Cancer immunotherapy holds enormous potential in combating malignant tumors. Messenger ribonucleic acid (mRNA) has emerged as a widely studied tool in recent years, functioning within the cytoplasm and eliminating the risk of unintentional insertion or mutation that was associated with plasmid DNA (pDNA) in the past. mRNA possesses the ability to encode tumor antigens (TAs), immune cell receptor cytokines, and antibodies. However, due to the inherent instability of mRNA's structure, there is a need to develop effective delivery systems. Lipid nanoparticles (LNPs) have become a crucial medium for mRNA delivery in cancer immunotherapy, offering protection to mRNA and enhancing intracellular delivery efficiency.
LNPs for mRNA Delivery
LNPs can serve as drug delivery systems for encapsulating small molecules, nucleic acids, small interfering RNA (siRNA), and mRNA. They are composed of ionizable lipids, helper lipids, cholesterol, polyethylene glycol (PEG) lipids, and mRNA. The characteristic feature of ionizable lipids is their pH sensitivity. Due to their head groups' ability to change charge, they can transition from a positively charged state at low pH to a neutral state at physiological pH. This property allows them to form "mRNA ionizable cationic lipid" complexes that stabilize and protect mRNA in a pH-dependent manner. Another key component, PEG-lipids, prolongs circulation and enhances stability by preventing macrophage activation and phagocytosis. The choice of PEG-lipids depends on the molar mass of PEG and lipid length, which can impact targeted delivery and cellular uptake efficiency. Helper lipids and cholesterol play crucial roles in the formation of LNPs, controlling LNP fluidity or rigidity. Particularly, cholesterol can influence effective LNP delivery and distribution and, through specific modifications, can selectively target certain cell types.
Tumor Immunotherapy Strategies Based on mRNA-LNPs
LNP-based cancer immunotherapy uses the following four main strategies.
(1) Activating the immune response by encoding TAs.
(2) Expressing antigen receptors, such as CAR (Chimeric Antigen Receptor) or T cell receptor (TCR).
(3) Encoding adjuvants to stimulate the immune system.
(4) Encoding immune-related proteins, such as cytokines and antibodies.
- Cancer Antigen Presentation
TA is a protein expressed in cancer cells, recognized as foreign by the immune system, and presented to T cells and B cells by antigen-presenting cells (APCs). This can induce a potent anti-cancer immune response. TA can exist in various forms, including full-length proteins, antigenic peptides, or pDNA encoding specific cancer antigens. Some studies have suggested that personalized neoantigens used in dendritic cell (DC) vaccines can have effective anti-tumor effects. In clinical trials, the feasibility of inducing an immune response in malignant melanoma using autologous tumor mRNA has been evaluated. DCs transfected with mRNA encoding TAs have become an effective cancer treatment strategy, showing long-term survival rates in clinical trials for brain cancer, prostate cancer, renal cell carcinoma, and melanoma. Notably, 50% of metastatic melanoma patients receiving DC vaccines alone or in combination with Interleukin-2 (IL-2) achieved long-term survival without severe adverse reactions. Given these anti-tumor effects, Oberli and colleagues optimized LNPs and demonstrated the intracellular delivery of mRNA-encoded TAs to APCs, promoting cytotoxic CD8+ T cell responses in a melanoma mouse model. In another study, in an OVA-bearing mouse model, LNP-OVA mRNA and C16-R848 effectively inhibited tumor growth. Sasaki and colleagues reported a method for optimizing LNPs using a microfluidic device to select the appropriate size and lipid composition. They delivered E.G7-OVA mRNA using A-11-LNP and compared it with two other LNP formulations, showing that A-11-LNP exhibited superior gene expression activity and maturation in DCs and demonstrated a significant anti-tumor therapeutic effect in the E.G7-OVA tumor model.
In another study, Chen and colleagues reported a lymph node-targeting mRNA vaccine based on LNPs, named 113-O12B, for cancer immunotherapy. In an in vivo model with B16F10-OVA, the targeted delivery of mRNA to the lymph nodes triggered a robust CD8+ T cell response to the encoded full-length OVA.
