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

Joanna Yang; Sabrina S Chen; Ethan Idnani; Sydney R Shannon; Kathryn Luly; Charina S Fabilane; D Scott Wilson; Jamie B Spangler; Stephany Y Tzeng; Jordan J Green

doi:10.1016/j.omta.2026.201708

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

Mol Ther Adv. 2026 Feb 28;34(2):201708. doi: 10.1016/j.omta.2026.201708. eCollection 2026 Jun 11.

Authors

Joanna Yang^{1

2}, Sabrina S Chen^{1

2}, Ethan Idnani^{1

2}, Sydney R Shannon^{1

2}, Kathryn Luly^{1

2}, Charina S Fabilane², D Scott Wilson^{1

2}, Jamie B Spangler^{1

2

3

4

5

6}, Stephany Y Tzeng^{1

2}, Jordan J Green^{1

2

3

4

5

7

8

9}

Affiliations

¹ Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.
² Translational ImmunoEngineering Center, Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.
³ Department of Chemical & Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
⁴ Department of Oncology and the Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.
⁵ Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.
⁶ Department of Molecular Microbiology & Immunology, Johns Hopkins University School of Public Health, Baltimore, MD 21205, USA.
⁷ Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
⁸ Department of Materials Science & Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
⁹ Institute for NanoBioTechnology and the Bloomberg∼Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD 21231, USA.

Abstract

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.

Keywords: T cell; bioengineering; cancer; gene delivery; gene therapy; immunoengineering; immunotherapy; melanoma; non-viral; plasmid.