-well plates (1 × 105 cells per well), and then the IFN-β of supernatant of second plate was measured after 48 h. Only LLC cells transferred supernatant from the LLC transduced with ad-IFN-β and Δ24RGD produced high levels of IFN-β (2.1 ng per 1.0 × 105 cells). This confirmed the release of intact ad-IFN-β from the cotransduced LLC and supports the current strategy. Data are represented as the mean±s.d. in triplicate wells.

Decreased tumorigenicity of LLC by cotransduction of ad-IFN-β and Δ24RGD

We examined the feasibility of LLC cells transduced with ad-IFN-β±Δ24RGD as a tumor vaccine by examining tumorigenicity. The LLC cells transduced with ad-IFN-β alone and Δ24RGD alone formed a tumor when injected into the subcutaneous tissue of the C57BL/6 mice. By contrast, the LLC cells transduced with ad-IFN-β and Δ24RGD formed very small tumors compared with the other groups (P<0.01; Figure 6). However, we confirmed that the LLC cells transduced with ad-IFN-β and Δ24RGD maintained their tumor-forming potential. Therefore, all LLC cells used as tumor vaccines were treated with radiation (1 Gy) just before injection to reduce the tumorigenicity, but maintain the ability to produce cytokines.

Figure 6
figure6

Decreased tumorigenicity by in vitro transduction with Δ24RGD and adenovirus expressing interferon-β (ad-IFN-β). The Lewis lung carcinoma (LLC) cells were transduced with Δ24RGD (100 multiplicity of infection (m.o.i.)), ad-IFN-β (100 m.o.i.) or Δ24RGD (100 m.o.i.)+ad-IFN-β (100 m.o.i.) and injected into the subcutaneous tissues of mice (1 × 106 per mouse, five mice in each group). The LLC cells transduced with Δ24RGD (100 m.o.i.) or ad-IFN-β (100 m.o.i.) formed a relatively large tumor; however, the LLC-Δ24RGD-ad-IFN-β resulted in very small tumor formation compared with the other groups (P<0.01 by analysis of variance test).

Treatment potential of LLC transduced with ad-IFN-β and Δ24RGD

We evaluated the tumor vaccine strategy with the LLC transduced with adenovirus on established tumors. The mice treated with the tumor vaccines (LLC) transduced with ad-IFN-β alone or Δ24RGD alone showed no difference in survival compared with the untreated mice. However, the mice treated with LLC transduced with ad-IFN-β and Δ24RGD showed a modest increase in survival compared with the other groups (P<0.05, log–rank (Mantel–Cox); Figure 7).

Figure 7
figure7

Survival prolongation in the mice treated with the Lewis lung carcinoma (LLC)-Δ24RGD-ad-interferon-β (IFN-β). The mice (n=5 in each group) with established lung cancer by subcutaneous injection of LLC cells (1 × 105 per mouse) were treated at day 7 with contralateral subcutaneous injection of tumor vaccines (LLC: 5 × 105 cells per mouse) transduced with Δ24RGD (conditionally replicating adenovirus (CRAd); 100 multiplicity of infection (m.o.i.)), adenovirus expressing IFN-β (ad-IFN-β) (100 m.o.i.) or Δ24RGD (100 m.o.i.)+ad-IFN-β (100 m.o.i.) and irradiated with 1 Gy. Control group was treated with untransduced, but irradiated LLC cells. The mice treated with LLC-Δ24RGD-ad-IFN-β showed a modest, but significant survival prolongation compared with the other groups (P<0.05 by log–rank, Mantel–Cox). Details are described in the text.

Enhanced cytotoxic T-cell response by cotransduction with Δ24RGD and ad-IFN-β

The results of the IFN-γ ELISPOT assay revealed that the LLC-stimulated splenocytes from the mice treated with LLC-ad-IFN-β or LLC-Δ24RGD-ad-IFN-β showed higher immune cell generation than the splenocytes from mice treated with LLC-Δ24RGD. Furthermore, vaccination with LLC-Δ24RGD-ad-IFN-β induced a 73% increase in the IFN-γ immune T-cell activation against LLC than the vaccination with LLC-ad-IFN-β (Figure 8). This finding suggests that enhanced cytotoxic T-cell activation by Δ24RGD+ad-IFN-β might be the mechanism underlying the survival prolongation observed in the treatment experiment.

