Effective gene therapy for medullary thyroid carcinoma using recombinant adenovirus inducing tumor-specific expression of interleukin-12

Abstract

No satisfactory treatment of metastatic medullary thyroid carcinoma (MTC) is available. Cell-specific gene therapy offers a new approach. We have constructed a recombinant replication-defective adenoviral vector expressing murine interleukin-12 (mIL-12), driven by a modified CALC-I promoter (TCP). This vector (AdTCPmIL-12) includes two separate cassettes encoding mIL-12 p35 or p40 subunit controlled by TCP inserted in the E1 region of adenovirus type 5. In vitro and in vivo reporter gene expression using TCP revealed its cell-specific activity. AdTCPmIL-12-infected rat MTC (rMTC) cells produced high amounts of functional mIL-12 cells in vitro, while other cell lines infected with AdTCPmIL-12 did not. AdTCPmIL-12-transduced rMTC cells completely lost their tumorigenicity in syngenic WAG/Rij rats. Direct injection of 1 × 109 plaque forming units of AdTCPmIL-12 into subcutaneous rMTC tumors in WAG/Rij rats caused tumor regression in over 60% of animals within 20 days. Rats cured of tumors did not develop tumors after re-injection of naive rMTC cells, demonstrating lasting immunity. Treatment with AdTCPmIL-12 of one tumor resulted in regression of an established tumor at a distant site. Moreover, intratumoral or intravenous injection of AdTCPmIL-12 did not induce evident toxicity. These results indicate AdTCPmIL-12 can contribute to effective and less toxic gene therapy of MTC.

Introduction

Medullary thyroid carcinoma derived from calcitonin-secreting thyroid C cells, causes about 5–10% of all thyroid tumors. Many cases develop invasive or metastatic disease, which are responsible for all deaths related to this disease.1 Beneficial therapy is limited at present to surgery. Both chemotherapy and radiotherapy have been extensively tested, but are often ineffective. The lack of any alternative to the standard treatments indicates the need for innovative methods.2

Successful trials of genetic cytokine immunotherapy have emerged as a promising approach for treating MTC,34 perhaps related to expression of tumor-associated antigens by MTC.5 IL-12, a 70 kDa disulfide-linked heterodimeric cytokine composed of 35-kDa (p35) and 40-kDa (p40) subunits, has been shown to generate potent antitumor and antimetastatic responses in various cancer models.67 IL-12 is a key factor in stimulation of NK and NKT cells8 and initiating Th1-type directed immune responses by activation of CD4+ and CD8+ cells.9 IL-12 also has the potential to promote NO production by macrophages.10 IL-12-activated cells, such as antigen presenting cells (APCs), or T cells, produce IFN-γ. Induction of IFN-γ is crucial for antitumor activity,11 due to anti-angiogenesis mediated by production of chemokine IP-10 and monokine Mig,1213 or by regulation of VEGF and MMPs.14 Direct effects of IL-12 on IL-12 receptor β-expressing tumor cells might contribute to T cell-mediated antitumor activity.1516

Systemic administration of recombinant IL-12 has shown antitumor effects including tumor regression and suppression of metastasis. However dose-dependent and schedule-related systemic toxicity has been a problem in animal studies and human clinical trials.17 Local expression of IL-12 using genetically engineered fibroblasts, gene gun, or viruses could circumvent such systemic toxicity.1819202122

E1-region deleted adenoviral vectors are popular and attractive tools for transduction of genes and allow local, highly efficient, but transient, gene expression in both dividing target cells and quiescent cells, without integration into the host genome.23

We found that rMTC tumors were strongly suppressed by intratumoral injection of E1-deleted recombinant adenovirus inducing mIL-12, driven by cytomegalovirus (CMV) promoter (AdCMVmIL-12).24 However, intratumorally injected adenoviral vector under control of CMV promoter could leak into the systemic circulation and induce unwanted expression of transgene in non-target organs, leading to serious side-effects.425 We cannot be sure that local injection of adenovirus using a promiscuous promoter is safe.

For low toxicity in non-target tissues, tissue-specific promoters have been utilized in cancer gene therapy.26 The CALC-I gene is predominantly expressed in thyroid C cells, inducing a high secretion of calcitonin (CT). This gene also exists in neuronal cells, resulting in secretion of calcitonin gene-related peptide (CGRP) by alternative splicing of the mRNA.27 We have reported an adenoviral vector using the CALC-I promoter and a tumor-selective alternative splicing system to intensify tissue-specific transgene expression in MTC cells.28

In this article, we describe the generation of AdTCPmIL-12, an adenovirus using a modified CALC-I promoter which is strongly active only in MTC cell lines, to drive expression of high levels of mIL-12. We demonstrate that this adenovirus vector shows antitumor efficacy, including an effect on tumors at a distant site in the rMTC animal model, and that treatment of tumor-bearing rats with this virus results in acquisition of long-lasting immunity against wild-type tumor cells. Further we show that AdTCPmIL-12 induces no toxicity. This approach could contribute to effective and less toxic MTC treatment.

