Introduction
Cancer immunotherapy is an experimental approach for treatment of cancer patients. It aims at evoking immune-based responses against malignant cells by activating and recruiting cells from the innate and adaptive immune system, including T cells that recognize tumor-specific antigens [
1]. Virus-mediated gene transfer has been widely used to enhance the susceptibility of cancer cells to immunotherapy. Therapeutic genes expressed by viral vectors included a broad number of immune modulators, such as e.g. granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interferons or CD40 ligand (CD40L) [
2‐
4]. These approaches have proven to be inefficient, however, since most tumors express weak tumor antigens and also lack co-stimulatory molecules necessary for induction of cellular immunity, and evade immune recognition. An additional major limitation of cancer immunotherapy has been the low rates of gene transfer.
Strategies to improve both, the potency of immune recognition of cancer cells and the efficacy of gene therapy are clearly required to successfully employ the promising concept of cancer immunotherapy. One way to enhance the duration of therapeutic gene expression is to increase viral spreading [
5], for example by replacing non-replicating therapeutic virus vectors with armed oncolytic viruses, which replicate selectively within cancers and also express therapeutic genes [
6‐
8]. Therapeutic genes include prodrug converting enzymes, suicide genes, and immunostimulatory proteins. The most widely used oncolytic viral vectors have been derived from non-integrating parental viruses, such as vaccinia virus, herpes simplex virus, measles virus and human Ad [
9]. Human Ads have an excellent safety profile in cancer gene therapy [
10]. In addition, they are easy to produce in large amounts, and efficient infection is possible with vectors derived from various serotypes or by tropism engineering [
11]. The latter is based on the availability of adequate methods to generate recombinant vectors of choice, including CRAds for cancer treatment [
12,
13].
The number of inserted therapeutic genes is, however, limited for Ad due to the packaging capacity of the viral capsid [
14]. One promising way to overcome this limitation is to use trans-complementing co-replication, where a CRAd is mixed with a second, E1-deleted and therefore replication-deficient (RD) vector expressing the therapeutic gene(s) of interest. In such a system, the E1A gene expressed by the first vector complements the second vector
in trans, which gives rise to efficient replication of both vectors, thereby strongly increasing the DNA copy number on a per cell basis. This concept was first confirmed by combining transduction of an E1-deleted Ad with transfection of a plasmid containing the E1 genes and subsequent production of progeny virus and enhanced viral transgene expression [
15,
16].
Subsequently, numerous variations of the binary virus system have been tested. The group of Alemany was the first to combine two defective viruses, a RD E1-deleted virus and a helper-dependent virus containing the E1 genes under the control of the liver tissue-specific promoter and demonstrated oncolytic spread following injection of a 1:1 mix into human hepatocarcinoma mouse xenograft model [
17]. Several groups used RD Ad vectors expressing reporter genes such as β-galactosidase, luciferase or eGFP to show enhancement of virus replication and cell spreading [
18‐
23]. More recently, this system has led to new exciting applications for non-invasive
in vivo imaging of tumor spread and assessment of Ad replication [
24‐
27]. Therapeutic genes utilized in binary vector systems included prodrug converting enzymes such as herpes simplex virus-thymidine kinase [
28,
29] and P450 enzyme [
21], suicide genes like Bcl-xs [
30], p53 [
31,
32], p27 [
25], tumor necrosis factor α-related apoptosis-inducing ligand [
23,
33‐
36], dominant-negative insulin-like growth factor-1R [
22], antiangiogenic soluble vascular endothelial growth factor receptor 2-Fc [
26,
37], and immunostimulatory proteins like GM-CSF [
33,
38], IL-2 and IL-12 [
39]. In most studies, co-administration of RD therapeutic vectors and CRAds was also tested in
in vivo xenotransplant models. Improved oncolytic efficacy was found and in some cases lead to complete and long lasting regression, which was not achieved when using the individual viral vectors alone [
21‐
26,
28,
29,
33‐
37,
39].
The currently available dual vector co-replication systems control the expression of the therapeutic genes by strong ubiquitous promoters lacking tissue or tumor specificity. In this study we suggested to combine a CRAd and a RD vector containing the same cancer cell-selective promoter, and to test, whether a strong enhancement of transgene expression can be achieved by trans-complementation. The results presented here show that when using the cancer cell-selective TETP promoter previously described for our melanoma/melanocyte-specific CRAd vector [
40] in combination with two novel RD vectors expressing IL-2 and CD40L from a bicistronic expression cassette, only moderate 3-fold enhancements of IL-2 or CD40L were obtained, whereas controls including the CMV promoter allowed much stronger expression enhancement in the Ad vector co-replication system. Possible reasons for this finding are discussed.
