Development of bicistronic plasmids and fusion proteins for clinical translation of tumor immune reprogramming

Delivery of 4-1BB ligand (4-1BBL) and interleukin 12 (IL-12) via poly(β-amino ester) (PBAE) nanoparticles (NPs) enables reprogramming of tumor cells into tumor-associated antigen-presenting cells (tAPCs), stimulating anti-tumor immune responses. Existing work on 4-1BBL/IL-12 NPs employs a dual-plasmid system and plasmid backbones containing antibiotic selection, both of which represent challenges for clinical translation. Production of two plasmids adds manufacturing complexity and costs, with amplification and purification processes required for both plasmids. Additionally, regulatory agencies discourage the use of antibiotic-resistance components in gene therapies due to concerns around rising antibiotic resistance. Here, we describe an approach to address manufacturing and regulatory challenges in translating 4-1BBL/IL-12 NPs into the clinic by engineering bicistronic plasmids co-expressing 4-1BBL and IL-12 via a T2A peptide or a (G₄S)₅ linker and utilizing smaller, antibiotic resistance-free nanoplasmid (NanoP) backbones. In vivo, we demonstrated bicistronic and NanoP-based tAPC NPs exhibited similar or improved survival compared to separate plasmid delivery of 4-1BBL and IL-12 in a B16-F10 tumor model. These results support the translation of bicistronic, antibiotic resistance gene-free plasmid strategies to advance tAPC PBAE-NPs and, more broadly, gene-delivered therapies toward the clinic.