Results

Construction of recombinant adenovirus expressing mIL-12

Murine IL-12 p35 and p40 cDNAs in pCA13 (pCAmIL-12/p35 and pCAmIL-12/p40) have been previously described.24 TCP, containing double tissue-specific enhancer elements (TSEs) upstream of a partial CALC-I promoter and exon 1, was obtained from p(TSE)2 CP1.GL3 by digestion with KpnI and HindIII. pCATCPmp35, or mp40, was constructed by insertion of the blunt-ended TCP fragment into the blunt-ended HindIII site of pCAmp35 or pCAmp40. pΔTCPmIL-12 was subcloned into pΔE1sp1B by transfer of the piece including TCPmp35 or mp40. AdTCPmIL-12 was generated by co-transfection of this shuttle vector with E1-deleted adenovirus backbone vector pJM17 into 293 cells by the calcium phosphate precipitation method (Figure 1a). To identify the correct construct, adenoviral DNA extracted from infected 293 cells was analyzed by digestion with HindIII. A band of about 6400 bp including the inserted gene was obtained Figure 1b. A fragment of mp35 (344bp) or mp40 (357bp), derived from mRNA of AdTCPmIL-12-infected rMTC cells, could be detected by RT-PCR analysis. These fragments were observed even in cells infected at 1 MOI. The fragment of β-actin (763 bp) was also seen in all reverse-transcribed samples Figure 1c.

Figure 1
figure1

Construction, molecular analysis and transgene transcription of AdTCPmIL-12. (a) The expression cassettes for mp35 and mp40 subunits were inserted into the E1 region of the adenovirus type 5 (Ad5) genome. Both cassettes are under control of modified CALC-I promoter ‘TCP’, which contains two tandemly arranged tissue specific enhancer elements (TSE) upstream of a partial CALC-I promoter with the CALC-I gene exon 1, and both are terminated by simian virus 40 polyadenylation signal (PA). Recombinant adenovirus was obtained by homologous recombination of the shuttle plasmid pΔTCPmIL-12 with adenovirus backbone vector pJM17 in 293 cells. (b) Rearrangement of HindIII restriction fragments confirmed the correct insertion on the transgene into the Ad5 E1 region. A 6400-bp inserted fragment was detected in AdTCPmIL-12 as compared with pJM17. (c) Expression of mp35 and mp40 in AdTCPmIL-12-infected rMTC cells. 1 × 106 rMTC cells were infected with AdTCPmIL-12 at the indicated MOIs. Total RNA was extracted 48 h later. 1 μg of total RNA was reverse-transcribed, followed by DNaseI treatment, and amplified for 30 cycles. PCR products were visualized in a 0.5-μg/ml ethidium-bromide 2% agarose gel. Expected band size is 344 bp for mp35, 356 bp for mp40 and 763 bp for β-actin. P, positive control for PCR using 5 ng of plasmid pΔTCPmIL-12; N, negative control for PCR without DNA.

Selective expression of luciferase in cell lines

To assess whether TCP could express a transgene in a cell-specific manner, we constructed recombinant adenoviruses containing the luciferase gene controlled by TCP (AdTCPluc), the original CALC-I promoter (AdCTluc), or the CMV promoter (AdCMVluc). Virus was used to infect rMTC, TT, Cos1, HeLa, C6, HepG2 and T98G cells at 100 MOI (Figure 2). AdCMVluc was strongly active in all cell lines as shown by high luciferase expression. AdTCPluc exhibited high activity in rMTC cells, equal to the activity of AdCTluc. AdTCPluc activity was 81.0% of AdCMVluc activity. In TT cells the percent activity of AdTCPluc related to AdCMVluc was 145.4% and that of AdCTluc was 4.7%. In HeLa cells AdTCPluc maintained cell specificity in comparison to AdCTluc. In C6 cells, known to be a CGRP-producing neuronal cell line, the activity of AdTCPluc was 28.3% of AdCMVluc. Weak activity of both of CALC-I promoter-driven viruses was detected in Cos1, HepG2 and T98G cells. These results show that TCP can induce cell-specific expression of a transgene in MTC cell lines.

Figure 2
figure2

Luciferase activity in transduced cell lines. (a) Two MTC cell lines and five non-MTC cell lines were infected with AdCTluc, AdTCPluc or AdCMVluc at 100 MOI. After incubation for 48 h with complete medium, the luciferase activity was assayed. The experiments were performed in triplicate. (b) The percent activity of AdCTluc or AdTCPluc was calculated in relation to AdCMVluc. Data are presented as mean ± s.d.

In vivo transfer of reporter gene driven by modified CALC-I promoter

Since the CALC-I promoter is active in neuronal cells, the transgene could be expressed in the brain. We were thus interested in evaluation of the activity of TCP in target and non-target organs. AdTCPluc or AdCMVluc were injected into tumor-bearing rats by the intratumoral or intraperitoneal route, and 4 days later the expression of the reporter gene was measured (Figure 3). After intratumoral injection, the transgene expression induced by TCP in tumors was 28.1% of that by CMV promoter, and expression was not detected in other organs, including the liver or brain. Intraperitoneal injection of the virus did not induce transduction in the rMTC tumors. However AdCMVluc injection resulted in overt expression in the liver after both intratumoral and intraperitoneal routes. These results indicate that the modified CALC-I promoter is functional in rMTC tumors, but has little activity in non-target organs after intratumoral or intraperitoneal injection.

