Gene Therapy advance online publication 6November2014; doi: 10.1038/gt.2014.95

P FForde1,3, L JHall2,3, Mde Kruijf1, M GBourke1, TDoddy1, MSadadcharam1 and D MSoden1

The current standard of care for cancer uses surgery, radiation and chemotherapy to achieve local tumour control and reduce the risk of disease recurrence. 1 Immunotherapy is potentially a new therapeutic pilar, which can complement the current standard of care and can reduce risk of disease recurrence.2, 3, 4, 5

Immunotherapy-based therapies have the potential to activate a tumour antigen-specific response, which can help to eradicate the tumour and reduce the risk of disease recurrence.6, 7, 8, 9 Delivering immunotherapies clinically can be achieved through a number of approaches including the use of gene therapy, which has many applications and methodologies already developed for cancer treatment.10, 11, 12, 13 For gene therapy to be successful, safe and efficient gene delivery is critical. 12 In current cancer gene therapy studies, viral vectors are used in the majority of gene delivery approaches, as they have high-efficiency transfection.14, 15 However, there are a number of significant drawbacks that include efficiency of production, host immunogenicity, integration and safety. 15, 16 An alternative option to viral vectors is plasmid DNA. Toxicity is generally very low, and large-scale production is relatively easy. 17 However, a major obstacle that has prevented the widespread application of plasmid DNA is its relative inefficiency in gene transfection.17, 18 Therefore, most applications for plasmid DNA have been limited to vaccine studies, with a few exceptions.18, 19 Consequently, methods that can significantly increase plasmid DNA transfection efficiency will greatly extend the utility of this promising mode of gene transfer. The technique of electroporation is widely used in vitro to effectively introduce DNA and other molecules into eukaryotic cells and bacteria. Application of short electrical pulses to the target cells permeabilises the cell membrane, thereby facilitating DNA uptake.20 A number of studies, preclinical and clinical, have shown highly successful responses with electroporated plasmid DNA encoding immune genes and also chemotherapeutic drugs.21, 22, 23, 24, 25 Recently, we have shown that applying electroporation to a range of tissue types using a new electroporation system, EndoVe in a large pig model. This will significantly enhance the application of electrogene therapy. 26

Several cytokines have demonstrated significant antitumour effects. Among these, granulocytemacrophage colony-stimulating factor (GmCSF) is one of the most potent, specific and long-lasting inducers of antitumour immunity. GmCSF can mediate its effect by stimulating the differentiation and activation of dendritic cells (DCs) and macrophages, and by increasing the antigen presentation capability.27 For optimal antigen presentation, engagement of the T-cell receptor with an antigen/major histocompatibility complex requires the costimulatory molecule such as B7-1 (CD80) and B7-2 (CD86). 27, 28 Subsequently, DC and macrophages process and present tumour antigens to T cells, and to both CD4+ and cytotoxic (CD8+) T cells, by augmenting the antitumour response.28 As such, GmCSF is particularly effective in generating systemic immunity against a number of poorly immunogenic tumours. 27

We recently characterised a non-viral vector therapy system: EEV plasmid (pEEV) with a vastly superior expression capacity when compared with a standard control vector. 26 The purpose of this study was to test the therapeutic potential of the pEEV system. We hypothesised that pEEV has the capability to reach the therapeutic threshold for the treatment of solid tumours. We present here the use of pEEV as a gene therapy vector carrying murine GmCSF and human b7-1 genes (pEEVGmCSF-b7.1). We used electroporation as a means to facilitate the delivery of the pEEV and assessed the efficacy and immune induction in primary and secondary responses to treatment in murine colon adenocarcinoma and melanoma cancer models.

