Tumor cell-based vaccines use tumor cells as a source of tumor-associated antigens. In our study, we aimed to develop and test a tumor vaccine composed of tumor cells killed by irradiation combined with in vivo interleukin-12 gene electrotransfer as an adjuvant. Vaccination was performed in the skin of B16-F10 malignant melanoma or CT26 colorectal carcinoma tumor-bearing mice, distant from the tumor site and combined with concurrent tumor irradiation. Vaccination was also performed before tumor inoculation in both tumor models and tumor outgrowth was followed. The antitumor efficacy of vaccination in combination with tumor irradiation or preventative vaccination varied between the tumor models. A synergistic effect between vaccination and irradiation was observed in the B16-F10, but not in the CT26 tumor model. In contrast, up to 56% of mice were protected from tumor outgrowth in the CT26 tumor model and none were protected in the B16-F10 tumor model. The results suggest a greater contribution of the therapeutic vaccination to tumor irradiation in a less immunogenic B16-F10 tumor model, in contrast to preventative vaccination, which has shown greater efficacy in a more immunogenic CT26 tumor model. Upon further optimization of the vaccination and irradiation regimen, our vaccine could present an alternative tumor cell-based vaccine.
Interleukin 12 (IL-12) is a cytokine used as a therapeutic molecule in cancer immunotherapy. Gene electrotransfer mediated delivery of IL-12 gene has reached clinical evaluation in the USA using a plasmid that in addition to IL-12 gene also carry an antibiotic resistance gene needed for its production in bacteria. In Europe however, European Medicines Agency recommends against the use of antibiotics during the production of clinical grade plasmids. We have prepared several antibiotic resistance gene-free plasmids using an antibiotic-free selection strategy called operator-repressor titration, including plasmids encoding mouse, canine and human IL-12 orthologues. The aim of this study was to evaluate the maintenance of these plasmids in bacterial culture and test their transfection efficiency using gene electrotransfer. Plasmid maintenance was evaluated by determining plasmid yields and topologies after subculturing transformed bacteria. Transfection efficiency was evaluated by determining the plasmid copy number, expression and cytotoxicity after gene electrotransfer to mouse, canine and human melanoma cells. The results demonstrated that our IL-12 plasmids without an antibiotic resistance gene are stably maintained in bacteria and provide sufficient IL-12 expression after in vitro gene electrotransfer; therefore, they have the potential to proceed to further in vivo evaluation studies.
Multimodal treatment approaches, such as radio-immunotherapy, necessitate regimen optimization and the investigation of the interactions of different modalities. The aim of this study was two-fold. Firstly, to select the most effective combination of irradiation and the previously developed tumor cell-based vaccine and then to provide insight into the immune response to the selected combinatorial treatment. The study was performed in immunologically different murine tumor models: B16F10 melanoma and CT26 colorectal carcinoma. The most effective combinatorial treatment was selected by comparing three different IR regimens and three different vaccination regimens. We determined the local immune response by investigating immune cell infiltration at the vaccination site and in tumors. Lastly, we determined the systemic immune response by investigating the amount of tumor-specific effector lymphocytes in draining lymph nodes. The selected most effective combinatorial treatment was 5× 5 Gy in combination with concomitant single-dose vaccination (B16F10) or with concomitant multi-dose vaccination (CT26). The combinatorial treatment successfully elicited a local immune response at the vaccination site and in tumors in both tumor models. It also resulted in the highest amount of tumor-specific effector lymphocytes in draining lymph nodes in the B16F10, but not in the CT26 tumor-bearing mice. However, the amount of tumor-specific effector lymphocytes was intrinsically higher in the CT26 than in the B16F10 tumor model. Upon the selection of the most effective combinatorial treatment, we demonstrated that the vaccine elicits an immune response and contributes to the antitumor efficacy of tumor irradiation. However, this interaction is multi-faceted and appears to be dependent on the tumor immunogenicity.
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