Non-Clinical In Vitro Evaluation of Antibiotic Resistance Gene-Free Plasmids Encoding Human or Murine IL-12 Intended for First-in-Human Clinical Study.

Spela Kos,Maja Cemazar,Tanja Jesenko,Gregor Sersa,Urska Kamensek,Andrej Rencelj,Masa Bosnjak,Urska Matkovic,Katarina Znidar,Bostjan Markelc,Rosana Hudej,Aneja Tuljak,Matjaz Peterka

doi:10.3390/pharmaceutics13101739

Abstract

Interleukin 12 (IL-12) is a key cytokine that mediates antitumor activity of immune cells. To fulfill its clinical potential, the development is focused on localized delivery systems, such as gene electrotransfer, which can provide localized delivery of IL-12 to the tumor microenvironment. Gene electrotransfer of the plasmid encoding human IL-12 is already in clinical trials in USA, demonstrating positive results in the treatment of melanoma patients. To comply with EU regulatory requirements for clinical application, which recommend the use of antibiotic resistance gene-free plasmids, we constructed and developed the production process for the clinical grade quality antibiotic resistance gene-free plasmid encoding human IL-12 (p21-hIL-12-ORT) and its ortholog encoding murine IL-12 (p21-mIL-12-ORT). To demonstrate the suitability of the p21-hIL-12-ORT or p21-mIL-12-ORT plasmid for the first-in-human clinical trial, the biological activity of the expressed transgene, its level of expression and plasmid copy number were determined in vitro in the human squamous cell carcinoma cell line FaDu and the murine colon carcinoma cell line CT26. The results of the non-clinical evaluation in vitro set the basis for further in vivo testing and evaluation of antitumor activity of therapeutic molecules in murine models as well as provide crucial data for further clinical trials of the constructed antibiotic resistance gene-free plasmid in humans.

Highlights

IntroductionInterleukin 12 (IL-12) is one of the most potent proinflammatory cytokines in the mediation of the antitumor activity of immune cells
A scalable production process for clinical grade plasmid manufacturing with relevant analytical methods was established, and thoroughly characterized plasmids were produced for non-clinical evaluation
The process is amenable to scale-up and cGMP manufacturing

Summary

Introduction

Interleukin 12 (IL-12) is one of the most potent proinflammatory cytokines in the mediation of the antitumor activity of immune cells. It has long been studied as a potential immunotherapeutic for cancer based on its ability to engage multiple effector mechanisms and reverse tumor-induced immunosuppression [1]. IL-12 induces Th1 cell differentiation, increases activation and cytotoxicity of T lymphocytes and natural killer (NK) cells and inhibits immunosuppressive cells, such as tumor-associated macrophages or myeloid-derived suppressor cells [1]. Most of the IL-12-induced effects are mediated by the secretion of interferon gamma (IFNγ), which itself exerts cytostatic and antiangiogenic activities and upregulates the major histocompatibility complex (MHC) I and Pharmaceutics 2021, 13, 1739.

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