In the current study, we investigated the therapeutic efficacy of pEEVGmCSF-b7.1 when compared with a standard vector also expressing GmCSF-b7.1. To test this, two tumour types (CT26 murine colorectal and B16F10 metastatic melanoma) were treated by electroporating tumours with pMG (standard plasmid backbone), pGT141GmCSF-b7.1 (standard plasmid therapy), pEEV (backbone) and pEEVGmCSF-b7.1 ( Figure 1). As expected, the volumes of all non-electroporated (untreated) CT26 tumours and those treated with the empty plasmids, pMG and pEEV, significantly increased (P<0.01) in size ( Figure 1a). However, we did observe that the empty pEEV plasmid inhibited growth of the CT26 tumour between days 8 and 12, which we have observed previously.26 Both therapeutic plasmids delayed the growth rate of the CT26 tumour. Importantly, the growth of pEEVGmCSF-b7.1-treated tumours was significantly more inhibited compared with pGT141GmCSF-b7.1-treated tumours (P<0.002) and untreated control tumours (P<0.0004). By day 26 post-treatment, the untreated and the pMG- and pEEV-treated groups of animals were euthanised because of tumour size ( Figure 1b). Although the standard therapy pGT141GmCSF-b7.1 did inhibit tumour growth, all animals from this group were killed by day 36 when the tumours reached the ethical size of 1.7cm3. One mouse was removed from the pEEVGmCSF-b7.1-treated group on day 36 and again at day 45 because of tumours exceeding the ethical size; however, the remaining 66% of the mice survived up until day 150 post-treatment when they were then killed for subsequent immune analysis. To further test the efficacy of pEEVGmCSF-b7.1 therapy, we used the B16F10 melanoma cell line because of its aggressive nature. Following the same experimental protocol as described for the CT26 model ( Figure 1c), we again observed that untreated tumours grew exponentially with the killing of the mice from day 12 onwards (because of tumour size). Again, we observed that pEEVGmCSF-b7.1 treatment delayed tumour growth when compared with the untreated group (P<0.0001) and pGT141GmCSF-b7.1 (P<0.0001). In terms of survival, the pMG, pEEV, untreated and pGT141GmCSF-b7.1-treated group of animals were killed by day 28 (Figure 1d). Notably, we observed an even greater survival efficacy for the pEEVGmCSF-b7.1 (when compared with the CT26 model) in that 100% mice survived and all remained tumour free for 150 days post-treatment until they were removed for subsequent immune analysis. Similar results were obtained in both tumour types treated based on a range of tumour sizes (Supplementary Figure S2). Taken together, these data indicate that pEEVGmCSF-b7.1 treatment is able to significantly reduce (in the CT26 model) or prevent (B16F10 model) primary tumour growth.

Therapeutic effect on established CT26 and B16F10 solid tumours. (a) Representative CT26 tumour growth curve: each Balb/C mouse was subcutaneously injected with 5 105 CT26 cells in the flank. On day 14 posttumour inoculation, tumours were treated with pMG (), pGT141GmCSF-b7.1 (), pEEV () and pEEVGmCSF-b7.1 () or untreated (). Six mice per groups were used and the experiment was performed two times. Tumour volume was calculated using the formula: V=ab2/6. Data are presented as the meanss.e.m. It was observed that the pEEVGmCSF-b7.1 therapy delayed the growth of the tumours most effectively in comparison with the other groups. At 17 days post-treatment, pEEVGmCSF-b7.1 significantly delayed tumour growth compared with untreated tumour (***P<0.0004) standard therapy vector pGT141GmCSF-b7.1 (**P<0.002). (b) Representative KaplanMeier survival curve of CT26-treated tumours was measured. Only mice treated with pEEVGmCSF-b7.1 survived. Sixty-six per cent of mice survived up to 150 days. All other groups were killed by day 36. (c) Representative growth curve of B16F10 tumour. Each C57BL/6J was subcutaneously injected with 2 x105 B16F10 cells in the flank of the mice. On day 15 posttumour inoculation, tumours were treated with pMG (), pGT141GmCSF-b7.1 (), pEEV () and pEEVGmCSF-b7.1 () or untreated (). Six mice per groups were used and the experiment was performed two times. At 12 days post-treatment, pEEVGmCSF-b7.1 significantly delayed tumour growth compared with untreated tumour (**P<0.0001) standard therapy vector pGT141GmCSF-b7.1 (*P<0.0001). (d) Representative KaplanMeier survival curve of B16F10 showing pEEVGmCSF-b7.1 had 100% survival up to 150 days post-treatment with all other groups killed by day 28. Similar results were obtained in two independent experiments.