- CAR Engineered Immune Cells
Engineered immune cell therapy has the potential to specifically target cancer cells for cancer treatment. CARs encoded by mRNA can be delivered to immune cells either in vitro or in vivo, where they are expressed on the cell surface to target cancer cells. Several clinical and preclinical studies have demonstrated the potential of mRNA-encoded CARs in cancer immunotherapy. Billingsley and colleagues have shown the feasibility of generating CAR-T cell therapies based on mRNA delivery platforms by developing ionizable lipids and optimizing LNP libraries with various combinations. LNPs encapsulating mRNA encoding CD19 CAR expressed CD19 CAR at levels similar to or higher than electroporation. CARs generated using this approach can also be applied to NK cell therapy, although viral vectors are currently more widely used, even though this method has greater potential.
One significant limiting factor of mRNA-based CARs is their relatively short duration of expression, which may restrict their therapeutic effectiveness. To overcome this challenge, researchers are exploring strategies to enhance mRNA stability and prolong CAR expression. These strategies include optimizing mRNA sequences and structures, optimizing LNP formulations to enhance intracellular delivery and release, with the aim of enabling sustained or repeated administration of mRNA-encoded CARs.
Immunogenic adjuvants play a role in modulating antigen recognition, upregulating co-stimulatory molecules, and inducing cytokine signaling. Toll-like receptors (TLRs) recognize conserved structures present in various pathogens and trigger innate immune responses, especially the production of Type I interferons (IFNs). Because TLR agonists can bridge innate and adaptive immune responses, they hold great promise as adjuvants in cancer therapy. Stimulator of Interferon Genes (STING) is a protein crucial in the innate immune response, and its activation triggers signaling cascades involving transcription factors like interferon regulatory factor 3 (IRF-3) and CD8+ T cell immune signals. Due to its central role in immune responses, STING has become a promising avenue for developing cancer immunotherapy.
In one study, combining LNP-encapsulated mRNA vaccines with an adjuvant (STING-V155M mutant of Interferon Gene Stimulator) enhanced immune responses in preclinical models and clinical research. This adjuvant was initially discovered in a patient with STING-associated vasculopathy of infancy (SAVI) and was found to significantly increase CD8+ T cells, enhance the immunogenic response to vaccines, and activate Type I interferon pathway via nuclear factor kappa B (NF-κB) and interferon-stimulated response elements (ISRE). When used in combination with an mRNA vaccine targeting human papillomavirus (HPV) cancer proteins, STING-V155M reduced tumor growth and improved survival in vaccinated mice, indicating the potential of mRNA-encoded adjuvants in cancer immunotherapy.
- Cytokines: Encoding Immune-Related Proteins
The IL-1 and IL-12 families collaborate to elicit anti-inflammatory and anti-tumor immune responses. The IL-1 family (comprising IL-1, IL-18, IL-33, IL-36, IL-37, and IL-38) participates in early immune responses following antigen invasion. IL-36, in particular, is associated with a favorable prognosis in cancer and stimulates APCs and T cells. The IL-12 family of cytokines serves as a bridge between innate and adaptive immunity, with IL-23 being a member that regulates immune responses and exhibits anti-tumor effects. Hewitt and colleagues designed an mRNA-LNP delivery system encoding OX40, IL-36, and IL-23 for mono- and combination therapy against tumors. Delivery of the three mRNAs (OX40, IL-36, and IL-23) via LNPs effectively activated DCs and T cells, significantly enhancing anti-cancer effects compared to individual mRNA treatments. These strategies triggered both innate and adaptive immune responses, re-attacking tumors, and effectively preventing tumor recurrence. In another study, the use of mRNA-LNPs encoding cytokines for anti-cancer therapy was demonstrated. These cytokines (IL-12, IL-27, and GM-CSF) acted synergistically, increasing T cell survival in the tumor microenvironment (TME) and promoting memory T cells through IFN-γ and IL-10. This combination had a significant tumor-suppressive effect in a melanoma model without toxicity. The combination of IL-12 and IL-27 attracted B cells, macrophages, CD4+/CD8+ T cells, and NK cells, demonstrating the potential of mRNA-LNP delivery systems for multiple cytokines to mobilize immune cells and provide effective therapy.
