Prodrug-tethered lipid nanoparticles for synergistic mRNA cancer immunotherapy

Introduction: Messenger RNA (mRNA)-based immunotherapy has emerged as a breakthrough strategy for cancer treatment. However, phenotypic regulation of T cells between activated and exhausted stages remains a significant challenge. Thus, a potential approach to improve anti-cancer T cell activity could be to simultaneously deliver interleukin (IL-12), to stimulate effector T cells, and indoleamine 2,3-dioygenase (IDO) inhibitor, to suppress T cell exhaustion. Here, we designed prodrug ionizable lipid nanoparticles (pLNPs), via a novel library of prodrug ionizable lipids (pILs), consisting of an intracellularly cleavable IDO inhibitor within the ionizable lipid and encapsulating mRNA transcript for IL-12 to achieve synergistic cancer immunotherapy.

Materials and

Methods: A library of pILs was synthesized using SN2 reaction between prodrug cores and epoxide-terminated lipid tails and then used to formulate pLNPs by microfluidic mixing with mRNA. The library of pILs was then formulated into pLNPs using standard lipid excipients and screened in vitro and in vivo using FLuc mRNA cargo. After obtaining the lead pIL, the efficacy of lead pIL for strong IL-12 mRNA transfection was further confirmed by ELISA and flow cytometry. At the same time, the capability of pLNP for drug release when triggered by GSH was monitored by HPLC. Finally, the treatment efficacy of pLNP encapsulating IL-12 mRNA was evaluated in a subcutaneous tumor model of colon adenocarcinoma.

Results, Conclusions, and Discussions: A library of pILs was generated from seven prodrug amine heads and three epoxide-terminated lipid tails to formulate distinct pLNPs (Fig. 1A). In vitro and in vivo screening of pLNPs encapsulating reporter firefly luciferase (FLuc) mRNA revealed a top-performing pIL, G0-SS-AA-C12 (Fig. 1B,C). Substitution of FLuc mRNA with IL-12 mRNA in G0-SS-AA-C12 pLNPs resulted in both strong IL-12 transfection of cancer cells and indoximod release, leading to T cell activation and reversal of T cell exhaustion (Fig. 1A). In a subcutaneous tumor model of colon adenocarcinoma, we demonstrated that pLNPs encapsulating IL-12 mRNA cleared primary tumors (Fig. 1D) by eliciting effector T cell infiltration while reducing T cell exhaustion. In terms of survival, PBS-treated mice succumbed to tumor burden before Day 45, and all G0-SS-AA-C12 IL-12 pLNP-treated mice survived to our study endpoint compared to other control groups (Fig. 1E). In summary, we engineered a novel pIL library coupled to an IDO inhibitor and formulated an pLNP platform with the top-performer encapsulating a therapeutic cytokine (IL-12) mRNA for solid tumor regression via mRNA-based cancer immunotherapy.

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