Figure 3
figure3

Tissue-specific activity of TCP promotor in vivo. 5 × 109 p.f.u. of AdTCPluc or AdCMVluc were injected intratumorally (i.t.) or intraperitoneally (i.p.) into rMTC tumor-bearing rats. Four days after virus injection animals were killed to obtain the organ samples and luciferase activity was determined. Data are presented as mean ± s.d. of four animals per group.

Highly cell-specific expression of AdTCPmIL-12 in rMTC cells

To confirm the functional secretion of mIL-12, ConA-blast proliferation assay was performed. Supernatant was harvested from cells infected with AdTCPmIL-12, AdCMVmIL-12 or AdTCPluc. Cytotoxicity due to virus itself was not observed at the indicated MOIs (data not shown). ConA-blasts responded to the supernatant of cells infected with AdTCPmIL-12, depending on the dose of infected virus. The stimulatory activity was completely neutralized with anti-IL-12 antibody (5 μg/ml) (data not shown). Peak expression was reached at 148 757 units/106 cells/24 h between 48 h and 72 h. C6 produced mIL-12 after infection with AdTCPmIL-12, but the highest amount was at the most 24 575 units/106 cells/24 h from 48 h to 72 h (Figure 4a). To evaluate cell-specific expression of mIL-12, rMTC, Cos1, HeLa, C6 and T98G cells were infected with AdTCPluc, AdCMLmIL-12 and AdTCPmIL-12. Supernatants were collected 48 h after infection. The production of IL-12 derived from AdTCPmIL-12 was 3.8 times higher than that from AdCMVmIL-12 at 100 MOI in rMTC cells. In other cell lines, a very low level of expression of IL-12 was seen from AdTCPmIL-12 as compared with AdCMVmIL-12 Figure 4b. These results indicate AdTCPmIL-12 has the ability to produce high amounts of functional mIL-12 in a rMTC cell-specific fashion.

Figure 4
figure4

Production of mIL-12 in infected cell lines. 4 × 105 cells were infected with AdTCPmIL-12 for 1 h. Infected cells were incubated in 3 ml of complete medium, followed by three times washing with serum-free medium. Supernatants were harvested at indicated time-points. ConA-activated splenocyte proliferation assay was performed in triplicate and IL-12 bioactivity in the supernatants was determined from a standard curve obtained using recombinant mIL-12. Supernatant from AdTCPluc infected cells served as negative control. (a) Dynamic production of mIL-12 in rMTC cells or C6 cells infected at 100 MOI. (b) Total amount of functional mIL-12 in cell lines infected at 20 or 100 MOI for 48 h. Data are presented as mean ± s.d.

AdTCPmIL-12 completely suppresses tumorigenicity of rMTC cells

To evaluate tumorigenicity, a mixture of virus-infected and naive cells was inoculated subcutaneously into rats. No rat that received AdTCPmIL-12-infected cells generated a tumor despite co-injection of naive cells. However, one of six rats treated with AdCMVmIL-12 developed a tumor. All control rats generated tumors within 6 days after injection of cells and the tumors grew progressively (Figure 5). These results show that AdTCPmIL-12 can completely suppress the tumorigenicity of rMTC cells.

Figure 5
figure5

Tumorigenecity of infected rMTC cells in syngenic rats. After Rat MTC cells were infected with mock (), AdTCPluc (•), AdCMVmIL-12 (□) or AdTCPmIL-12 (▪) at 20 MOI for 1 h in vitro, 1.5 × 106 infected cells mixed with an equal number of wild-type cells were injected subcutaneously into WAG/Rij rats. Animals were observed every 2 days for development of tumors. The evolution of tumor size is shown, and the fraction of rats without tumor development within 24 days is indicated. Error bars show s.d. Differences between the AdTCPmIL-12-infected group and mock or AdTCPluc-infected group were statistically significant (*, P < 0.001). There is no significant difference between AdTCPmIL-12 infected and AdCMVmIL-12 infected group.

In vivo AdTCPmIL-12-transduced rMTC cells produce high amounts of mIL-12

We wanted to make sure of the level of mIL-12 produced in rMTC cells infected with viruses in vivo. Two days after injection of virus into pre-established tumors, infected cells were obtained from the tumor and incubated for 24 h in vitro. We measured the level of mIL-12 in the incubation fluid by ConA-blast proliferation assay. During culture of in vivo AdTCPmIL-12-infected cells, they produced 65 977 units/106 cells of mIL-12 per 24 h (Figure 6).

Figure 6
figure6

IL-12 production in rMTC cells infected in vivo. 1 × 109 p.f.u. of virus was injected into rMTC tumors in WAG/Rij rats. Two days later the tumor was excised and 1.5 × 107 cells were incubated in 15ml complete medium for 24 h in vitro. ConA-blast proliferation assay was performed using the harvested supernatants.