As already indicated, for optimal cancer therapy, robust immune responses must be induced; thus, to determine immune cell recruitment, we performed a comprehensive immune population profile of spleens and tumours 72h post-treatment. CT26 tumour mice treated with pEEVGmCSF-b7.1 had a significantly greater percentage of splenic CD19+ (B cells), DX5+/CD3+ (natural killer T (NKT) cells), DX5+/CD3 (NK cells) and CD8+ (cytotoxic T cells) as shown in Table 1. Within the tumour environment, we observed that the percentage of all cell types examined (with the exception of CD4+ cells (T cells)) were significantly greater in pEEVGmCSF-b7.1-treated tumours when compared with untreated tumours. Importantly, when the therapeutic plasmid treatments were compared, we observed that pEEVGmCSF-b7.1-treated mice had significantly more splenic and tumour CD19+ cells (P<0.05) and significantly greater number of tumour DX5+/CD3 (P<0.001), F4/80+ (macrophages) (P<0.001) and CD8+ (P<0.001) cells than control pGT141GmCSF-b7.1-treated mice. We observed a similar immune profile for the B16F10-treated mice ( Figure 2b). Splenic and tumour CD19+ (P<0.001), DX5+/CD3+ (P<0.01), DX5+/CD3 (P<0.01), CD11c+ (DCs) (P<0.001), F4/80 (P<0.001) and CD8+ (P<0.001) cells were all significantly higher for the pEEVGmCSF-b7.1-treated mice than for untreated animals. However, we did not observe any significant differences in CD4+ or T-cell receptor +/CD3+ ( T cells) (data not shown). Notably, when the standard pGT141GmCSF-b7.1 therapy was compared with pEEVGmCSF-b7.1, the percentage of CD19+ (P<0.001), DX5+/CD3+ (P<0.01), CD11c+ (P<0.001) and CD8+ (P<0.001) cells were all significantly greater, indicating that pEEVGmCSF-b7.1 recruits a superior immune recruitment locally at the tumour site. The spleen data had a similar trend as the tumour data with the percentage of CD19+ (P<0.01), DX5+/CD3+ (P<0.001), CD11c+ (P<0.001), F4/80 (P<0.001) and CD8+ (P<0.001) cells all significantly greater in the pEEVGmCSF-b7.1-treated mice when compared with the standard therapy. These data indicate that treatment with pEEVGmCSF-b7.1 induces robust recruitment of innate and adaptive immune cell populations in both colorectal and metastatic melanoma models.

Percentage of the respective T cells found locally at the site of the B16F10 tumours treated with pMG, pEEV, pGT141GmCSF-b7.1 and pEEVGmCSF-b7.1 or untreated. (a) Represents data obtained for the CD4+CD25+FoxP3+cells and (b) CD4+CD25FoxP3+ cells. Data represent the mean of the respective cells. Error bars show s.d. from four animals. The asterisks (*) indicate significant values of *P <0.05 and **P <0.01 as determined by one-way analysis of variance (ANOVA) following Bonferronis multiple comparison of pEEVGmCSF-b7.1 compared with untreated tumour. The asterisks () indicate significance values of P<0.05 as determined by one-way ANOVA following Bonferronis multiple comparison of pEEVGmCSF-b7.1 compared with the standard vector pGT141GmCSF-b7.1. The asterisks () indicate significant values of P<0.05 as determined by one-way ANOVA following Bonferronis multiple comparison of pEEVGmCSF-b7.1 compared with pMG. The asterisks (*) indicate significant values of *P<0.05 as determined by one-way ANOVA following Bonferronis multiple comparison of untreated compared with pEEV. Similar results were obtained in two independent experiments.

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