Adenovirus-Mediated Expression of Interleukin-12 Induces Natural Killer Cell Activity and Complements Adenovirus-Directed gp75 Treatment of Melanoma Lung Metastases

Edward A. Hirschowitz and Ronald G. Crystal

Division of Pulmonary and Critical Care Medicine, New York Hospital-Cornell Medical Center, New York, New York

	Abstract

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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 (10⁸ PFU) reduced tumor burden to 45 ± 7% of controls (P < 0.01), and AdIL12 administration (10⁸ PFU, intraperitoneally) reduced the number of metastases to 43 ± 7% of controls (P < 0.01). The combination of Adgp75 (10⁸ PFU, intraperitoneally) and AdIL12 (10⁸ 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.

	Introduction

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Interleukin-12 (IL-12), normally expressed by antigen-presenting cells, including macrophages/monocytes, dendritic cells, and B cells, has a variety of effects on augmenting cellular immunity, and has significant potential as an anticancer agent (1, 2). IL-12 activates and expands the numbers of natural killer cells (NK cells), serving to destroy tumor cells, and particularly premetastatic, circulating tumor cells that express low levels of major histocompatibility complex I (MHC I) molecules (3, 4). IL-12 also has direct effects on CD8⁺ lymphocytes, and is capable of activating T-helper-1 (Th1) cells and inducing secretion of interferon- (IFN-) (5). Because IFN- in turn potentiates the Th1 phenotype, there is further augmentation of the development of cytotoxic T lymphocytes (CTL) through subsequent IL-2 secretion, as well as enhancement of antigen presentation via MHC II on the surface of antigen-presenting cells, and in some cases, enhancement of MHC I expression on the surface of tumor cells (9).

We have previously described the potential for adenovirus (Ad)-vector-mediated transfer of the melanoma antigen gp75 (Adgp75) to effectively immunize syngeneic mice against a challenge with murine B16 melanoma cells endogenously expressing gp75 (12). The dominant effector mechanism in these studies appears to have been T lymphocytes. The study reported here sought to evaluate the potential for treatment with Adgp75 alone of preestablished pulmonary metastases, and the ability of Ad-mediated transfer of murine IL-12 complementary DNA (cDNA) to enhance this effect. In this context, we have compared the effects of Adgp75 (12), an adenovirus vector expressing IL-12 (AdIL12) (13), and of a combination of these vectors, in a murine model of preestablished pulmonary metastases of melanoma. Interestingly, the data suggested that Ad-vector-mediated IL-12 expression activates NK cells in vivo, and that this contributes to the suppression of pulmonary metastases of B16 melanoma.

	Materials and Methods

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Adenovirus Vectors

All Ad vectors used in the study were E1, E3 vectors based on the Ad5 genome. The Adgp75 vector expresses the murine melanoma antigen gp75 under control of the cytomegalovirus (CMV) early/intermediate promoter/enhancer (12). The AdIL2 vector (a gift of Frank Graham, McMaster University, Ontario, Canada) expresses the p35 and p40 subunits of murine IL-12 under control of the CMV immediate early promoter (13). The AdIL12 vector was validated for production of a functional IL-12 heterodimer through use of a phytohemagglutinin (PHA)-blast assay and stimulation of NK activity in vitro (13). The control vector included Adgal, expressing the Escherichia coli -galactosidase (gal) gene under control of the CMV promoter (14, 15). All vectors were amplified and titered as previously described (16).

Cell Line and Animal Model

The B16 murine melanoma cell line is a weakly immunogenic tumor that expresses the gp75 protein in melanosomes within the cells and on the cell surface (17). The B16 cell line is known to express low levels of MHC I molecules on its surface in tissue culture (18). The cell line is syngeneic with the C57BL/6 mouse, and reliably produces pulmonary metastases after being intravenously injected (22). To establish metastases, we injected 10⁵ B16 cells into the lateral tail vein of C57BL/6 mice (Charles River, Wilmington, MA). The number of metastases were quantified by counting black tumor nodules on the lung surface as described by Poste and colleagues (22).