AdTCPmIL-12 treatment reduced tumor growth and initiated long-lasting immunity in vivo

The efficacy of cancer gene therapy by mIL-12 driven by TCP was tested by direct intratumoral injection of AdTCPmIL-12. AdTCPmIL-12 induced the same efficient antitumor effect on rMTC tumor, as did AdCMVmIL-12. Five of seven rats showed complete tumor regression 20 days after tumor treatment with AdTCPmIL-12, and two tumors were reduced in size by 75% compared with control. Tumors of mock or AdTCPluc-treated rats grew progressively (Figure 7).

Figure 7
figure7

Antitumor effect in vivo. Tumor bearing rats were treated intratumorally with 1 × 109 p.f.u. of mock (), AdTCPluc (•), AdCMVmIL-12 (□) or AdTCPmIL-12 (▪). Data are expressed as the mean tumor size of eight rats per group. Error bar shows s.d. Mean tumor size of AdTCPmIL-12-infected group and mock or AdTCPluc-infected group were statistically different (*, P < 0.001). There is no difference between AdTCPmIL-12-infected and AdCMVmIL-12-infected groups.

Twenty and 60 days after initial injection of virus, rats which had experienced tumor regression were challenged with wild-type rMTC cells. All rats cured by AdTCPmIL-12 and AdCMVmIL-12 injection obtained long-lasting immunity and no tumor developed after challenge (Table 1). These results indicate AdTCPmIL-12 can induce an efficient antitumor effect on rMTC tumors and produce lasting immunity against rMTC cells.

Table1 Re-challenge with wild-type rMTC cells

AdTCPmIL-12 treatment repressed the growth of distant tumor

To access the antitumor effect of AdTCPmIL-12 on a distant tumor, one of two tumors was treated with virus and tumor growth was monitored. Tumor at the untreated site regressed after continuous growth to day 6. Volume reduction of the tumor at the site treated with AdTCPmIL-12 was seen within 1 week after infection of virus (Figure 8). This result indicates that AdTCPmIL-12 has the ability to suppress not only the growth of directly treated tumor, but also growth of a distant tumor.

Figure 8
figure8

Regression of a distant rMTC tumor following adenovirus-mediated IL-12 gene therapy. WAG/Rij rats bearing tumors on each flank, were injected with 1 × 109 p.f.u. of mock (), AdTCPluc (•), AdCMVmIL-12 (□) or AdTCPmIL-12 (▪) into a tumor on the left flank. A tumor on right flank was left untreated. Data are expressed as the mean tumor size of six rats per group. The mean tumor sizes of the AdTCPmIL-12-infected group and mock or AdTCPluc-infected group was significantly different (*P < 0.001). There is no difference between AdTCPmIL-12-infected and AdCMVmIL-12-infected groups.

AdTCPmIL-12 does not cause toxicity

For estimation of the toxicity due to AdTCPmIL-12, serum mIL-12 levels, the amount of liver transaminases in blood and the weight of spleens, were monitored after intratumoral or intravenous (tail vein) injection of 1 × 109 p.f.u. of virus.

Two days after intratumoral injection of AdCMVmIL-12, serum mIL-12 was elevated, ranging from 524 pg/ml to 6199 pg/ml (mean 2458 pg/ml). Intratumoral injection of AdTCPmIL-12 also resulted in production of IL-12, with levels ranging from 53 pg/ml to 2046 pg/ml (mean 691 pg/ml). However, serum IL-12 was undetectable in the group treated with AdTCPmIL-12 intravenously, in contrast to production of IL-12 after intravenous administration of AdCMVmIL-12 (mean 3936.5 pg/ml) (Figure 9a).

Figure 9
figure9

Toxicity studies. 1 × 109 p.f.u. of virus was injected into a rat intratumorally or intravenously. (a) serum mIL-12 level 2 days after infection; (b) liver transaminase levels 7 days after infection; (c) weight of spleen 2 or 7 days after infection. Data are presented as mean ± s.d. of four animals per group.

Intravenous injection of AdCMVmIL-12 resulted in a moderate increase of GOT and GPT, whereas AdCMVmIL-12 injected intratumorally did not. AdTCPmIL-12 given by either route did not have any effect Figure 9b.

An overt increase of spleen size was observed in all animals treated with AdCMVmIL-12 7 days after infection. Animals treated withAdTCPmIL-12 did not show the elevation of spleen weight after either route of injection Figure 9c.

Histologically, an obvious periportal-sinusoidal lymphocytic accumulation was observed in livers of rats treated with AdCMVmIL-12 intravenously, while AdCMVmIL-12 injected intratumorally or AdTCPmIL-12 given by either route did not cause this effect. Enlarged red pulp with increased phagocytes and megakaryocytes was observed in spleens of animals given AdCMVmIL-12 intratumorally or intravenously, indicating extramedullary hematopoiesis. In animals given AdTCPmIL-12 intratumorally, we found similar, but less intense changes in the spleen. There were few histological changes in the group given AdTCPmIL-12 intravenously. Accumulation of plasma cells was found in pulmonary arteries of rats treated with intravenous AdCMVmIL-12, but not in those of the other groups. Histological abnormalities were not recognized in the kidney or thyroid in any group (data not shown).

