No abstract

An estimated 350 million people are living with chronic Hepatitis B virus (HBV) worldwide. Preventative HBV vaccination in infants has reduced the disease burden; however, insufficient immunization programs and access obstacles leave vulnerable populations at risk for infection in endemic regions. Gene electrotransfer (GET) using a noninvasive multielectrode array (MEA) provides an alternative platform for DNA vaccination in the skin. DNA vaccines are nonlive and nonreplicating and temperature stable unlike their counterparts. In addition, their simple engineering allows them to be manufactured quickly at a low cost. In the current work, we present the combination of GET and moderate heating for delivery of a DNA vaccine against HBV. Our laboratory has previously shown the synergy between moderate tissue preheating at 43°C and GET with the MEA as a means to reduce both the applied voltage and pulse number to achieve similar if not higher gene expression than GET alone. In this study, we expand upon this work, by optimizing the plasmid dose to achieve the highest level of expression. Using the reporter gene luciferase, we found that an intradermal injection of 100 lL at 1 mg/mL induced the highest expression levels across all tested GET conditions. We then evaluated our moderate heat-assisted GET platform for the intradermal delivery of a plasmid encoding Hepatitis B surface antigen (pHBsAg) via a prime and prime plus boost vaccination protocol. At 18 weeks, following the prime plus boost protocol, we observed that a high-voltage low-pulse GET condition with moderate heating (45 V 36 p+heat) generated antibodies against Hepatitis B surface antigen (HBsAb) at peak measuring 230-fold over injection of plasmid DNA alone with moderate heating. HBsAbs remained robust over the 30-week observation period. These data suggest that moderate heatassisted GET has the potential to induce strong immune responses, an attractive feature for development of an alternative vaccine delivery platform.

Gene-based approaches for protein replacement therapies have the potential to reduce the number of administrations. Our previous work demonstrated that expression could be enhanced and/or the applied voltage reduced by preheating the tissue prior to pulse administration. In the current study, we utilized our 16-pin multi-electrode array (MEA) and incorporated nine optical fibers, connected to an infrared laser, between each set of four electrodes to heat the tissue to 43 °C. For proof of principle, a guinea pig model was used to test delivery of reporter genes. We observed that when the skin was preheated, it was possible to achieve the same expression levels as gene electrotransfer without preheating, but with a 23% reduction of applied voltage or a 50% reduction of pulse number. With respect to expression distribution, preheating allowed for delivery to the deep dermis and muscle. This suggested that this cutaneous delivery approach has the potential to achieve expression in the systemic circulation, thus this protocol was repeated using a plasmid encoding Human Factor IX. Elevated Factor IX serum protein levels were detected by ELISA up to 100 days post gene delivery. Further work will involve optimizing protein levels and scalability in an effort to reduce application frequency.

Resistance to checkpoint-blockade treatments is a challenge in the clinic. Both primary and acquired resistance have become major obstacles, greatly limiting the long-lasting effects and wide application of blockade therapy. Many patients with metastatic melanoma eventually require further therapy. The absence of T-cell infiltration to the tumor site is a well-accepted contributor limiting immune checkpoint inhibitor efficacy. In this study, we combined intratumoral injection of plasmid IL-12 with electrotransfer and anti-PD-1 in metastatic B16F10 melanoma tumor model to increase tumor-infiltrating lymphocytes and improve therapeutic efficacy. We showed that effective anti-tumor responses required a subset of tumor-infiltrating CD8+ and CD4+ T cells. Additionally, the combination therapy induced higher MHC-I surface expression on tumor cells to hamper tumor cells escaping from immune recognition. Furthermore, we found that activating T cells by exposure to IL-12 resulted in tumors sensitized to anti-PD-1 treatment, suggesting a therapeutic strategy to improve responses to checkpoint blockade.

Immune checkpoint inhibitors (ICIs) have been demonstrated to have significant clinical benefits. Effectiveness of ICIs have been limited by both primary and acquired resistance. In addition, absence of T cell infiltration within the tumor site is one of the major obstacles limiting ICIs efficacy against solid tumors. Interleukin-12 (IL-12) is a potent immune stimulator that has been shown to stimulate growth and survival of T cells as well as NK cells. Utilizing a B16.F10 melanoma model in C57Bl/6 mice, we observed a significant reduction in T regulatory cells and myeloid derived suppressor cells while T effector cells were significantly increased within the tumor microenvironment (TME) following intratumor delivery of a plasmid encoding IL-12 using gene electrotransfer. Monotherapy with plasmid IL-12 delivered with gene electrotransfer (pIL-12 GET) resulted in prolonged disease-free survival as well as long term immune memory. To determine the robustness of this therapeutic approach and to evaluate the responsiveness in combination with anti-PD1, we used a two-tumor model consisting of a subcutaneous B16F10 tumor and B16F10 cells expressing luciferase injected via the intraperitoneal route in a C57Bl/6 mouse. Intratumor delivery of pIL-12 GET as a monotherapy resulted in reduction or elimination of the subcutaneous tumor. However, the monotherapy approach was only successful in reducing the peritoneal spread in about 50% of the mice. When pIL-12 GET was combined with anti-PD1 administered via an intraperitoneal injection not only was there an elimination of the subcutaneous tumor, but it resulted in the elimination of intraperitoneal metastatic growth. The elimination of peritoneal spread was confirmed utilizing an In Vivo Imaging System. Observations on day 60 revealed background levels of luminescence in 90% of mice treated with the combination therapy thus confirming long-term disease-free survival of these mice. This level of response was not seen in mice treated with anti-PD-1 alone or with simple injection of pIL-12. In addition, the combination therapy resulted in an enhanced memory response which protected against growth of new tumors following challenge. In addition, exposure of tumor cells to IL-12 resulted in upregulation of MHC class 1 and PDL1 expression. The results have this study suggest that the combination of pIL-12 GET combined with ICIs could be utilized as an effective combination therapy for melanoma. Citation Format: Richard Heller, Megan Scott, Cathryn Mangiamele, Jody Synowiec, Guilan Shi. Gene therapy approach to suppress metastasis in a mouse melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4210.