Treatment of Pulmonary Metastasis Using Adenovirus-Mediated Immune Modulation

Treatment groups included mice receiving Adgp75 (10⁸ PFU, intraperitoneally, n = 5), AdIL12 (10⁸ PFU, intraperitoneally, n = 5), or a combination of Adgp75 and AdIL12 (10⁸ PFU, intraperitoneally, for each vector, n = 5) 2 d after tumor cell challenge. Therapeutic response was compared with controls receiving Adgal (2 × 10⁸ PFU, intraperitoneally, n = 5) 2 d after tumor cell injection or mice receiving no therapy (n = 7). Lungs were removed 14 d after tumor cell delivery, and the number of tumor nodules was counted under a dissecting microscope as previously described (12).

In Vitro Detection of NK Activity after AdIL12 Delivery In Vivo

To assess mice for Ad-vector-induced NK cells, we isolated mononuclear cells from spleens of naive mice and mice receiving Adgp75 or AdIL12 as described earlier (n = 3). Spleens were minced and ground, sheared with a 19-gauge needle, and passed through a 200-µm mesh to remove fibrous tissue. Cells were pelleted and then resuspended in Dulbecco's modified Eagle's medium (GIBCO BRL, Grand Island, NY), 10% fetal calf serum (GIBCO BRL), and penicillin 100 µg/ml and streptomycin 100 U/ml (GIBCO BRL) (5-8 × 10⁷ cells/spleen). Live lymphocytes were separated from dead cells and red blood cells by using Ficoll-Paque (Pharmacia, Piscataway, NJ) density separation, and were washed and then resuspended in complete medium. Splenocytes were derived from mice 7 d after intraperitoneal administration of AdIL12 (10⁸ PFU), and were evaluated for cytolysis of YAC-1 murine hybridoma cells (American Type Culture Collection [ATCC], Rockville, MD), which are a specific target for NK cells (5, 23), with H2-matched murine fibroblasts (C57SVH2^b) serving as a negative control, and with B16 cells from in vitro culture, using a lactate dehydrogenase (LDH) release cytotoxicity assay (Promega, Madison, WI). Splenocytes were harvested as described earlier, and were incubated with 10⁴ YAC-1 cells in increasing effector-to-target cell ratios (0:1, 10:1, 50:1, 100:1) for 6 h at 37°C. LDH was assayed in the supernatant by optical density (OD) measurement at 490 nm (24). Target cell lysis was calculated as: (OD of sample  OD with spontaneous release of LDH from target cells  OD with spontaneous release of LDH from effector cells) × 100/(OD with maximal release of LDH from target cell  OD with spontaneous release of LDH from target cell) (24).

Adoptive Therapy

Splenic mononuclear cells from mice receiving Adgp75 (10⁸ PFU, intraperitoneally, n = 5), Ad-null mice (10⁸ PFU, intraperitoneally, n = 5), and naive mice were isolated 10 d after intraperitoneal delivery of Ad vectors (25). Spleens harvested from each group were minced and ground, sheared with a 19-gauge needle, and passed through a 200-µm mesh to remove fibrous tissue. Live lymphocytes were separated from dead cells and red blood cells by using Ficoll-Paque density separation, and were washed and then resuspended in Hanks' balanced salt solution (GIBCO BRL). Mononuclear spleen cells from mice receiving Adgp75 (n = 3), Adgp75 + AdIL12 (n = 3), or naive mice (n = 3) were pooled in tissue culture, and nonadherent splenocytes were collected at 3 h for adoptive transfer. Before adoptive transfer, a portion of splenocytes from mice receiving Adgp75 + AdIL12 were depleted of NK cells in vitro (n = 3) through use of a monoclonal anti-NK-cell antibody (5E6; Pharmingen, Piscataway, NJ) and rabbit compliment (Cedarlane Hornby, Ontario, Canada) (23). NK cell depletion was confirmed by trypan blue staining of dead cells with an in vitro cytotoxicity assay showing abrogation of cytolysis of NK-cell- specific YAC-1 target cells. At 1 d before tumor cell challenge, splenocytes (10⁷) were injected into the tail vein in a complementary treatment group of three mice, and tumor burden was assessed 14 d after tumor cell delivery. All groups were compared with control mice receiving no therapy (n = 5).

Statistical Analysis

All data are presented as mean ± SEM, and all comparisons were made with the unpaired, two-tailed Student's t test.