These results indicate that AdTCPmIL-12 given by intratumoral or intravenous injection does not cause evident toxicity in comparison to AdCMVmIL-12.

Discussion

IL-12 is a key cytokine in T cell-mediated antitumor immunity. Adenovirus-mediated local expression of IL-12 driven by CMV promoter is a promising approach for treatment of cancers. However, virus may disseminate to non-target organs via the systemic circulation, leading to undesired toxicity.

Our goal is the establishment of effective and non-toxic gene therapy against MTC. To develop a strategy utilizing the advantages of adenoviral vector and antitumor efficacy of IL-12, we generated an adenovirus with a tissue-specific promoter inducing mIL-12 expression. We used a modified CALC-I promoter, TCP. This promoter was constructed from two tandemly arranged tissue-specific enhancer elements (TSEs), and a minimal proximal CALC-I promoter, with exon 1 of the CALC-I gene. The TSE is a DNA sequence located approximately 1 kb upstream from the transcription start site of CALC-I gene, and includes an Ets-like response element and a CANNTG E-box motif (E2) which are related to cell-specific transcription of CALC-I gene. Peleg et al reported that two copies of E2, inserted in front of a minimal CALC-I promoter, increased the promoter activity in the CT-positive MTC cell line TT. Furthermore, it was revealed that this cell-type specific component of the enhancer interacts with the ubiquitous HLH and the thyroid C cell-specific OB2 transcription factors.3031 Messina et al showed that the TCP promoter induced 14 times greater luciferase expression than did the full-length natural promoter in TT cells, and had little activity in non-MTC cells by transfection assay. In our in vitro study using an adenovirus with the TCP promoter expressing luciferase, we also detected high luciferase expression in both MTC cell lines. However, there is a difference in the ratio of expression of AdTCPluc to AdCTluc in the two cell lines. Although a low ratio in rMTC cells may mean that the TSE is unnecessary for enhancement of gene expression, we believe that the TSE is valuable for increasing cell specificity in rMTC cells. The activity of TCP was decreased as compared with that of the whole CALC-I promoter in HeLa and C6 cell line.

The TCP promoter had little activity in non-MTC cells in vitro, except for the neuronal cell line C6, which produces endogenous CGRP. We examined in vivo transfer of high doses (5 × 109 p.f.u.) of AdTCPluc by intratumoral or intraperitoneal injection of virus to rule out expression of the transgene in the central nervous system. As compared with CMV promoter, the modified promoter TCP had no activity in non-target tissues, although virus-injected intratumorally is presumably disseminated to tissues such as liver. No activity of either promoter was seen in the brain. Possibly virus was not disseminated to brain. Most organs carry both the coxsackie adenovirus receptor (CAR) and alphav-integrin, necessary for adenoviral transfer into tissues, but expression of the receptors is insufficient for vector transfer in vivo because of the anatomical barrier.33 Huard et al found the central nervous system could not be transduced by any route of administration of high doses (more than 5 × 109 p.f.u.) of CMV promoter-driven luciferase-encoding adenovirus, presumably because of the blood–brain barrier.

We generated a recombinant adenovirus containing the cDNAs for the mIL-12 p35 and p40 subunits in the E1-deleted virus, each under control of TCP. The functional production of biologically active mIL-12 by adenovirus-infected rMTC cells was assayed using ConA-activated mouse splenocytes. AdTCPmIL-12 induced expression of high amounts of biologically active mIL-12 in a rMTC-specific manner, as compared with AdCMVmIL-12. Simultaneous expression of both subunits is crucial for obtaining biological activity, while dimerization of mp40 blocks IL-12 function.35 Adenovirus carrying mp40 cDNA alone had no in vivo antitumor activity in a bladder cancer model because of this dimerization.38 Several groups have constructed an adenoviral vector containing both subunits linked by internal ribosome entry sites (IRES) to prevent overproduction of mp40. Bramson et al constructed a CMV promoter-driven adenoviral vector carrying the mp35 subunit cDNA in the E1 region of adenovirus type 5 and mp40 subunit cDNA in the E3 region. They detected dimeric IL-12 and some trimer (mp35+mp35+mp40 or mp40+mp40+mp35) by immunoprecipitation analysis. Thus a one promoter–one expression cassette system is also useful to produce IL-12 bioactivity. We found that AdTCPmIL-12 was able to induce high functional expression of mIL-12 in specific target cells, overcoming a possible suppressive effect of mp40 dimers. Production of bioassayable mIL-12 by AdTCPmIL-12 was superior to that by AdCMVmIL-12 in vitro infected rMTC cells, in contrast to the equal promoter activity of AdTCPluc and AdCMVluc. The exact reason for this difference is uncertain, but may be related to mRNA processing. Transcripts of AdTCPmIL-12 contain mRNA from exon 1 of the CALC-I gene, while that of AdCMVmIl-12 does not. This difference may affect post-transcriptional regulation of IL-12 biosynthesis in rMTC cells. In C6 cells a low level of mIL-12 expression was seen, but the lack of viral dissemination to the CNS may render this phenomenon unimportant.

