The landscape of cancer immunotherapy has seen transformative advancements with the advent of lipid nanoparticle (LNP)-encapsulated mRNA. By delivering immunomodulatory proteins directly into tumors, this localized strategy avoids systemic toxicity while unleashing a precise immune assault on cancer cells. A novel approach combining mRNA for interleukin-21 (IL-21), interleukin-7 (IL-7), and 4-1BB ligand (4-1BBL) within LNPs, dubbed Triplet LNP, demonstrates unprecedented therapeutic synergy in eliminating tumors and building long-lasting immunity.

Traditionally, systemic delivery of immunotherapies such as IL-21 or 4-1BB agonists has been plagued by dose-limiting toxicities and poor tumor-specific effects. Triplet LNP bypasses these hurdles by localizing immunostimulatory activity within the tumor microenvironment (TME). This innovation ignites CD8+ T-cell responses, enhances their cytotoxic potential, and synergizes with checkpoint blockade therapies, showcasing its versatility across different tumor models.

IL-21, a key member of the γ-chain cytokine family, plays a pivotal role in enhancing the effector functions of CD8+ T cells and natural killer (NK) cells. Alone, IL-21 demonstrates limited clinical efficacy due to its short half-life and insufficient immune activation. However, when paired with IL-7 and 4-1BBL, its immunostimulatory potential multiplies.

IL-7 primarily sustains naïve CD8+ T cells and drives their differentiation into memory T cells, fortifying long-term immune defenses. It counteracts T-cell exhaustion, a common obstacle in the TME, where immunosuppressive factors diminish the efficacy of immune therapies. Meanwhile, 4-1BBL provides a critical co-stimulatory signal that enhances T-cell proliferation, survival, and effector cytokine production. Together, these molecules create a dynamic immunological environment capable of activating tumor-specific CD8+ T cells and increasing their infiltration into tumors.

This three-component synergy is critical. In preclinical models, Triplet LNP treatment achieves tumor regression rates far exceeding those of monotherapies or dual combinations. Importantly, the therapy confers long-term protection against tumor rechallenge, highlighting its ability to generate immunological memory.

Intratumoral injection of Triplet LNP initiates a cascade of events within the TME. The nanoparticles, composed of ionizable lipids and mRNA cargo, efficiently deliver their payload to both immune (CD45+) and non-immune (CD45-) cells within tumors. Particularly, tumor-associated macrophages (TAMs) and dendritic cells (DCs) demonstrate the highest uptake and expression of the delivered mRNA.

DC activation is a cornerstone of Triplet LNP efficacy. Upon uptake, conventional type 2 dendritic cells (cDC2s) increase the expression of maturation markers such as CD80 and CCR7, indicating readiness to migrate toward tumor-draining lymph nodes (tdLNs). In tdLNs, these activated DCs interact with naïve and stem-like CD8+ T cells, facilitating their differentiation into effector cells primed to infiltrate tumors.

The role of tumor-draining lymph nodes is critical. Blocking CD8+ T-cell trafficking from tdLNs to tumors eliminates the therapeutic efficacy of Triplet LNP, underscoring the importance of this interstitial communication. Triplet LNP enhances this coordination, increasing the frequency of tumor-specific CX3CR1+ CD8+ T cells in the blood—a potential biomarker for therapeutic response.

The Triplet LNP fundamentally reshapes the immune landscape within tumors. Following treatment, CD8+ T cells dominate the TME, outpacing immunosuppressive TAMs. These T cells not only infiltrate tumors in higher numbers but also exhibit heightened functional states, characterized by increased production of granzyme B, IFN-γ, and TNF-α—cytokines essential for tumor cell destruction.

Exhausted T-cell subsets, which typically limit immune responses in chronic TME conditions, are transformed. The treatment induces expression of exhaustion markers (e.g., PD-1, LAG-3) alongside cytotoxic signatures, indicating a functional exhaustion state with preserved anti-tumor activity. Transcriptomic analyses reveal expanded T-cell clusters enriched in genes regulating cytotoxicity, T-cell activation, and migration.

Interestingly, while NK cells also exhibit enhanced functionality post-treatment, their role in tumor clearance is dispensable. Depleting CD8+ T cells, however, abolishes the therapeutic efficacy of Triplet LNP, proving that CD8+ T cells are the primary mediators of this immunotherapy.

One of the most compelling findings of Triplet LNP therapy is its ability to eradicate untreated distal tumors, a phenomenon known as the abscopal effect. This systemic anti-tumor immunity arises from CD8+ T-cell activation in the tdLNs and subsequent trafficking of tumor-specific T cells to secondary tumor sites.

In models of subcutaneous and orthotopic tumors, local Triplet LNP treatment effectively reduced the growth of distant, non-injected tumors. Similarly, in a lung pseudometastasis model, the therapy significantly reduced metastatic burden and improved survival rates. This demonstrates that localized delivery can induce widespread systemic immunity, offering potential for metastatic cancer treatment.

Systemic delivery of 4-1BB agonists has been marred by severe hepatotoxicity, limiting their clinical application. Triplet LNP addresses this challenge through localized administration, minimizing off-target effects while maximizing tumor-specific activity. Across multiple studies, mice treated with Triplet LNP showed no significant weight loss, elevated liver enzymes, or long-term liver inflammation. This favorable safety profile positions Triplet LNP as a compelling alternative to conventional immunotherapies.

Translating preclinical successes to clinical efficacy is the ultimate goal of Triplet LNP therapy. Human tumor biopsy analyses reveal that LNPs efficiently transfect both immune and non-immune cells, particularly macrophages. This finding validates the potential of Triplet LNP in human settings, where TAMs dominate the immune microenvironment.

Higher expression levels of IL-21, IL-7, and TNFSF9 (encoding 4-1BBL) are associated with improved survival outcomes across multiple cancer types, further supporting the clinical relevance of this approach. Moving forward, integrating Triplet LNP with immune checkpoint blockade therapies, such as anti-PD-1, holds promise for overcoming resistance in refractory cancers.

The development of Triplet LNP encapsulating mRNA for IL-21, IL-7, and 4-1BBL marks a paradigm shift in immunotherapy. By leveraging precise intratumoral delivery, this approach overcomes the limitations of systemic immunostimulatory therapies, enabling potent and durable anti-tumor immunity. Its ability to activate CD8+ T cells, reshape the immune microenvironment, and induce systemic responses positions it as a formidable tool in the fight against cancer.

As research progresses toward clinical trials, Triplet LNP stands poised to redefine the boundaries of cancer treatment, offering new hope for patients with otherwise limited options.

Study DOI: https://doi.org/10.1038/s41467-024-54877-9

Engr. Dex Marco Tiu Guibelondo, B.Sc. Pharm, R.Ph., B.Sc. CpE

Editor-in-Chief, PharmaFEATURES

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