	Results

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Therapeutic Effect of Ad Vectors against Pulmonary Metastases

To evaluate their therapeutic effect, Adgp75, AdIL12, and Adgp75 + AdIL12 were given to mice at 2 d after delivery of B16 tumor cells. Control groups receiving intraperitoneal Adgal (2 × 10⁸ PFU) had similar numbers of metastases as those of naive animals (P < 0.1, all comparisons) (Figure 1). In contrast, intraperitoneal administration of Adgp75 (10⁸ PFU) 2 d after intravenous delivery of 10⁵ B16 melanoma cells was able to reduce tumor burden to 45 ± 7% of controls; P < 0.01). AdIL12 (10⁸ PFU, intraperitoneally) reduced the number of metastases to 43 ± 7% of controls (P < 0.01 versus control; P > 0.1 versus Adgp75). The combination of Adgp75 (10⁸ PFU, intraperitoneally) and AdIL12 (10⁸ PFU, intraperitoneally) provided further significant protection (15 ± 3%; P < 0.01 compared with naive control; P < 0.01 compared with Adgp75 and AdIL12 alone).

View larger version (13K): [in this window] [in a new window]   Figure 1.   Therapeutic effect of Adgp75, AdIL12, and Adgp75 + AdIL12 on preexisting pulmonary metastases of B16 melanoma cells. Therapeutic groups were compared with a group treated with Adgal and with naive controls. The vectors were administered 2 d after intravenous administration of the tumor cells. The lungs were evaluated for tumor burden 14 d after establishment of metastases. The data, representing n = 5 animals per group, are expressed as the mean ± SE of the number of tumor nodules in the various treatment groups.

MHC I-Independent Lysis by NK Cells Stimulated In Vivo with AdIL12

NK cells lyse targets in an MHC I-independent fashion, with MHC I molecules being an inhibitory signal for NK-dependent lysis (3, 21). Since delivery of IL-12 augments both NK and CTL activity, we evaluated the NK activity against B16 cells, YAC-1 cells (specific target for NK cells), and C57SV-H2^b cells (normally expressing MHC I, and used as a negative control), of splenocytes from mice receiving AdIL12 in vitro. The data showed that pooled splenocytes from mice receiving AdIL12 were capable of lysing B16 cells in a dose-dependent fashion, resembling, although to a lesser extent, YAC-1 cell lysis (Figures 2A and 2B). In contrast, there was insignificant lysis of C57SVH2^b cells (Figure 2C). The data suggested that NK activity can be induced by in vivo delivery of AdIL12 but not of Adgp75, and that B16 cells are sensitive to NK-dependent lysis in vitro.

View larger version (14K): [in this window] [in a new window]   Figure 2.   Assessment of Ad-vector-treated mice for cytotoxic cells directed against YAC-1, B16 cells, and an H2 matched fibroblast cell line (C57SVH2b). Splenocytes were derived from mice receiving AdIL12, others receiving Adgp75, and naive controls. Mononuclear splenocytes from three mice in each group were harvested, pooled, and incubated with each target at increasing effector-to-target cell ratios. The data are expressed as percent lysis of the target cells. (A) YAC-1. (B) B16 cells. (C ) C57SVH2b cells, used as a negative control.

Adoptive Transfer

To explore the role of cellular effectors in the therapeutic effect seen with Ad-vector-based immunotherapy, we treated groups of naive mice with Adgp75, AdIL12, and a combination of Adgp75 and AdIL12. After 10 d, splenocytes from these animals were harvested and plated to remove macrophages. To assess the contribution of NK cells to antitumor immunity, we divided the splenocytes from mice treated with Adgp75 + AdIL12, with a subgroup being depleted of NK cells through use of an NK-cell specific antibody and complement in vitro. The depletion of NK cells was confirmed by the abrogation of YAC-1 target cell lysis in an in vitro cytotoxicity assay. Each group of macrophage-deficient splenocytes was adoptively transferred to naive mice (10⁷ cells/mouse) 1 d before B16 tumor cell challenge, and reduction in these animals' tumor burden was compared with that of naive control animals. The data showed that adoptive transfer of control cells (pooled splenocytes from naive mice) offered no therapeutic advantage (P > 0.1 compared with naive mice injected with B16 cells and given no therapy). In contrast, recipients of cells from mice receiving Adgp75 showed 44 ± 6% of control metastases, and recipients of cells from mice receiving AdIL12 showed 67 ± 2% of control metastases (P < 0.01, each compared with control). Transfer of splenocytes from mice receiving AdIL12 and Adgp75 was associated with a reduction in the number of metastases to 26 ± 3% of the control value. With depletion of NK cells, tumor burden was significantly reduced (54 ± 3% of naive control; P < 0.01 for Adgp75 + AdIL12 versus naive control), suggesting a role for NK cells as well as T cells in the antitumor effect seen with the combination of the two vectors (Figure 3).