In our tumorigenicity study, in vitro infection by AdTCPmIL-12 prevented tumor formation by infected rMTC cells mixed with half wild-type cells. In vivo tumor treatment with AdTCPmIL-12 and AdCMVmIL-12 demonstrated suppression of tumor growth beginning 2 days after infection, and over 60% of rats treated with AdTCPmIL-12 had tumor regression within 20 days, accompanied by high secretion of mIL-12. Rats cured by AdTCPmIL-12 treatment obtained lasting immunity against rMTC cells. Furthermore AdTCPmIL-12 had antitumor efficacy on a treated tumor and an untreated distant tumor, reflecting the inhibitory effect of IL-12 on metastatic tumors.6

Antitumor effects of IL-12 by adenoviral transfer were reported in a variety of cancer models.37383940414243 In a colon carcinoma model, 76% of mice infected intratumorally with AdCMVIL-12 showed complete regression within 10 days, accompanied with local secretion of IL-12 and IFN-γ. Antitumor effects on tumors at a distant site and acquired immunity were confirmed.41 In a poorly immunogenic prostate cancer model, direct intratumoral injection of adenovirus expressing mIL-12, in a dose of 5 × 107 to 3 × 108 p.f.u., brought significant growth suppression and increase survival. Furthermore pre-established lung metastases were suppressed efficiently by 1 × 108 p.f.u. of virus.42 In a model of hepatocellular carcinoma (HCC), 50% of rats with a single tumor implanted in the liver showed complete tumor regression after intratumoral injection of 5 × 109 p.f.u. AdCMVIL-12, accompanied by transient elevation of serum IL-12 level and continuous high levels of serum IFN-γ. In rats with two independent tumor nodules in the liver, injection in one tumor with AdCMVIL-12 induced complete regression of treated tumors in half of infected rats and also of untreated tumors in half of the animals.43

Our observed antitumor efficacy is consistent with these recent reports. However, in most previous reports recombinant adenovirus driven by the CMV promoter was used. It was recently reported that adenovirus-mediated heat-inducible IL-12 expression, driven by a hsp70 promoter, targeted gene expression to desired tissues.44 However, as far as we know, this is the first report that adenoviral transfer of IL-12-encoded gene under control of tissue-specific promoter is effective in targeted cancer gene therapy.

High doses of IL-12 can induce toxicity1745 and immune suppression related to IL-10 induction.46 We investigated serum IL-12 levels 2 days after viral infection. Intratumoral injection of AdTCPmIL-12 resulted in an elevation of various levels of serum IL-12. IL-12 binds with high affinity to heparin/heparan sulfate glycosaminoglycans on the cell surface and in the extracellular matrix. This binding is likely to retain IL-12 close to its tissue sites of secretion.47 This may indicate that high level of IL-12 in blood is not always required for antitumor activity. When our results of tumor treatment studies are considered, we find elevation of IL-12 suitable for inducing antitumor activity, but without toxicity. Intravenous injection of AdTCPmIL-12 did not change the amount of serum IL-12, in striking contrast to the obvious elevation of IL-12 after injection of AdCMVmIL-12. By PCR or RT-PCR analysis, we could not detect viral DNA or RNA from the transgene in tumors of rats treated with either virus intravenously (data not shown). These findings suggest that AdCMVmIL-12 induces the extratumoral expression of mIL-12, whereas AdTCPmIL-12 does not. Therefore AdTCPmIL-12 must be more safe than AdCMVmIL-12. We believe that the slightly greater mean amounts of IL-12 secretion induced by intratumorally injected AdCMVmIL-12, than by intratumorally injected AdTCPmIL-12, may reflect this extratumoral expression.

Some of the major toxicities of IL-12 are hepatosplenomegaly and liver inflammation, mainly due to induction of IFN-γ4849 and inducible expression of adhesion molecules.50 We thus measured the level of liver transaminases in blood, and spleen weight. Intratumoral or intravenous injection of AdTCPmIL-12 did not result in elevation of liver transaminases or an increase of spleen weight, in contrast to AdCMVmIL-12. The histological findings are consistent with these data. Plasma cell accumulation was found only in pulmonary arteries of rats treated with AdCMVmIL-12 intravenously. Although it was reported that recombinant IL-12 could give rise to pulmonary edema with pleomorphic septal infiltrates in a study using the squirrel monkey,51 we are not sure that our finding is related to the biological effect of secreted IL-12.

We did not see evidence of abnormal brain function. We could not detect viral DNA in brain by PCR analysis in any treated group (data not shown), or see abnormal behavior (such as continuous spinning), suggestive of neuronal dysfunction.