Figure 8
figure8

Interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay. Mice were injected with irradiated the Lewis lung carcinoma (LLC)-Δ24RGD (100 multiplicity of infection (m.o.i.)), LLC-adenovirus expressing IFN-β (ad-IFN-β) (100 m.o.i.) or LLC-Δ24RGD (100 m.o.i.)-ad-IFN-β (100 m.o.i.; 1.0 × 106 cell per mice, three mice in each group). The splenocytes were harvested from the minced spleen after 2 weeks. After stimulation with irradiated LLC (50Gy), IFN-γ production from the splenocytes was measured in a 96-well plate coated with anti-mouse INF-γ antibody. The IFN-γ ELISPOT was measured according to the manufacturer's protocol. Details are described in the text. The splenocytes from the mice treated with LLC-Δ24RGD-ad-IFN-β showed significantly higher (73% more) IFN-γ spots than the LLC-ad-IFN-β (P<0.01; by Kruskal–Wallis test). LLC-Δ24RGD alone showed only a few IFN-γ spots.

Discussion

Cancer is a systemic disease that is characterized by metastasis to multiple organs. Most cancer gene therapies, however, have focused on local treatment. This might be one of the reasons why gene therapy clinical trials have failed to produce significant results. Genetic immunotherapy might provide a method that solves these problems. Immunotherapy affects all cancer cells throughout the human body; thus, the impact is systemic. However, in spite of the initial expectations, many genetic immunotherapy protocols have also failed to show significant results in the clinical setting.3, 4, 24, 25, 26 The absence or paucity of tumor antigens and inadequate immune stimuli might be, in part, the reasons for previous failures.

In this study, we focused on the release of tumor antigens by CRAd, and on the amount and duration of cytokines produced by the ad-cytokine and released into the tumor microenvironment to attract a variety of immune cells. These factors were predicted to facilitate the host immune system that would detect the tumor antigens and then generate a tumor-specific immune response against the tumor antigens resulting in the eradication of the tumors systemically. When we designed this study, we planned to use immune-competent mice and mouse cancer cell lines. However, most CRAds have been designed to infect and replicate in human cancer cells. This situation presented us with uncertainty that had to be addressed.

First, we had to determine whether the CRAd (Δ24RGD) could replicate in the mouse cancer cell line (LLC). Jogler et al.27 reported that the CRAd could replicate only in porcine cells with efficiency close to that of human cells. The cells from all other species, including mice, were not permissive to the CRAd.27 Thomas et al.28 found that Syrian hamster cells were also permissive to the CRAd and suggested the Syrian hamster for the study of oncolytic adenoviral vectors. However, some mouse breast cancer cells were also permissive to the CRAd 29. In this study, mouse lung cancer cells (LLC) and human lung cancer cells (A549) were transduced with Δ24RGD at different concentrations and exhibited cell lysis. The LLC showed significant lysis with Δ24RGD over 100 m.o.i.; however, the A549 showed almost complete lysis at 5 m.o.i. (Figure 1). Therefore, these experiments confirmed that the Δ24RGD replicated and induced cell lysis in the LLC, even though it was more resistant and required higher concentrations of adenovirus than the human lung cancer cell line.

In addition, it was unclear whether cotransduction of the Δ24RGD and E1-deleted adenovirus in the mouse cancer cell line would induce the replication of E1-deleted adenovirus, as previously shown in human cancer cell lines.17, 18 Theoretically, E1-deleted adenovirus could replicate with the aid of the mutant E1 protein produced by Δ24RGD in the pRB/p16 inactivated cells. However, we did not know whether this would occur in the mouse cancer cell line. Guo et al.29 previously reported that Δ24 aided in the replication of ad-TRAIL in mouse breast cancer cells (4T1). Several experiments were carried out to investigate this question. The combined transduction of E1-deleted adenovirus containing the luciferase reporter gene (ad-luc) and Δ24RGD in the LLC produced a 50-fold increase in luciferase compared with the single transduction (Figure 2). This finding strongly suggests that ad-luc can replicate in LLC with the aid of Δ24RGD. In addition, we carried out the same experiment with ad-IFN-β. The cotransduction of Δ24RGD and ad-IFN-β also enhanced the production of IFN-β in the LLC by almost 100-fold. This finding indirectly confirmed that the mutant E1 from Δ24RGD aided in the replication of cotransduced ad-IFN-β in LLC. Furthermore, induction of IFN-β production by media transfer from LLC-Δ24RGD-ad-IFN-β to untransduced LLC confirmed the release of ad-IFN-β from the LLC-Δ24RGD-ad-IFN-β. A greater and more prolonged production of IFN-β definitely aided in the induction of the antitumor immune response.