View larger version (16K): [in this window] [in a new window]   Figure 3.   Evaluation of the effect of adoptive transfer of splenocytes from Ad-vector-treated mice to naive mice prior to B16 tumor cell challenge. Mice received the Adgp75 vector and/or AdIL12 vector. Purified splenocytes harvested at Day 10 from each group were given (107 PFU, intravenously) to naive mice, followed after 24 h by intravenous (105 × cells) challenge with B16 melanoma cells. Splenocytes from a group of animals that had previously received Adgp75 + AdIL12 were treated with anti-CD56 monoclonal antibody + rabbit complement, to deplete NK cells. Confirmation of NK depletion was assessed by abrogation of cytotoxicity against NK cell-specific target YAC-1 cells. The NK-depleted splenocytes (compared with controls) were then given to the naive mice 24 h before tumor challenge. Tumor burden was measured as reduction in pulmonary metastasis compared with control at Day 14. The data represent mean ± SE for n = 3 mice (AT = adoptive transfer).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Previous studies in our laboratory have shown that delivery of Adgp75 to C57BL/6 mice can protect them from tumor cell challenge with B16 cells expressing gp75 (12). The current study explored the potential of this approach in a model of preexisting pulmonary metastases, and further explored the therapeutic effects of Ad-mediated expression of murine IL-12 cDNA both alone and in combination with Adgp75. The data showed that Adgp75 and AdIL12 were similarly effective at reducing the tumor burden in mice with preestablished tumor. The combination of Adgp75 and AdIL12 provided further significant therapeutic benefit in comparison both with controls and with either therapy alone. Importantly, adoptive transfer studies suggest that the effects of combined therapy depend on both T-cell-mediated immunity and NK-cell- mediated cytotoxicity.

gp75 Antigen in Immunotherapy

The human and murine gp75 antigen (also referred to as the tyrosinase related protein-1; TRP-1) are highly homologous, 75-kD glycoproteins encoded in the murine brown locus (26). Both human and murine gp75 are abundant in pigmented melanocytes and nevi, primary melanoma, and metastatic melanoma, and are not expressed in other tissues (29). The potential of the gp75 antigen for immunotherapy is suggested by the finding that a modified form of the murine gp75 protein, as well as the human gp75 protein (80.2% homologous to the murine gp75 protein), are effective at inducing protective immunity against B16 pulmonary metastases (30). Interestingly, the unmodified wild- type murine gp75 protein does not induce protective immunity against challenge with B16 melanoma cells, whereas Ad-mediated delivery of gp75 cDNA in vivo does protect against B16 melanoma tumor cell challenge (30). This therapeutic effect appears to depend on T-cell-mediated immunity (12), and may be related to the high efficiency of gene transfer afforded by Ad vectors in vivo, to sustain expression of the gp75 transgene, and to the host cellular immune responses known to be directed against Ad vector transgenes (25, 31). The data in the present study show that this approach is also effective in treating established metastases, although the antitumor effects are less dramatic than with Adgp75 administration before tumor cell challenge.

Activation of NK Cells with Ad-Mediated Expression of IL-12

Although the therapeutic responses of preexisting melanoma tumors to Adgp75 are significant, they are incomplete. Immune responses induced by Adgp75 may be too weak to eradicate the tumor, with escape of tumor clones manifested by continued tumor growth (19). One mechanism that may be involved in such incomplete responses is the downregulation of MHC I molecules on the surfaces of B16 cells, with a concomitant decrease in tumor antigen presentation and limitations in the ability of antigen-specific T cells to recognize tumor cells (19). The B16 murine melanoma growing in vivo may represent a heterogenous mix of high and low expressors of MHC I molecules (18, 19, 23, 38). There is experimental evidence suggesting that B16 cells, which show low levels of MHC I, are susceptible to destruction by NK cells (18, 19, 20, 39), and it is therefore logical that enhanced NK cell activity, with efficacy against cells expressing low levels of MHC I, would compliment T-cell immunity induced by Adgp75 (19).