In our model AdTCPmIL-12 did not show evidence of systemic toxicity. However, we need to remember that genetic or epigenetic variability can cause idiosyncratic adverse effects in response to IL-12 gene therapy.52

In conclusion, a recombinant adenovirus inducing IL-12 driven by tissue-specific promoter, produced large amounts of functional mIL-12 in an MTC-specific manner. This vector showed efficient in vivo antitumor effects on MTC tumors, including an effect on tumors at a distant site, without toxicity. Treatment of tumor bearing rat with this virus resulted in acquisition of long-lasting immunity against wild-type tumor cells. This approach can contribute to effective and less toxic cancer genetic immunotherapy of MTC and possibly provide a much needed therapy for this illness.

Materials and methods

Cell culture

The rat MTC cell line 6-23 was maintained in DMEM supplemented with 15% horse serum. The human MTC cell line TT was grown in RPMI1640 containing 15% fetal bovine serum (FBS). Cos1 cells (monkey kidney cell line), HeLa cells (human uterine cervical carcinoma cell line), HepG2 cells (human hepatocellular carcinoma cell line) and C6 cells (rat glioma cell line) were maintained in DMEM with 10% FBS. T98G cells (human glioblastoma cell line) were grown in MEM with 10% FBS, 100 mM sodium pyruvate and 100 mM nonessential amino acids. The human embryo kidney 293 cell line was maintained in MEM with 10% calf serum. All culture media were supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. All cell lines were maintained at 37°C in a humidified atmosphere at 5% CO2. All cell culture reagents were purchased from Mediatech Inc (Herndon, VA, USA) or GIBCO BRL (Gaithersburg, MD, USA).

WAG/Rij rats were bred at the Carlson Biocontainment Suit under standard conditions based on the Guidelines of the Animal Resource Center.

Recombinant adenoviruses

The plasmids pCA13 and pΔE1sp1B were purchased from Microbix Biosystems Inc (Ontario Canada, M8Z3A8). p(TSE)2CP1.GL3 is a plasmid containing TCP, which consists of two tandemly arranged tissue-specific enhancer elements (TSEs) upstream of a PCR product encompassing nucleotides −128–+104 of the human CALC-I promoter. This plasmid was the kind gift of Dr Bruce G Robinson (University of Sydney). The adenovirus backbone vector pJM17 was kindly provided by Dr S Refetoff (The University of Chicago).

The detailed method for construction of AdTCPmIL-12 in described in Results.

For the shuttle plasmid pΔTCPluc, a cassette containing luciferase gene inserted downstream of TCP was obtained from p(TSE)2CP1.GL3 by digesting with KpnI and AccI, and then subcloned into the EcoRV site of pΔE1sp1B after blunt-ending with T4 DNA polymerase.

To construct the shuttle plasmid pΔCTluc, TCP in pΔTCPluc was replaced by the construct of the whole human CALC-I promoter including CALC-I gene exon 1. This promoter was obtained by digestion of the plasmid pΔDCTkozak28 using HindIII and BssHII After blunt-ending of the promoter and digesting plasmid pΔTCPluc with KpnI and HindIII, the promoter was inserted into this plasmid.

For the shuttle plasmid CMVluc, luciferase gene was obtained from p(TSE)2CP1.GL3 by digesting with HindIII and XbaI, and ligated into the same sites of pCA13.

AdCMVmIL-12 was constructed as previously described.24

Recombinant adenovirus was generated by co-transfection of shuttle vector with pJM17 into 293 cells using the calcium phosphate precipitation technique. Incorporation of the expression cassette into the isolated recombinant adenovirus was confirmed by digestion with restriction enzymes. Recombinant adenovirus was rescued and propagated in 293 cells. Virus was purified by double cesium chloride gradient ultracentrifugation. Viral titer was determined by plaque assay using 293 cells. Purified virus was stored in 10% glycerol at −80°C for further use.

Generation of rMTC tumor

Rat MTC cells were washed and resuspended in serum-free DMEM at a density of 1.5 × 107 /ml. 100 μl of cell suspension was injected subcutaneously into 4- to 6-week-old WAG/Rij rats. Palpable tumors developed within 7 to 10 days.

Detection of mp35 and mp40 mRNA in virus-infected rMTC cells

rMTC cells (1 × 106) were infected with AdTCPmIL-12 at a multiplicity of infection (MOI) of 0 to 100 for 1 h and then incubated 48 h in six-well plates. Total RNA was isolated from the cells using TRIZOL (GIBCO BRL). 1 μg of total RNA was used for cDNA synthesis followed by treatment with DNaseI (GIBCO BRL). cDNA synthesis was performed by SuperScript First-Strand Synthesis System for RT-PCR (GIBCO BRL). 0.5 to 1 μl of the cDNA mixture was subjected to PCR using specific oligonucleotide primers for mp35,53 mp40,54 and β-actin as a positive control.55 PCR amplification was performed by using 30 cycles comprising 60 s of denaturation at 94°C, 90 s of annealing at 57°C and 60 s of extension at 72°C.