Discussion
Strategies to improve experimental cancer therapy include co-delivery of RC oncolytic Ads together with RD vectors expressing therapeutic genes [
17,
21,
22,
26,
28,
30,
31,
33‐
39]. There is a large choice of candidate therapeutic genes that has been evaluated, including tumor-suppressor, immunomodulatory or cytotoxic genes. Depending on the type of cytotoxic genes used, premature death of transduced cells was found to reduce replication of oncolytic viruses, thereby antagonizing the therapeutic efficacy [
54]. Genes with indirect antitumor effects such as immune stimulators might thus be advantageous. We have shown earlier that the combination of IL-2 and CD40L had an improved efficacy over the use of single agents, when applied for direct
in situ therapy or vaccination therapy in a mouse melanoma model [
44]. Moreover, we showed that intratumoral injection of an IL-2-expressing Ad vector could induce tumor regression in patients with advanced melanoma [
3]. The currently 287 ongoing or closed gene therapy protocols utilizing Ad vectors for cancer treatment include 20 protocols with IL-2, and 11 protocols with CD40L as therapeutic gene, respectively. Five protocols, mainly for treatment of leukemia, include combined expression of IL-2 and CD40L
http://www.wiley.co.uk/genmed/clinical/.
Utilization of a binary vector system as compared to direct therapeutic expression from single oncolytic viruses poses disadvantages, including more demanding vector manufacturing and clinical handling. On the other side, possible advantages compared to armed oncolytic vectors include their flexible application in combination with an increase of the overall therapeutic gene capacity of the delivery system. For example, a single CRAd can be combined with numerous therapeutic RD vectors, which have been tested previously as single agents. Alternatively, efficacy of different CRAds could be compared side-by-side in combination with the same therapeutic vector. The binary vectors may also be safer than single viruses, as the amount of vector expressing the therapeutic gene can be delayed to later rounds of virus application, and can, in addition, be dose-adjusted. Dissemination of the therapeutic vector, however, remains a risk, in particular if strong ubiquitous promoters are used to control expression. Thorne et al., e.g., found that co-injection of an oncolytic AdΔ24 together with RD CMV promoter/enhancer-controlled luciferase vector into xenotransplanted tumors not only gave rise to enhanced and more durable gene expression in the tumor tissue, but also led to secondary, weaker bioluminescence in the liver [
26]. Thus, additional safety measures are warranted for this kind of approach.
Here we suggest to utilize our previously described melanoma/melanocyte-specific TETP [
40] to control expression of CD40L/IL-2 from a bicistronic expression vector. About 30 tissue-specific mammalian promoters have been evaluated in RC CRAds [
47], and many more were tested for their fidelity to express transgenes from RD vectors (reviewed in [
46]). The quest for such promoters was brought up following the findings that
in vivo, expression from vectors containing the ubiquitously active and strong CMV promoter resulted in efficient transduction that peaked within a few days, but frequently became undetectable by one month after injection. These findings were ascribed to either elimination of viral genomes by the immune system or to methylation-induced promoter silencing, and it was suggested that this could be overcome by using tissue-specific mammalian promoters [
55,
56].
We found that replacement of the CMV enhancer promoter with the TETP allowed tight melanoma-specific transgene expression, as IL-2 expression levels amounted to a minimal 149-fold to a maximal 5652-fold higher expression levels in melanoma cells compared to HeLa cells. This is comparable with earlier reports, where a recombinant Ad expressing β-Gal under the control of two copies of the mouse tyrosinase enhancer in combination with a 770 bp mouse tyrosinase promoter gave rise to 100- to 200-fold higher expression in melanoma cells compared to non-melanoma cells [
57]. In our experiments, a comparison of TETP-controlled transgene expression levels with those derived from CMV-controlled vectors revealed, depending on cell line and transgene, 5 to- 40-fold lower expression levels in melanoma cells. This is in line with findings that tissue-specific promoters frequently turned out to be relatively weak promoters compared to CMV or other strong viral promoters [
58]. A slightly different version of tyrosinase promoter/enhancer combination has been claimed to reach, at least in a very limited number of melanoma cells, comparable expression levels as from CMV promoter [
57], whereas others found only maximal 1.3% of the CMV promoter-driven transcriptional activity [
59]. In our hands, TETP-controlled luciferase expression was found to give rise to about 10- to 20-fold higher expression levels compared to SV40 promoter [
40].
Variations in the expression levels of IRES upstream and downstream genes have been observed previously. In one study, IRES-dependent second gene expression has been reported to range from 6 to 100% of the first gene expression. This was found to be influenced by several factors, including the choice of optimal/nonoptimal Kozak sequence of the ATG start codon, as well as the particular cell type used for expression [
48]. In contrast, in IRES-encoding RNA viruses, yields of translation product from the downstream IRES-dependent cistron were also found to be higher than from the upstream cistron [
60].