- Antibodies: Encoding Immune-Related Proteins
Antibody therapy has shown remarkable efficacy in clinical settings, but it still has certain limitations. Stability issues, the complexity of large-scale manufacturing, and treatment costs can hinder its widespread application. One approach to address the limitations and challenges of conventional antibodies is to deliver mRNA encoding antibodies for in vivo antibody production. HER2 antibody (i.e., trastuzumab) is a prime example of targeted cancer treatment. When trastuzumab binds to HER2 on cancer cells, it exerts its anticancer effects by blocking cancer cell proliferation and survival pathways. Building on this mechanism, Rybakova and colleagues utilized LNP delivery to express trastuzumab-encoding mRNA. When injected into mice, the concentration of trastuzumab antibody expressed in the serum gradually increased until it disappeared after 7 days. This suggests that mRNA-LNP delivery encoding antibodies can serve as an alternative to traditional antibody therapy.
Sahin's team designed mRNA encoding RiboMABs targeting CD3 and Claudin6 (CLDN6), one of the tumor-associated antigens (TAAs). After injecting CD3×CLDN6-RiboMAB LNPs into mice, they monitored the concentration of these mRNA-derived antibodies in the serum over time. The mRNA gradually decreased within 144 hours, while the antibody proteins rapidly disappeared after 6 hours of dosing. This study demonstrates that low-dose mRNA-based on LNP can be repeatedly administered and replicated, allowing for continuous antibody production. This overcomes the limitations of the short half-life of antibody therapy and showcases its potential clinical applicability.
In another study by Thran and colleagues, they explored the use of rituximab (a CD20 antibody used for targeted lymphoma therapy) and investigated the utility of chemically modified mRNA in passive immunity. They designed LNP-encapsulated mRNA encoding rituximab and evaluated its anti-tumor effects. In an in vivo lymphoma model, the group treated with LNP mRNA therapy showed a higher tumor suppression rate and survival rate compared to the group treated with recombinant rituximab antibody therapy. Furthermore, a single injection of mRNA-LNP was sufficient to achieve rapid, stable, and sustained serum antibody titers, providing preventive and therapeutic protection against deadly rabies infections or botulinum toxin poisoning. These findings suggest that mRNA-LNP encoding antibodies offer better delivery and therapeutic efficacy compared to their recombinant protein counterparts.
Conclusion and Future Outlook
In summary, mRNA-based therapeutic strategies have garnered significant attention in recent years due to their simplicity of manufacture and the ability to encode proteins without genomic mutations. However, the instability of mRNA structures necessitates effective carriers or delivery systems to enhance intracellular uptake. With the emergence of nanoparticle-targeted delivery technologies, LNPs have become an innovative delivery platform capable of improving mRNA stability and intracellular delivery efficiency, making them highly promising candidates for cancer immunotherapy. mRNA encoding cancer antigens, CARs, adjuvants, cytokines, and antibodies has demonstrated potential in reducing tumor growth, further underscoring the potential of LNP-facilitated mRNA-based cancer immunotherapy. As research and development in this field continue to advance, we can anticipate the emergence of more effective, personalized, and safer treatment approaches. These advancements hold the promise of improving treatment outcomes for cancer patients, mitigating adverse effects of therapy, and enhancing overall quality of life. Ultimately, these iterative improvements will continue to elevate the efficacy of treatment and provide new hope and opportunities in cancer therapy based on mRNA.
References
- Han, J.; et al. Lipid nanoparticle-based mRNA Delivery Systems For Cancer Immunotherapy. Nano Convergence.2023, 10(36).