P403 Intratumoral administration of a multigene construct by electroporation can effectively modulate anti-tumor response in a murine B16.F10 model Shawna Shirley1, Anandaroop Mukhopadhyay1, Christoph Burkart1, Jocelyn Wright1, Robert Pierce2, Jean Campbell2, David Canton1 1Oncosec Medical Incorporated, San Diego, CA, USA 2Fred Hutchinson Cancer Research Center, Seattle, WA, USA Background Immunomodulatory cytokines, such as interleukin-12 (IL-12) are attractive candidates for cancer immunotherapy. IL-12 is a pro-inflammatory cytokine with potent anti-tumor effects; however, systemic administration of IL-12 shows limited clinical efficacy and dose-associated toxicity. In preclinical and clinical studies, intratumoral (IT) delivery of IL-12 plasmid DNA by electroporation (EP) can provide a safe and effective alternative for efficacious dosing. To augment the effects of IL-12, we developed a DNA plasmid platform that allows for delivery of agents that modulate multiple immune pathways as well as tumor- or patient-specific neoantigens. Polycistronic IL-12 Immune Modulator plasmid (PIIM) is a single plasmid encoding IL-12, and a fusion of Flt3L to an antigen. Flt3L is a ligand that stimulates dendritic cell (DC) maturation and enhances antigen processing and presentation. The encoded antigen can be a viral or shared antigen, or a patient-specific neoantigen, which enables customization to patient populations, as well as providing an aid to monitoring antigen-specific immune response(s) that can be correlated to patient outcomes. Here we demonstrate the first functional characterization of PIIM. Methods Two PIIM constructs were created for functional characterization: PIIM-OVA (IL12~Flt3L-OVA) for mouse experiments, and PIIM-NYESO1 (IL12~Flt3L-NYESO1) for testing in human cells. The expression and functional activity of PIIM components were determined. Treated tumors and spleens were assessed for transcriptional changes by NanoString® and phenotypic changes by flow cytometry. Systemic effects of PIIM were assessed using a syngeneic two-tumor model of B16.F10 in which only one tumor received IT-PIIM-EP while the other contralateral lesion remained untreated. Results PIIM-OVA and PIIM-NYESO1 secrete functional IL-12p70, Flt3L-OVA and Flt3L-NYESO1 fusion proteins as assessed by ELISA, flow and cell-based assays. PIIM promotes DC maturation and antigen-specific T cell proliferation both ex vivo and in vivo. Hydrodynamic-based gene delivery of PIIM-OVA lead to splenomegaly and significantly increased splenic CD11c+ DCs. Furthermore, IT-PIIM-EP lead to generation of splenic OVA-specific CD8s and increased APM gene expression. When introduced intratumorally in a mouse two-tumor model, IT-PIIM-EP delays B16.F10 tumor growth in both treated and contralateral tumors compared to untreated controls resulting in increased overall survival. Conclusions PIIM represents a novel approach to cancer immunotherapy. A combination of functional immune modulators can be expressed locally in the tumor microenvironment that increase inflammatory infiltrate, enhances antigen presentation and produces a systemic T cell response specific to the antigen encoded on the plasmid. This customizable approach has the potential to improve therapeutic outcome by enhancing adaptive-immunity and addressing patient-specific neoantigens needs. mouse experiments, and PIIM-NYESO1 (IL12~Flt3L-NYESO1) for testing in human cells. The expression and functional activity of PIIM components were determined. Treated tumors and spleens were assessed for transcriptional changes by NanoString® and phenotypic changes by flow cytometry. Systemic effects of PIIM were assessed using a syngeneic two-tumor model of B16.F10 in which only one tumor received IT-PIIM-EP while the other contralateral lesion remained untreated. Results PIIM-OVA and PIIM-NYESO1 secrete functional IL-12p70, Flt3L-OVA and Flt3L-NYESO1 fusion proteins as assessed by ELISA, flow and cell-based assays. PIIM promotes DC maturation and antigen-specific T cell proliferation both ex vivo and in vivo. Hydrodynamic-based gene delivery of PIIM-OVA lead to splenomegaly and significantly increased splenic CD11c+ DCs. Furthermore, IT-PIIM-EP lead to generation of splenic OVA-specific CD8s and increased APM gene expression. When introduced intratumorally in a mouse two-tumor model, IT-PIIM-EP delays B16.F10 tumor growth in both treated and contralateral tumors compared to untreated controls resulting in increased overall survival. Conclusions PIIM represents a novel approach to cancer immunotherapy. A combination of functional immune modulators can be expressed locally in the tumor microenvironment that increase inflammatory infiltrate, enhances antigen presentation and produces a systemic T cell response specific to the antigen encoded on the plasmid. This customizable approach has the potential to improve therapeutic outcome by enhancing adaptive-immunity and addressing patient-specific neoantigens needs.