When we used the AdCMV-eGFP/wtAd5 binary vector system, robust trans-complementation-mediated reporter expression enhancement was obtained in melanoma and non-melanoma cells. In our eight cell types tested, four revealed enhancements in the range of 3 to 10, and four had enhancements larger than 10, with a maximal 363-fold for SW480 cells on day 4. Such a strong variation of trans-complementation-mediated enhancement has been found in other studies, and may depend on cell type used, exact transgene vector: CRAd ratio, time point of individual virus additions (variable in some studies), trans-activation of the CMV promoter by cellular and viral gene products [
51], and most importantly, also the type and dynamic range of analysis system utilized for quantification. Enhancement values of > 50, e.g., were obtained by others when using luciferase assays [
22,
24,
28], ELISA [
38,
39], and virus progeny titers [
19] which have the largest dynamic range.
When replacing the transgene vector AdCMV-eGFP with AdCMV-CD40L or AdCMV-IL-2, 49- and 131-fold trans-complementation-mediated transgene expression enhancement was found in melanoma M000301 cells, when combined with wtAd5, and 53- and 288-fold enhancement when combined with AdΔEP-TETP. To our surprise, combination of the newly established AdTETP-CD40L/IL-2 with AdΔEP-TETP or wtAd5 revealed an unexpectedly low trans-complementation-mediated expression enhancement of maximal 3.3 fold for the more sensitive IL-2 expression analysis. Possible reasons for this low enhancement effect could include competition for virus binding, similar entry pathways or post entry factors such as transcription factors or nuclear domains for replication. We consider entry competition unlikely, since trans-complementation with CMV-promoters from Ad5 capsids was highly efficient. All viruses tested here utilize the Coxsackie virus B Ad receptor as attachment receptor and αv integrins as entry receptors [
61].
The unique difference between the AdCMV-eGFP and AdTETP-CD40L/IL-2 vectors relates to the sequences of the CMV-eGFP and TETP-CD40L/IL-2 expression cassettes, whereas the rest of the viral genome is identical (Fig.
1). Transcription factors could be a limiting factor if they bound in a rate-limiting manner to the TETP sequence (but not to the CMV enhancer/promoter sequence), and at the same time also to one of the viral promoters. The human tyrosinase promoter sequence consists of the M-box (a conserved element found in other melanocyte-specific promoters) containing a first E-box motif, an SP1 site, and a highly conserved CR2 element comprising a second E-box motif and an overlapping octamer element [
62]. Both E-boxes were demonstrated to bind the basic helix-loop-helix transcription factor Mitf, which is essential for melanocyte differentiation. Of note, a related E-box motif is also contained in the Ad major late promoter (MLP), which usually is bound by USF, another ubiquitous member of the basic helix-loop-helix transcription factor family [
63]. Based on band shift assays, it was found that Mitf also could bind to the E-box of the MLP, and conversely, the USF also could bind to the M-box of melanocyte-specific promoters [
53]. Similarly, band shift assays with an oligonucleotide containing the SP1 motif of the tyrosinase promoter were competed by a SP1 motif found in the Ad EII late promoter [
53]. The tyrosinase enhancer sequence is less clearly characterized, but was suggested to contain a binding site for a member of the fos family transcription factor [
64]. The strong CMV enhancer/promoter contains multiple transcription factor binding sites, including the ubiquitous Sp1 family of transcription factors, NF-κB, retinoic acid nuclear receptors and CREB/ATF [
65]. Thus, depending on the cell type and expression levels of members of the basic helix-loop-helix transcription factor or SP1 family, competition for such factor(s) could possibly contribute to the lower enhancement of the Ad trans-complementation system found here.
However, a more likely explanation relates to the findings by the group of Goding, which reported that ectopic expression of Ad E1A resulted in down regulation of Mitf in mouse melan-a melanoma cells, leading to repression of TRP-1 and tyrosinase levels and subsequent loss of pigmentation [
52]. This repression could be relieved by over expression of Mitf. We confirmed down regulation of Mitf transcripts in human M000301 melanoma cells following infection with E1A-expressing wtAd5 and AdΔEP-TETP, but not with E1A-deleted AdCMV-lacZ. Whether this is the exclusive mechanism to explain lack of strong enhancement, or whether there is an additive effect together with the above discussed competition for transcription factors remains to be further studied. In addition, as strong and specific delivery of therapeutic genes is one of the main goals of cancer therapy, it may be of importance for the general usage of the binary Ad expression system to investigate whether this finding is unique to the TETP system tested here, or whether it also occurs with other tissue- or cell type-specific promoters.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
ACF and SH designed the experiments, generated the Ad vectors and carried out the transduction studies. VL performed the IL-2 ELISA, and OS carried out the RT-PCR measurements. SH coordinated the study and wrote the manuscript. RD and UFG participated in the design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.