In this context, one approach to improving the antitumor effects of Adgp75 is through Ad-mediated IL-12 expression (13, 40). IL-12 is a cytokine derived from activated macrophages and dendritic cells, which leads to activation and proliferation of NK cells (5, 45). Consistent with this concept is that fresh splenocytes pooled from naive mice receiving AdIL12 showed dose-dependent cytolytic activity against NK-cell specific YAC-1 target cells, which express lower levels of MHC I (23, 45) than cells treated with Adgp75 alone and naive controls. Furthermore, in accordance with published reports of decreased MHC I expression by B16 cells, fresh splenocytes from naive AdIL12-treated mice were able to lyse B16 targets at high effector-to-target ratios.

Augmentation of Adgp75 Immunization with AdIL12

IL-12 also has direct effects on T-cell-mediated immunity (45, 46). It can activate CD8+ lymphocytes, and in the presence of antigen presentation on cell-surface MHC II can activate Th1 cells and induce IFN- secretion (5, 9, 49, 50). IFN- has myriad effects, including potentiation of the Th1 phenotype and increasing antigen presentation via unregulated MHC II on the surfaces of antigen-presenting cells, and in some cases MHC I on the surfaces of tumor cells (10, 38). Additionally, NK cells can express IFN- (50, 51). Recombinant IL-12 has proven antitumor effects when used alone (5, 49, 52) and in combination with tumor antigen (56), but has dose-limiting toxicities (57). In vivo gene therapy limits the expression of a cytokine such as IL-12, yielding higher concentrations of cytokine at the site of gene delivery that avoid potential side effects of systematically administered cytokines (58, 59). Gene-mediated expression of cDNA for IL-12 induces efficient antitumor effects when tumor cells are transduced with this cDNA ex vivo (48, 52, 54, 55, 62), when IL-12 is expressed locally after gene transfer to established tumor cells in vivo (40), and when IL-12 is expressed at sites distant from tumor growth, such as the skin (65, 66).

The present study expands the potential of Ad-mediated IL-12 production to augment antitumor effects of an Ad-mediated, gene-based, tumor-antigen-specific vaccine. Although Adgp75 and AdIL12 show similar significant activity against established metastases, combination of the two vectors has superior antitumor activity than does either alone. Additionally, adoptive transfer of splenocytes from mice receiving Adgp75, AdIL12, or Adgp75 + AdIL12 produces relative reductions in tumor burden resembling that seen with in vivo vector administration alone. Interestingly, splenocytes from mice receiving Adgp75 ± AdIL12, from which NK cells are depleted prior to adoptive transfer, produce a partial reversal of tumor response, suggesting that NK cell activation is responsible for part of the improved efficacy seen with the combination of AdIL12 and Adgp75. From these experiments it not possible to determine the ability of AdIL12 to enhance antigen presentation or T-cell response as compared with that of Adgp75 alone, but the experiments do suggest that the combination of AdIL12 and Adgp75 provides superior therapeutic efficacy through antigen-specific immunization and antigen-nonspecific (MHC I-independent) mechanisms. This approach may therefore be rational for tumors of types similar to B16 melanoma.

	Footnotes

Address correspondence to: Edward A. Hirschowitz, M.D., Division of Pulmonary and Critical Care Medicine, The New York Hospital-Cornell Medical Center, 520 East 70th Street, ST505, New York, NY 10021.

(Received in original form May 6, 1998 and in revised form September 22, 1998).

Acknowledgments: The authors thank Hassan Naama and Carlo Russo for their helpful discussion, and N. Mohamed for help in preparation of the manuscript. These studies were supported in part by Grant R01 CA75192 from the National Institutes of Health; by the Will Rogers Memorial Fund (White Plains, NY); and by GenVec, Inc. (Rockville, MD).

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