Finally, we investigated whether increased IFN-β production and cytolysis induced an immune response for tumor rejection. The injection of LLC-Δ24RGD-ad-IFN-β formed markedly smaller tumors than the LLC-Δ24RGD and LLC-ad-IFN-β; however, the tumorigenicity was not completely abolished. Therefore, we used irradiated LLC in the next treatment experiment to reduce the tumorigenicity, but maintain cytokine production. In most previous clinical trials using a tumor vaccine, the tumor cells were retrieved from the original tumor and genetically modified according to specific protocols. Finally, the cells were irradiated to abolish the tumorigenicity and injected into different sites.4, 5, 30 Similarly, we designed treatment experiments to be carried out on established tumors, and injection of irradiated LLC-Δ24RGD-ad-IFN-β was used as a tumor vaccine and injected at different sites. The goal was to stimulate a systemic immune response. Treatment with the LLC-Δ24RGD-ad-IFN-β induced a modest, but significant increase in survival compared with LLC-Δ24RGD and LLC-ad-IFN-β. A more potent CTL response, measured by the IFN-γ ELISPOT assay, was induced by LLC-Δ24RGD-ad-IFN-β than LLC-ad-IFN-β (Figure 8). This modest survival benefit was somewhat disappointing. These results could be explained by the unirradiated LLC-Δ24RGD-ad-IFN-β maintaining its tumorigenicity. However, if we use this strategy with other potent immune cytokines, such as granulocyte macrophage colony-stimulating factor (GM-CSF) or interleukin (IL)-12, perhaps a stronger antitumor immune reaction would be found.

Our findings showed that the replicating potential of Δ24RGD in mouse cancer cells was very weak compared with its potential in human cells. If we applied this strategy to human cancer cells, we would expect more potent cell lysis leading to the release of tumor antigens, and a greater production of cytokines released into the tumor microenvironment. Many previous reports on oncolytic adenovirus carrying cytokine genes, such as GM-CSF,15, 31 MDA-7/IL-2413, 16, 32 and IL-12,16 studied for genetic immunotherapy showed improved antitumor immunity by oncolysis and co-expressing cytokines. Some of the previous studies used human cancer cell lines in immunodeficient mice.13, 32, 33 However, these findings are difficult to interpret because we could not expect the generation of adequate antitumor immune response in immunodeficient mice. Furthermore, oncolytic adenoviruses carrying cytokine genes have some limitations. For example, the insertion of cytokines into E3 reduced the replicating potential of CRAd.34

In contrast to previous experiments, our study had three unique findings. First, we used two different adenoviruses, CRAd and the adenovirus with E1 replaced with cytokine cDNA instead of a single CRAd carrying cytokine gene. This combination induced tumor-restricted replication of the replication-defective ad-IFN-β. Second, we studied these vectors in immunocompetent animal models. Theoretically, any E1-deleted adenovirus expressing different cytokines or immune molecules, such as major histocompatibility complex molecules and immune costimulatory molecules (B7), could combine with CRAd. Furthermore, combination of multiple adenoviruses with CRAd is feasible. Finally, we chose to inject the vaccine at many different sites instead of the usual intratumoral injection; this was carried out to simulate the clinical situation of advanced cancer in patients with cancer spread throughout the body.

In conclusion, cotransduction of Δ24RGD and ad-IFN-β into the same cancer cells resulted in the replication-defective adenovirus (ad-IFN-β) replicating with the aid of mutant E1 produced by Δ24RGD; this resulted in release into the tumor microenvironment and the infection of adjacent tumor cells. A high local concentration of IFN-β and the local release of tumor antigens by CRAd induced strong antitumor immunity. Therefore, this combination strategy of ad-cytokines and CRAd might be useful for further study. Future experiments are planned to find the ideal combination for induction of the most potent antitumor immune response for the eradication of pre-existing tumors.

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Acknowledgements

This work is supported by the Health & Medical Technology R&D program of Korea (A060195).

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Correspondence to C-T Lee.

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Park, MY., Kim, D., Jung, H. et al. Genetic immunotherapy of lung cancer using conditionally replicating adenovirus and adenovirus-interferon-β. Cancer Gene Ther 17, 356–364 (2010). https://doi.org/10.1038/cgt.2009.78

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Keywords

  • conditionally replicating adenovirus
  • adenovirus-IFN-β
  • lung cancer
  • genetic immunotherapy

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