Adenoviral transduction of luciferase gene

For in vitro study, cells infected with either AdCTluc, AdTCPluc or AdCMVluc at 0 to 100 MOI, were plated in 24-well plates at a density of 2 × 104 per well. The luciferase activity was determined by using lysates harvested at 48 h (72 h for TT) after infection in the Luciferase Assay System (Promega, Madison, WI, USA).

To evaluate in vivo dissemination of virus, we injected 5 × 109 p.f.u. of virus in 100 μl of serum-free DMEM or 0.9% sodium chloride solution directly into a rMTC tumor or percutaneously into the peritoneal cavity. Four days later rats were killed. The organs were taken and immediately frozen in liquid nitrogen. 200 mg portions of each tissue were homogenized in 1ml of cell culture lysis reagent (Promega) and incubated 15 min at room temperature. Cell debris was pelleted by centrifugation. The supernatant was transferred into another microcentrifuge tube and frozen at –80°C until assay.

IL-12 proliferation assay using ConA-blasts

The ability of concanavalin A (ConA)-activated mouse splenocytes to respond to mIL-12 has been previously described.56 C57BL/6 splenocytes were incubated in tissue culture medium (TCM) containing 2% heat-inactivated FBS, 30 U/ml human recombinant IL-2 (kindly provided by Dr Jose Quintan, The University of Chicago) and 2 μg/ml ConA (Sigma Chemical, St Louis, MO, USA) at a density of 1 × 106 cells/ml. ConA-blasts were harvested after 3 days of culture for assay. The blasts were resuspended in TCM with 5% heat-inactivated FBS at a concentration of 4 × 105 cells/ml. Aliquots of 50 μl were distributed into 96-well plates (Costar 3596) and 50 μl of serially diluted standard mIL-12 (kindly provided by Dr Thomas Gajewski, The University of Chicago), or samples from culture supernatants, were added. The plates were incubated 48 h. 0.5 μCi of 3H-thymidine (ICN Pharmaceutical, Costa Mesa, CA. USA) was added to each well during the final 18 h of incubation. The cells were then harvested on to glass fiber filters and incorporated tritium was determined. Samples were tested in triplicate. Polyclonal anti-mIL-12 antibody (R&D Systems, Minneapolis, MN, USA) was used to neutralize biological activity of mIL-12. A standard curve using recombinant mIL-12 was included in each experiment and the concentration of mIL-12 in the supernatants was determined by comparison to it. One unit of mIL-12 is defined as the amount of IL-12 producing half-maximum proliferation of the Th1 clone HDK1.57 The specific activity of IL-12 was 5 × 106 U/mg.

Tumorigenicity of AdTCPmIL-12 transduced rMTC cells

Rat MTC cells were infected with virus at 20 MOI for 1 h in vitro, and a 1:1 mixture of infected and uninfected cells (1.5 × 106) was then injected subcutaneously into rats. Tumor size was measured with a caliper in two dimensions every 2 days. The volume was calculated by the formula: width2 × length × 0.5.

Direct intratumoral injection of the recombinant adenovirus

1 × 109 p.f.u. of virus in 100 μl serum-free DMEM was injected into tumors on the flanks of rats. Tumor size was monitored every 2 days by measuring two perpendicular tumor diameters.

Injection of wild type rMTC cells into rats previously treated and tumor free

rMTC cells (1.5 × 106) were injected on the opposite flank of rats which had achieved tumor regression in the tumor treatment study. Wild-type rMTC cells were injected 20 or 60 days after the initial treatment with virus. Tumorigenicity was observed for another 20 days.

Distant effect on tumor regression by injection of recombinant adenovirus

Viruses (1 × 109 p.f.u.) were injected into one tumor of rats bearing a tumor on each flank. The size of both tumors was measured by caliper every 2 days.

mIL-12 assay ELISA

Serum mIL-12 level was determined by mouse IL-12 (p70) OptE1A Set (Pharmingen, San Diego, CA, USA), following a standard procedure.

Liver transaminase assay

Both glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) level in blood were measured with a commercial quantitative colormetric kit (Sigma), following the advised protocol.

Histology

Rat tissues were harvested 7 days after infection of virus and fixed with Zamboni's fixative solution immediately. The specimens, embedded in paraffin and sectioned, were stained with hematoxylin and eosin.

Statistical analysis

The statistical significance of findings was determined by ANOVA and Tukey's HSD procedure. P < 0.05 was considered significant.

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Acknowledgements

We are grateful to our colleague Masaki Takara for giving us helpful suggestions, and to Miss Myrna Zimberg for her excellent secretarial assistance. This work was supported by a Center of Excellence award from Knoll Pharmaceuticals and by the David Wiener Research Fund.

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Correspondence to LJ DeGroot.

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Yamazaki, M., Zhang, R., Straus, F. et al. Effective gene therapy for medullary thyroid carcinoma using recombinant adenovirus inducing tumor-specific expression of interleukin-12. Gene Ther 9, 64–74 (2002) doi:10.1038/sj.gt.3301617

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Keywords

  • gene therapy
  • adenovirus
  • medullary thyroid carcinoma
  • interleukin-12
  • calcitonin
  • tissue-specific promoter

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